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Background: Several studies have reported the roles of Trichinella spiralis extracellular vesicles in immune regulation and pathogen diagnosis. Currently, the T. spiralis muscle larvae excretory/secretory product (Ts-ML-ES) is the antigen recommended by the International Commission on Trichinellosis (ICT) for serological diagnosis of trichinellosis. However, it can only be used to detect middle and late stages of infections, and cross-reactions with other parasite detections occur. Therefore, there is a need to identify antigens for specific detection of early stage trichinellosis., Methods: Extracellular vesicles of T. spiralis muscle larvae (Ts-ML-EVs) were isolated by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry and western blot. Ts-ML-EVs protein profiles were analyzed by LC-MS/MS proteomics for identification of potential antigens (Ts-TTPA). Ts-TTPA were cloned into pMAL-c5X vector and expressed as recombinant proteins for evaluation of potential as detected antigens by western blot and ELISA., Results: Isolated Ts-ML-EVs were round or elliptic (with diameters between 110.1 and 307.6 nm), showing a bilayer membrane structure. The specific surface markers on the Ts-ML-EVs were CD81, CD63, enolase and the 14-3-3 protein. A total of 53 proteins were identified by LC-MS/MS, including a variety of molecules that have been reported as potential detection and vaccine candidates. The cDNA of Ts-TTPA selected in this study has a total length of 1152 bp, encoding 384 amino acids with a molecular weight of 44.19 kDa. It contains a trypsin domain and can be recognized by anti-His antibody. It reacted with swine sera infected with 10,000 T. spiralis at 15, 25, 35 and 60 days post-infection (dpi). At 10 μg/ml, this antigen could detect T. spiralis antibodies from the swine sera at 13 dpi. There were no cross-reactions with the swine sera infected with other parasites including Clonorchis sinensis, Toxoplasma gondii, Taenia suis, Ascaris suis and Trichuris suis., Conclusions: This study identifies potential early stage detection antigens and more thoroughly characterizes a serine protease domain-containing protein. Extracellular vesicle proteins may be explored as effective antigens for the early stage detection of trichinellosis., (© 2023. The Author(s).)

Trichinella spiralis (T. spiralis) derived extracellular vesicles (EVs) have been proposed to play a key role in regulating the host immune responses. In this study, we provided the first investigation of EVs proteomics released by T. spiralis muscle larvae (ML). T. spiralis ML EVs (Ts-ML-EVs) were successfully isolated and characterized by transmission electron microscopy (TEM) and western blotting. Using liquid chromatograph mass spectrometer (LC-MS/MS) analysis, we identified 753 proteins in the Ts-ML-EVs proteome and annotated by gene ontology (GO). These proteins were enriched in different categories by GO, kyoto encyclopedia of genes and genomes (KEGG) and domain analysis. GO enrichment analysis indicated association of protein deglutathionylation, lysosomal lumen and serine-type endopeptidase inhibitor activity with proteins which may be helpful during parasite-host interaction. Moreover, KEGG enrichment analysis revealed involvement of Ts-ML-EVs proteins in other glycan degradation, complement and coagulation cascades, proteasome and various metabolism pathways. In addition, BALB/c mice were immunized by subcutaneous injection of purified Ts-ML-EVs. Ts-ML-EVs group demonstrated a 23.4% reduction in adult worms and a 43.7% reduction in ML after parasite challenge. Cellular and humoral immune responses induced by Ts-ML-EVs were detected, including the levels of specific antibodies (IgG, IgM, IgE, IgG1 and IgG2a) as well as cytokines (IL-12, IFN-γ, IL-4 and IL-10) in serum. The results showed that Ts-ML-EVs could induce a Th1/Th2 mixed immune response with Th2 predominant. This study revealed a potential role of Ts-ML-EVs in T. spiralis biology, particularly in the interaction with host. This work provided a critical step to against T. spiralis infection based on Ts-ML-EVs., Competing Interests: The authors have declared that no competing interests exist.

Single-cell EV (extracellular vesicle) secretion analysis is emerging for a better understanding of non-genetic cellular heterogeneity regulating human health and diseases through intercellular mediators. However, the requirements of expensive and bulky instrumentations hinder its widespread use. Herein, by combining gold nanoparticle-enhanced silver staining and the Poisson distribution, we reported the use of a home-use scanner to realize high-throughput single-cell EV secretion analysis without cell counting. We applied the platform to analyze the secretions of different EV phenotypes with the human oral squamous cell carcinoma cell line and primary cells from patients, which generated single-cell results comparable with those of the immunofluorescence approach. Notably, we also realized the quantification of the number of EVs secreted from every single cell using their respective titration curves obtained from population samples, making it possible to directly compare different EV phonotypes in regard to their secretion number, secretion rate, and so forth. The technology introduced here is simple, easy to operate, and of low cost, which make it a potential, easily accessible, and affordable tool for widespread use in both basic and clinical research.

