Investigating sepsis immunomodulation and the role of vasopressor therapies using monocyte functional assays

In sepsis, monocytes exhibit the differential immune response states of ‘priming’ (increased responsiveness to secondary stimuli) and ‘deactivation’ (reduced responsiveness, depressed expression of HLA-DR and CD86, increased PDL-1), which may reflect the opposing hyperinflammatory and immune-suppressive systemic conditions. In an era where the number and phenotype of circulating leucocytes are used to guide sepsis immune therapy investigation, there is debate as to whether sepsis immunity is differentially regulated within the blood and tissue compartments of the body. Adding to this uncertainty is the fact that core support therapies, such as vasopressor resuscitation, may have an immunomodulatory effect during sepsis. We hypothesised that monocytes undergo trans-endothelial reprogramming during migration between vascular and tissue compartments and that this is a crucial determinant of sepsis immunity and its clinical monitoring. Further, we hypothesised that noradrenaline and vasopressin have a role in the functional and phenotypic modification of monocytes during sepsis. Our aims were to 1) develop an in-vitro model of priming and deactivation using healthy volunteer (HV) monocytes; 2) to test the direct effects of noradrenaline and vasopressin on monocytes in comparison to sera from the VAsopressin versus Noradrenaline as Initial therapy in Septic sHock (VANISH) trial; 3) develop a human lung microvascular endothelial cell (HLMVEC) transwell model of monocyte migration and associated phenotypic changes during sepsis. The major findings of this work were that in combination vasopressin and noradrenaline suppressed LPS-induced TNF release in non-pretreated and primed HV monocytes. In contrast, when HV monocytes were incubated with VANISH patient sera we found no difference in surface marker expression or LPS-induced TNF release. Conditioning of monocytes with vasopressin or noradrenaline was found to enhance their migration in an uncoated transwell assay. Lastly, we successfully developed an HLMVEC-coated transwell migration assay able to detect changes in pre- and post-migration monocyte phenotype.Open Acces

The regulation and biological activity of cell surface virulence determinants in model opportunist aerobic and anaerobic bacterial pathogens

Burkholderia cepacia is an aerobic Gram- negative bacterium originally described as the cause of soft rot in onions but now recognised as a serious human pathogen most notably in patients with cystic fibrosis (CF), the most common, fatal inherited disease affecting Caucasian populations. Bacteroides fragilis, a Gram- negative commensal associated with the mucosal surface of the human colon, is the most frequently isolated anaerobic bacterium from clinical specimens and is increasingly implicated as an important source of endotoxin in gut-derived sepsis. Previously, both organisms were considered to pose little hazard to human health and consequently, their pathogenesis and virulence factors are poorly understood. The cell surface components of a bacterium are classic virulence determinants and the influence of growth environment on the plasticity of the bacterial surface is well -established. This thesis considers the environmental regulation and biological activity of putative virulence factors for both organisms.Initial studies focused on the expression and antigenicity of the cell surface components; lipopolysaccharide (LPS), exopolysaccharide (EPS) and outer membrane from several representative strains. To determine the influence of growth environment, these components were investigated under different culture conditions and extraction methods. Results demonstrated that as with other Gram- negative organisms, the composition of outer membrane proteins of both B. cepacia and B. fragilis were influenced by cultural conditions with bacteria inducing or repressing protein structures presumably to influence overall permeability and survival. The EPS and LPS of B. fragilis also varied with environmental growth condition. Interestingly, extraction method was found to influence the LPS structure of B. cepacia including the loss of the distinctive rough LPS phenotype of an 'epidemic' strain.B. cepacia and B. fragilis exhibited a greater biological activity than previously recognised both in terms of endotoxicity and cytokine induction. For B. cepacia the capacity to induce the proinflammatory cytokines TNF -a and IL -8 from several cell types was significantly, and unexpectedly, higher compared to the other major CF pathogen, Pseudomonas aeruginosa. This enhanced inflammatory potential of B. cepacia was not due to a more efficient LPS signalling pathway. As both CF pathogens appeared to induce TNF -a in a similar manner, the combined effect of both species was examined. Surprisingly, when P. aeruginosa was present in increasing amounts compared to B. cepacia, cytokine levels were down -regulated. These results indicate that B. cepacia has a major potential to cause immune - mediated damage and concurrent colonisation with P. aeruginosa may modulate this effect. For B. fragilis cytokine levels were compared to Escherichia coli, a facultative anaerobic commensal considered of great importance in gut- derived sepsis due to its extremely active LPS. TNF -a levels induced by B. fragilis were 20 -fold lower than E. coli. However, considering the predominance of Bacteroides species in the gut, outnumbering facultative organisms by 20 -300 fold, results imply that as a population B. fragilis may possess as much biological potential as E. coli. Thus, B. fragilis may play a vital role in gut- derived sepsis. The relevance of these findings to the understanding of B. cepacia in CF and of B. fragilis in sepsis is discussed

Proceedings of the FAA-NASA Symposium on the Continued Airworthiness of Aircraft Structures

This publication contains the fifty-two technical papers presented at the FAA-NASA Symposium on the Continued Airworthiness of Aircraft Structures. The symposium, hosted by the FAA Center of Excellence for Computational Modeling of Aircraft Structures at Georgia Institute of Technology, was held to disseminate information on recent developments in advanced technologies to extend the life of high-time aircraft and design longer-life aircraft. Affiliations of the participants included 33% from government agencies and laboratories, 19% from academia, and 48% from industry; in all 240 people were in attendance. Technical papers were selected for presentation at the symposium, after a review of extended abstracts received by the Organizing Committee from a general call for papers

Investigation into the role of monocyte tumour necrosis factor-alpha converting enzyme as a regulator of the inflammatory response in sepsis

Sepsis consists of both the systemic inflammatory response syndrome (SIRS) and the compensatory anti-inflammatory response syndrome (CARS). How these differential response states are regulated is yet to be fully elucidated. Tumour necrosis factor-alpha (TNF) is one of the principal cytokines involved in mediating SIRS. TNF is released from cells by tumour necrosis factor-alpha converting enzyme (TACE), this enzyme is responsible for the ectodomain cleavage of a number of other substrates relevant to inflammation including both TNF receptors and the adhesion molecule L-selectin. How TACE contributes to, and functions in, SIRS and CARS is not yet known. My objective was to investigate TACE activity and associated substrate shedding in monocytes, specifically how the enzyme behaved in the context of in vitro models that I designed to induce states of priming and tolerance. I then obtained in vivo samples from critically ill patients to determine whether there were similarities between the TACE activity profiles found in patient cells, and volunteer cells placed in the in vitro models. My aims were: 1) Determine how TACE activity profiles were altered when sequential inflammatory stimuli were utilised in a two-hit model of sepsis designed to induce states of priming and tolerance and 2) To perform a clinical study to investigate TACE behaviour in the context of critical illness. I successfully refined a method of isolating primary monocytes from healthy volunteers and patients that allowed determination of TACE activity profiles. Furthermore, I demonstrated that the LPS-TACE axis was reset in the context of a two-hit LPS model and in sepsis. I found evidence of differential signalling pathway reprogramming in monocytes taken from patients with infectious and non-infectious SIRS. Finally, I demonstrated that the monocyte TACE response to LPS is dependent on cell contact. These data provide new insights into monocyte inflammatory function during the immune response

Mechanisms of Vascular Disease: A Reference Book for Vascular Specialists

New updated edition first published with Cambridge University Press. This new edition includes 29 chapters on topics as diverse as pathophysiology of atherosclerosis, vascular haemodynamics, haemostasis, thrombophilia and post-amputation pain syndromes