Neutrophil Microvesicles Restrict the Phlogistic Activation of Macrophages

PhDReleased in response to cellular activation, microvesicles are a major vector mechanism for the delivery of protein, nucleic acid and bioactive lipid payloads in local tissues and plasma. Large numbers of microvesicles (including those from neutrophils) are found within inflammatory sites, such as the rheumatoid synovium. Human neutrophil microvesicles promote tissue protection, and in some cases repair, by affecting function and phenotype of other inflammatory cells. Of these, tissue macrophages are central to the recovery of homeostasis after an inflammatory insult. The data herein indicate that microvesicles released by activated neutrophils impede lipopolysaccharide and interferon gamma-induced \M1-like" polarisation of macrophages via phosphatidylserine (PtdSer) exposure, and induce annexin A1-dependent release of transforming growth factor beta (TGFb). Macrophages treated with these vesicles stimulate the production of cartilage matrix from chondrocytes, and are unable to induce an inflammatory phenotype in fi broblasts. The efficacy of these vesicles is reproduced in two in vivo models of acute inflammation, zymosan-induced peritonits and K/BxN serum-transfer arthritis. Finally, the possibility of using both autologous, and cell-line-derived microvesicles as pharmacodynamic tools is explored. Microvesicles generated from neutrophils from patients with rheumatoid arthritis are found to be protective, and can outcompete the pro-inflammatory effects of both platelet microvesicles, and those isolated from synovial fluid of patients with rheumatoid arthritis. By building on the observation that anxA1 on microvesicles stimulates TGFb release in macrophages, a cell line was transfected to release anxA1+ microvesicles, and their e ects compared to those of their wild type counterparts

Confirmation of Galba truncatula as an intermediate host snail for Calicophoron daubneyi in Great Britain, with evidence of alternative snail species hosting Fasciola hepatica

BACKGROUND: Fasciola hepatica is a highly prevalent parasite infecting livestock in Great Britain, while Calicophoron daubneyi is an emerging parasite within the GB livestock industry. Both F. hepatica and C. daubneyi require an intermediate host snail to complete their life-cycles and infect ruminants; however, there has been no confirmation of the intermediate host of C. daubneyi in GB, while there are questions regarding alternative host snails to Galba truncatula for F. hepatica. In this study, PCR was used to identify C. daubneyi hosting snail species on Welsh pastures and to identify any alternative snail species hosting F. hepatica. FINDINGS: Two hundred and sixty four snails were collected between May-September 2015 from six farms in mid-Wales known to have livestock infected with C. daubneyi and F. hepatica. Fifteen out of 134 G. truncatula were found positive for C. daubneyi, one of which was also positive for F. hepatica. Three snail species were found positive for F. hepatica [18/134 G. truncatula, 13/52 Radix balthica, and 3/78 Potamopyrgus antipodarum (New Zealand mud snail)], but no evidence of C. daubneyi infection in the latter two species was found. CONCLUSION: This study indicates that G. truncatula is a host for C. daubneyi in GB. Galba truncatula is also an established host of F. hepatica, and interactions between both species at intermediate host level could potentially occur. Radix balthica and P. antipodarum were found positive for F. hepatica but not C. daubneyi. This could indicate a role for alternative snail species other than G. truncatula in infecting pastures with F. hepatica in GB. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-015-1271-x) contains supplementary material, which is available to authorized users

Extracellular Vesicles Derived From Human Corneal Endothelial Cells Inhibit Proliferation of Human Corneal Endothelial Cells

Corneal endothelial cells (CEnCs) are a monolayer of hexagonal cells that are responsible for maintaining the function and transparency of the cornea. Damage or dysfunction of CEnCs could lead to blindness. Human CEnCs (HCEnCs) have shown limited proliferative capacity in vivo hence, their maintenance is crucial. Extracellular vesicles (EVs) are responsible for inter- and intra-cellular communication, proliferation, cell-differentiation, migration, and many other complex biological processes. Therefore, we investigated the effect of EVs (derived from human corneal endothelial cell line–HCEC-12) on corneal endothelial cells. HCEC-12 cells were starved with serum-depleted media for 72 h. The media was ultracentrifuged at 100,000xg to isolate the EVs. EV counting, characterization, internalization and localization were performed using NanoSight, flow cytometry, Dil labeling and confocal microscopy respectively. HCEC-12 and HCEnCs were cultured with media supplemented with EVs. Extracted EVs showed a homogeneous mixture of exosomes and microvesicles. Cells with EVs decreased the proliferation rate; increased apoptosis and cell size; showed poor wound healing response in vitro and on ex vivo human, porcine, and rabbit CECs. Thirteen miRNAs were found in the EV sample using next generation sequencing. We observed that increased cellular uptake of EVs by CECs limit the proliferative capacity of HCEnCs. These preliminary data may help in understanding the pathology of corneal endothelial dysfunction and provide further insights in the development of future therapeutic treatment options

