Immuno-analysis of microparticles: Probing at the limits of detection

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ∼60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30-40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data

Exploring experimental cerebral malaria pathogenesis through the characterisation of host-derived plasma microparticle protein content

© 2016 The Author(s). Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection responsible for thousands of deaths in children in sub-Saharan Africa. CM pathogenesis remains incompletely understood but a number of effectors have been proposed, including plasma microparticles (MP). MP numbers are increased in CM patients' circulation and, in the mouse model, they can be localised within inflamed vessels, suggesting their involvement in vascular damage. In the present work we define, for the first time, the protein cargo of MP during experimental cerebral malaria (ECM) with the overarching hypothesis that this characterisation could help understand CM pathogenesis. Using qualitative and quantitative high-throughput proteomics we compared MP proteins from non-infected and P. berghei ANKA-infected mice. More than 360 proteins were identified, 60 of which were differentially abundant, as determined by quantitative comparison using TMT TM isobaric labelling. Network analyses showed that ECM MP carry proteins implicated in molecular mechanisms relevant to CM pathogenesis, including endothelial activation. Among these proteins, the strict association of carbonic anhydrase I and S100A8 with ECM was verified by western blot on MP from DBA/1 and C57BL/6 mice. These results demonstrate that MP protein cargo represents a novel ECM pathogenic trait to consider in the understanding of CM pathogenesis

DIANNEXIN DOWN-MODULATES TNF-INDUCED ENDOTHELIAL MICROPARTICLE RELEASE BY BLOCKING MEMBRANE BUDDING PROCESS.

BACKGROUND: Microparticles are now recognised as true biological effectors with a role in immunopathology through their ability to disseminate functional properties. Diannexin, a homodimer of annexin V, binds to PS with a higher affinity and longer blood half-life than the monomer, inhibits prothrombinase complex activity thereby diminishing coagulation and reperfusion injury mediators and prevent microvesicle-mediated material transfer. Our aim was to determine if Diannexin could modulate microparticle production by endothelial cells by interacting with the phosphatidylserine exposure occurring during the release of these vesicles. RESULTS: In this study we showed that fluorescently labelled Diannexin binds to calcimycin-activated endothelial cells but not to resting cells. After overnight incubation, Diannexin enters cells and their released MP carry Diannexin. Some Diannexin seems to be processed via early endosomes and later is found in lysosomes. Both unlabelled Diannexin and fluorescent Diannexin inhibit MP release from TNF-activated endothelial cells. However, Diannexin treatment does not prevent endothelial activation by TNF. In addition, the inhibitory effect of Diannexin on MP release could be observed when cells were pre-, concomitantly or post-treated with cytokines. Scanning electron microscopy showed differences in the numbers and types of protuberances at the cell surface when cells were treated or not with Diannexin. Finally, there is no apparent congruency between fluorescent Diannexin labelling and surface protuberances as shown by correlative microscopy. CONCLUSIONS: Altogether these data suggest that Diannexin can inhibit endothelial vesiculation by binding PS present either at the cell surface or at the level of the inner leaflet of the plasma membrane

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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How do patients and their family members experience the transition from peritoneal dialysis to incentre haemodialysis? A multisite qualitative study in England and Australia.

BACKGROUND: While numerous studies have explored the patient experience of dialysis or other end-stage kidney disease (ESKD) treatments, few have explored the process of transitioning between dialysis modalities. This study aimed to develop an in-depth understanding of patient and caregiver perceptions and experiences of the transition from peritoneal to haemodialysis (HD) and to identify ways in which transitions can be optimised. METHODS: Fifty-four in-depth, semi-structured interviews were undertaken at six study sites across the West Midlands, UK (n = 23), and Queensland, Australia (n = 31). Thirty-nine participants were patients with ESKD; the remainder were family members. An inductive analytical approach was employed, with findings synthesised across sites to identify themes that transcended country differences. RESULTS: Of the 39 patient transitions, only 4 patients reported a wholly negative transition experience. Three cross-cutting themes identified common transition experiences and areas perceived to make a difference to the treatment transition: resistance to change and fear of HD; transition experience shared with family; and bodily adjustment and sense of self. CONCLUSION: Although each transition is unique to the individual and their circumstances, kidney care services could optimise the process by recognising these patient-led themes and developing strategies that engage with them. Kidney care services should consider ways to keep patients aware of potential future treatment options and present them objectively. There is potential value in integrating expert support before and during treatment transitions to identify and address patient and family concerns

Professionalising the 'resilience' sector in the Pacific Islands region: formal education for capacity building

Increasingly practitioners and policy-makers recognise the importance of integrating disaster risk management (DRM) and climate change adaptation (CCA). This approach has been adopted by the European Union Pacific Technical Vocational Education and Training on Sustainable Energy and Climate Change Adaptation Project (EU PacTVET) project. A key barrier to improving national resilience to disaster risk and climate change impacts has been identified as a lack of capacity and expertise resulting from the absence of sustainable accredited and quality-assured training programmes. TVET modules and tools developed under the EU PacTVET project will establish a community of practitioners supporting community resilience in Oceania

