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BACKGROUND: Infections with Ascaris lumbricoides and Trichuris trichiura remain significant contributors to the global burden of neglected tropical diseases. Infection may in particular affect child development as they are more likely to be infected with T. trichiura and/or A. lumbricoides and to carry higher worm burdens than adults. Whilst the impact of heavy infections are clear, the effects of moderate infection intensities on the growth and development of children remain elusive. Field studies are confounded by a lack of knowledge of infection history, nutritional status, presence of co-infections and levels of exposure to infective eggs. Therefore, animal models are required. Given the physiological similarities between humans and pigs but also between the helminths that infect them; A. suum and T. suis, growing pigs provide an excellent model to investigate the direct effects of Ascaris spp. and Trichuris spp. on weight gain. METHODS AND RESULTS: We employed a trickle infection protocol to mimic natural co-infection to assess the effect of infection intensity, determined by worm count (A. suum) or eggs per gram of faeces (A. suum and T. suis), on weight gain in a large pig population (n = 195) with variable genetic susceptibility. Pig body weights were assessed over 14 weeks. Using a post-hoc statistical approach, we found a negative association between weight gain and T. suis infection. For A. suum, this association was not significant after adjusting for other covariates in a multivariable analysis. Estimates from generalized linear mixed effects models indicated that a 1 kg increase in weight gain was associated with 4.4% (p = 0.00217) decrease in T. suis EPG and a 2.8% (p = 0.02297) or 2.2% (p = 0.0488) decrease in A. suum EPG or burden, respectively. CONCLUSIONS: Overall this study has demonstrated a negative association between STH and weight gain in growing pigs but also that T. suis infection may be more detrimental that A. suum on growth.

Aims: To provide a reliable, reproducible and centrifuge-free filtration protocol for clarification of large volumes of bacterial cultures. Methods and Results: Four experiments were designed to compare different techniques enabling clarification of Escherichia coli cultures using as a benchmark the concentration and quality of bacterial outer membrane vesicles (OMVs). The experiments were designed to examine the performance of different extraction methods on large volume (≥1 L) filtrations of bacterial culture media. Performance parameters included filtration flow rates, sterility testing and characterization of the filtrates by: (i) SDS-PAGE, (ii) cryogenic transmission electron microscopy, (iii) nanoparticle tracking analysis and (iv) Qubit protein quantification. The experiments revealed that: (i) addition of the filter aid Diatomaceous Earth to the bacterial cultures improved filtration flow rates significantly and eliminated the need for centrifugation prior to filtration; (ii) sterile filtration was successful as no bacterial passage was identified through the membrane filter; (iii) centrifuge-free filtrates contained an increased amount of OMVs compared to centrifuged filtrates. Conclusions: In comparison to conventional centrifuge-based protocols, the clarification method presented has universal applicability for a broad range of microbial extraction procedures, regardless of the volume of culture harvested. Moreover, the decreased amount of OMVs presented in the filtrates following centrifugation step provides an additional argument in favour of a centrifuge-free approach. Significance and Impact of the Study: Sterile filtration is a universal method for the clarification of bacterial cultures. Common challenges related to filtration include filter clogging and long processing times, due to limited centrifugation capacity, which can affect product quality. The proposed protocol is likely to ensure a highly effective filtration process and could be a nov

Emerging evidence suggests that immune cells not only communicate with each other through cytokines, chemokines, and cell surface receptors, but also by releasing small membranous structures known as extracellular vesicles (EVs). EVs carry a variety of different molecules that can be taken up by recipient cells. Parasitic worms are well known for their immunomodulatory properties, but whether they can affect immune responses by altering EV-driven communication between host immune cells remains unclear. Here we provide evidence that stimulation of bone marrow-derived macrophages (BMDMs) with soluble products of Trichuris suis (TSPs), leads to the release of EVs with anti-inflammatory properties. Specifically, we found that EVs from TSP-pulsed BMDMs, but not those from unstimulated BMDMs can suppress TNFα and IL-6 release in LPS-stimulated BMDMs and BMDCs. However, no polarization toward M1 or M2 was observed in macrophages exposed to EVs. Moreover, EVs enhanced reactive oxygen species (ROS) production in the exposed BMDMs, which was associated with a deregulated redox homeostasis as revealed by pathway analysis of transcriptomic data. Proteomic analysis identified cytochrome p450 (CYP450) as a potential source of ROS in EVs from TSP-pulsed BMDMs. Finally, pharmacological inhibition of CYP450 activity could suppress ROS production in those BMDMs. In summary, we find that TSPs can modulate immune responses not only via direct interactions but also indirectly by eliciting the release of EVs from BMDMs that exert anti-inflammatory effects on recipient cells.