Detection of Galba truncatula, Fasciola hepatica and Calicophoron daubneyi environmental DNA within water sources on pasture land, a future tool for fluke control?

Abstract Background: Increasing trematode prevalence and disease occurrence in livestock is a major concern. With the global spread of anthelmintic resistant trematodes, future control strategies must incorporate approaches focusing on avoidance of infection. The reliance of trematodes on intermediate snail hosts to successfully complete their life-cycle means livestock infections are linked to the availability of respective snail populations. By identifying intermediate snail host habitats, infection risk models may be strengthened whilst farmers may confidently apply pasture management strategies to disrupt the trematode life-cycle. However, accurately identifying and mapping these risk areas is challenging. Methods: In this study, environmental DNA (eDNA) assays were designed to reveal Galba truncatula, Fasciola hepatica and Calicophoron daubneyi presence within water sources on pasture land. eDNA was captured using a filter-based protocol, with DNA extracted using the DNeasy? PowerSoil? kit and amplified via PCR. In total, 19 potential G. truncatula habitats were analysed on four farms grazed by livestock infected with both F. hepatica and C. daubneyi. Results: Galba truncatula eDNA was identified in 10/10 habitats where the snail was detected by eye. Galba truncatula eDNA was also identified in four further habitats where the snail was not physically detected. Fasciola hepatica and C. daubneyi eDNA was also identified in 5/19 and 8/19 habitats, respectively. Conclusions: This study demonstrated that eDNA assays have the capabilities of detecting G. truncatula, F. hepatica and C. daubneyi DNA in the environment. Further assay development will be required for a field test capable of identifying and quantifying F. hepatica and C. daubneyi infection risk areas, to support future control strategies. An eDNA test would also be a powerful new tool for epidemiological investigations of parasite infections on farms.publishersversionPeer reviewe

Identification of factors associated with Fasciola hepatica infection risk areas on pastures via an environmental DNA survey of Galba truncatula distribution using droplet digital and quantitative real-time PCR assays

Abstract Environmental DNA (eDNA) is a powerful tool for identifying the spatial and temporal presence and density of species in a range of aquatic habitats. The analysis of eDNA has a wide range of application, one of which may be to inform of Fasciola hepatica infection risk on pastures based on the detection of its eDNA as well as that of its intermediate snail host, Galba truncatula eDNA. Here, droplet digital PCR (ddPCR) and quantitative real‐time PCR (qPCR) assays were developed to detect the eDNA of F. hepatica, and its intermediate snail host, G. truncatula in water samples collected from pastures grazed by cattle and/or sheep. Environmental factors associated with species presence, as detected via an eDNA survey, were identified using zero‐inflated linear mixed models. Sixty‐four habitats were sampled across six farms in Ceredigion, Wales, UK, with ddPCR and qPCR identifying 42 and 33 habitats to be positive for G. truncatula eDNA, respectively. G. truncatula eDNA was significantly less likely to be detected in habitats fully shaded by trees, those that contained black or dark brown soils and habitats that contained deep water pools (p < 0.05). Significantly higher G. truncatula eDNA concentrations were observed in habitats that tend to dry up during Summer (i.e., temporary habitats) (p < 0.05). ddPCR also identified five habitats to be positive for F. hepatica eDNA; however, questions remain regarding the utility of F. hepatica eDNA detection due to a lack of specificity toward infective F. hepatica larval stages. The results of this study inform of factors which influences G. truncatula distribution and ecology on pastures and also provided practical information for farmers to aid F. hepatica control in their flocks and herds