HDL Interfere with the Binding of T Cell Microparticles to Human Monocytes to Inhibit Pro-Inflammatory Cytokine Production

BACKGROUND: Direct cellular contact with stimulated T cells is a potent mechanism that induces cytokine production in human monocytes in the absence of an infectious agent. This mechanism is likely to be relevant to T cell-mediated inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. Microparticles (MP) generated by stimulated T cells (MPT) display similar monocyte activating ability to whole T cells, isolated T cell membranes, or solubilized T cell membranes. We previously demonstrated that high-density lipoproteins (HDL) inhibited T cell contact- and MPT-induced production of IL-1beta but not of its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra). METHODOLOGY/PRINCIPAL FINDINGS: Labeled MPT were used to assess their interaction with monocytes and T lymphocytes by flow cytometry. Similarly, interactions of labeled HDL with monocytes and MPT were assessed by flow cytometry. In parallel, the MPT-induction of IL-1beta and sIL-1Ra production in human monocytes and the effect of HDL were assessed in cell cultures. The results show that MPT, but not MP generated by activated endothelial cells, bond monocytes to trigger cytokine production. MPT did not bind T cells. The inhibition of IL-1beta production by HDL correlated with the inhibition of MPT binding to monocytes. HDL interacted with MPT rather than with monocytes suggesting that they bound the activating factor(s) of T cell surface. Furthermore, prototypical pro-inflammatory cytokines and chemokines such as TNF, IL-6, IL-8, CCL3 and CCL4 displayed a pattern of production induced by MPT and inhibition by HDL similar to IL-1beta, whereas the production of CCL2, like that of sIL-1Ra, was not inhibited by HDL. CONCLUSIONS/SIGNIFICANCE: HDL inhibit both MPT binding to monocytes and the MPT-induced production of some but not all cytokines, shedding new light on the mechanism by which HDL display their anti-inflammatory functions

In vitro assessment of adsorbents aiming to prevent deoxynivalenol and zearalenone mycotoxicoses

The high prevalence of the Fusarium mycotoxins, deoxynivalenol (DON) and zearalenone (ZON) in animal feeds in mild climatic zones of Europe and North America results in considerable economic losses, as these toxins affect health and productivity particularly of pigs from all age groups. The use of mycotoxin adsorbents as feed additives is one of the most prominent approaches to reduce the risk for mycotoxicoses in farm animals, and to minimise carry-over of mycotoxins from contaminated feeds into foods of animal origin. Successful aflatoxin adsorption by means of different substances (phyllosilicate minerals, zeolites, activated charcoal, synthetic resins or yeast cell-wall-derived products) has been demonstrated in vivo and in vitro. However, attempts to adsorb DON and ZON have been less encouraging. Here we describe the adsorption capacity of a variety of potential binders, including compounds that have not been evaluated before, such as humic acids. All compounds were tested at realistic inclusion levels for their capacity to bind ZON and DON, using an in vitro method that resembles the different pH conditions in the gastro-intestinal tract of pigs. Mycotoxin adsorption was assessed by chemical methods and distinct bioassays, using specific markers of toxicity as endpoints of toxicity in cytological assays. Whereas none of the tested substances was able to bind DON in an appreciable percentage, some of the selected smectite clays, humic substances and yeast-wall derived products efficiently adsorbed ZON (>70%). Binding efficiency was indirectly confirmed by the reduction of toxicity in the in vitro bioassays. In conclusion, the presented test protocol allows the rapid screening of potential mycotoxin binders. Like other in vitro assays, the presented protocol combining chemical and biological assays cannot completely simulate the conditions of the gastro-intestinal tract, and hence in vivo experiments remain mandatory to assess the efficacy of mycotoxin binders under practical conditions

Parasitoid Increases Survival of Its Pupae by Inducing Hosts to Fight Predators

Many true parasites and parasitoids modify the behaviour of their host, and these changes are thought to be to the benefit of the parasites. However, field tests of this hypothesis are scarce, and it is often unclear whether the host or the parasite profits from the behavioural changes, or even if parasitism is a cause or consequence of the behaviour. We show that braconid parasitoids (Glyptapanteles sp.) induce their caterpillar host (Thyrinteina leucocerae) to behave as a bodyguard of the parasitoid pupae. After parasitoid larvae exit from the host to pupate, the host stops feeding, remains close to the pupae, knocks off predators with violent head-swings, and dies before reaching adulthood. Unparasitized caterpillars do not show these behaviours. In the field, the presence of bodyguard hosts resulted in a two-fold reduction in mortality of parasitoid pupae. Hence, the behaviour appears to be parasitoid-induced and confers benefits exclusively to the parasitoid