Background: Exosomes have been implicated in tumour progression and metastatic spread. Little is known of the effect of mechanical and innate immune interactions of malignant cell-derived exosomes on endothelial integrity, which may relate to increased extravasation of circulating tumour cells and, therefore, increased metastatic spread. Methods: Exosomes isolated from non-malignant immortalized HCV-29 and isogenic malignant non-metastatic T24 and malignant metastatic FL3 bladder cells were characterized by nanoparticle tracking analysis and quantitative nanomechanical mapping atomic force microscopy (QNM AFM) to determine size and nanomechanical properties. Effect of HCV-29, T24 and FL3 exosomes on human umbilical vein endothelial cell (HUVEC) monolayer integrity was determined by transendothelial electrical resistance (TEER) measurements and transport was determined by flow cytometry. Complement activation studies in human serum of malignant and non-malignant cell-derived exosomes were performed. Results: FL3, T24 and HCV-29 cells produced exosomes at similar concentration per cell (6.64, 6.61 and 6.46×104 exosomes per cell for FL3, T24 and HCV-29 cells, respectively) and of similar size (120.2 nm for FL3, 127.6 nm for T24 and 117.9 nm for HCV-29, respectively). T24 and FL3 cell-derived exosomes exhibited a markedly reduced stiffness, 95 MPa and 280 MPa, respectively, compared with 1,527 MPa with non-malignant HCV-29 cell-derived exosomes determined by QNM AFM. FL3 and T24 exosomes induced endothelial disruption as measured by a decrease in TEER in HUVEC monolayers, whereas no effect was observed for HCV-29 derived exosomes. FL3 and T24 exosomes traffic more readily (11.6 and 21.4% of applied exosomes, respectively) across HUVEC monolayers than HCV-29 derived exosomes (7.2% of applied exosomes). Malignant cell-derived exosomes activated complement through calcium-sensitive pathways in a concentration-dependent manner. Conclusions: Malign, Background: Exosomes have been implicated in tumour progression and metastatic spread. Little is known of the effect of mechanical and innate immune interactions of malignant cell-derived exosomes on endothelial integrity, which may relate to increased extravasation of circulating tumour cells and, therefore, increased metastatic spread.Methods: Exosomes isolated from non-malignant immortalized HCV-29 and isogenic malignant non-metastatic T24 and malignant metastatic FL3 bladder cells were characterized by nanoparticle tracking analysis and quantitative nanomechanical mapping atomic force microscopy (QNM AFM) to determine size and nanomechanical properties. Effect of HCV-29, T24 and FL3 exosomes on human umbilical vein endothelial cell (HUVEC) monolayer integrity was determined by transendothelial electrical resistance (TEER) measurements and transport was determined by flow cytometry. Complement activation studies in human serum of malignant and non-malignant cell-derived exosomes were performed.Results: FL3, T24 and HCV-29 cells produced exosomes at similar concentration per cell (6.64, 6.61 and 6.46×104 exosomes per cell for FL3, T24 and HCV-29 cells, respectively) and of similar size (120.2 nm for FL3, 127.6 nm for T24 and 117.9 nm for HCV-29, respectively). T24 and FL3 cell-derived exosomes exhibited a markedly reduced stiffness, 95 MPa and 280 MPa, respectively, compared with 1,527 MPa with non-malignant HCV-29 cell-derived exosomes determined by QNM AFM. FL3 and T24 exosomes induced endothelial disruption as measured by a decrease in TEER in HUVEC monolayers, whereas no effect was observed for HCV-29 derived exosomes. FL3 and T24 exosomes traffic more readily (11.6 and 21.4% of applied exosomes, respectively) across HUVEC monolayers than HCV-29 derived exosomes (7.2% of applied exosomes). Malignant cell-derived exosomes activated complement through calcium-sensitive pathways in a concentrat