Extracellular vesicles (EVs) are membranous containers released by cells that are powerful agents of intercellular communication. EVs have been described for various parasites and are associated with tissue inflammation. Several studies have demonstrated that parasite EVs can have either pro- or anti-inflammatory impacts, depending on the type of parasite. To evaluate the immunomodulatory properties of EVs produced by Trichinella spiralis (T. spiralis), we established a mouse model with dextran sulphate sodium (DSS)-induced colitis. The muscle larvae of T. spiralis were cultured in vitro and the released EVs were isolated by ultracentrifugation. T. spiralis EVs (Ts-EVs) were characterized according to morphology, size and constituent surface proteins (CD63, Enolase and Hsp70). Mice were treated with water containing 3% DSS after last intraperitoneal injection of Ts-EVs. Disease activity index (DAI), macroscopic and histopathological scores of Ts-EVs group was lower than DSS group. And Ts-EVs prevented the increase in the expression of TNF-α, IFN-γ, IL-17A and IL-1β observed in the colon of DSS-treated mice. In contrast, upregulation of IL-4, IL-10, TGF-β and IL-13 expression was detected in Ts-EVs+DSS group. In addition, Ts-EVs increased the infiltration of alternatively activated (M2) macrophages into the colon. The expression of CD206 (M2 marker) in the mesenteric lymph nodes (MLN) of mice with colitis increased in Ts-EVs+DSS group. Furthermore, Ts-EVs interfered with both the NF-κB and MAPK signalling pathways. Taken together, our findings demonstrate that Ts-EVs can affect the development of inflammation in DSS-induced colitis by inhibiting M1 macrophage polarization, due to their immunomodulatory ability., (Copyright © 2020. Published by Elsevier B.V.)

Helminths are masters at modulating the host immune response through a wide variety of versatile mechanisms. These complex strategies facilitate parasite survival in the host and can also be exploited to prevent chronic immune disorders by minimizing excessive inflammation. Extracellular vesicles (EVs) are small membrane-bound structures secreted by helminths which mediate immune evasion during parasite infection. The goal of this study was to investigate the immunoregulatory properties of Trichinella spiralis EVs ( Ts -EVs) in a murine model of colitis. We found that Ts -EVs significantly ameliorated 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. Ts -EVs alleviated intestinal epithelium barrier damage, markedly reduced pro-inflammatory cytokine secretion and neutrophil infiltration, and upregulated immunoregulatory cytokine expression in colon tissue. Ts -EVs also modulated the adaptive immune response by influencing T-cell composition. The numbers of Th1 and Th17 cells in MLNs, as well as the expression levels of Th1/Th17-associated cytokines and transcription factors in colon were reduced. In contrast, Th2 and Treg cells were increased after Ts -EVs treatment. Furthermore, sequencing of EV-derived microRNAs (miRNAs) indicated that an array of miRNAs was involved in the regulation of the host immune response, including inflammation. These findings expand our knowledge of host-parasite interactions, and may help design novel and effective strategies to prevent parasite infections or to treat inflammatory diseases like IBD. Further studies are needed to identify the specific cargo molecules carried by Ts -EVs and to clarify their roles during T. spiralis infection., (Copyright © 2020 Yang, Liu, Liu, Zhang, Shi, Jia, Zhu, Jia, Liu and Bai.)

Cell–cell interactions are the fundamental behaviors to regulate cellular activities. A comprehensive evaluation of intercellular interactions requires direct profiling of various signaling behaviors simultaneously at the single‐cell level, which remains lacking. Herein, an integrative single‐cell secretion analysis platform is presented to profile different secreted factors (four proteins, three extracellular vesicles (EV) phenotypes), spatial distances, and migration information (distances and direction) simultaneously from high‐throughput paired single cells using an antibody‐barcode microchip. Applying the platform to analyze the tumor–stromal and tumor–immune interactions with the human oral squamous cell carcinoma (OSCC) cell lines and primary OSCC cells reveals that the initial distances between cells would determine their migratory distances and direction to approach stable organization. The cell–cell in close proximity enhances protein secretions while attenuating EV secretions. Migration has a more profound correlation with protein secretions than EV secretions, in which absolute migration distance affects protein secretions significantly but not the direction. These findings highlight the significance of spatial organization in regulating cell signaling behaviors and demonstrate that the integrative single‐cell secretion profiling platform is well‐suited for a comprehensive dissection of intercellular communication and interactions, providing new avenues for understanding cell–cell interaction biology and how different signaling behaviors coordinate within the tumor microenvironment. [ABSTRACT FROM AUTHOR]

• EVs-miR-106b from MSC alleviates neuronal injury in PD mice. • EVs-miR-106b from MSC promotes autophagy and inhibits neuronal apoptosis in PD. • miR-106b targets and negatively regulates CDKN2B expression. • CDKN2B reverses the promoting effects of EVs-miR-106b on neuronal autophagy. • EVs-miR-106b inhibits CDKN2B expression and alleviates neurological damage in PD. Parkinson's disease (PD) is the second most frequent neurodegenerative disorder, and autophagy dysfunction is involved in the pathogenesis of PD. Mesenchymal stem cells (MSC)-derived extracellular vesicles (EVs) have been established as an attractive therapeutic tool, since they can serve as biological nanoparticles with beneficial effects in PD. Herein, the study aimed to investigate the effects of EVs derived microRNA (miR)-106b on autophagy of neurons in PD. Following the development of a mouse model of PD, we conducted behavior test, TUNEL assay and HE staining to verify the success of modeling. Afterward, MSC-derived EVs were extracted and identified. In hippocampal tissues and neurons of PD mice, miR-106b was poorly expressed, while CDKN2B was highly expressed. miR-106b shuttled by MSC-derived EVs increased neuronal survival, autophagy, LC3II/LC3I ratio and Bcl-2 protein expression, while inhibited neuronal apoptosis and Bax expression in PD mice. It was also confirmed that CDKN2B is a downstream target of miR-106b. Overexpression of CDKN2B reversed the protective effects of miR-106b-containing EVs on neurons in mice with PD. Collectively, miR-106b-containing EVs alleviate neuronal apoptosis and enhance neuronal autophagy in PD by downregulating CDKN2B. [ABSTRACT FROM AUTHOR]