Excretory-secretory Products Research Articles

Disruption of Epithelial Barrier of Caco-2 Cell Monolayers by Excretory Secretory Products of Trichinella spiralis Might Be Related to Serine Protease.

The physical barrier is composed of epithelial cells which are joined together through intercellular connections. It serves to prevent pathogenic microorganisms from departing the intestinal lumen to invade the host. The excretory secretory (ES) products of Trichinella spiralis are critical for invasion. However, whether ES products of T. spiralis can act on the intestinal barrier is still unknown. In this study, the role of ES products of T. spiralis muscle larvae (Ts-ML-ES) in host invasion was studied by establishing an in vitro cell monolayers model. Barrier integrity analysis by a transmembrane resistance test and a paracellular permeability assay revealed that the Ts-ML-ES was able to destroy barrier function. It occurred via a reduction in the expression of tight junction (TJ) proteins, which was induced by serine protease. Furthermore, Western bolt analysis indicated that Ts-ML-ES reduced the expression of TJ proteins via the MAPK signaling pathway. Based on these data, we conclude that serine protease are likely the main factors from Ts-ML-ES that affect host intestinal barrier integrity by reducing the expression of TJs via the P38-MAPK signaling pathway. Serine protease in Ts-ML-ES might be a key invasion factor in T. spiralis.

Toxocara canis and Toxocara cati Somatic and Excretory-Secretory Antigens Are Recognised by C-Type Lectin Receptors.

Toxocara canis and Toxocara cati, the worldwide occurring intestinal roundworms of canids and felids, represent an important public health threat due to various disease manifestations in humans. Host recognition of pathogens is mediated by pattern recognition receptors (PRRs). Myeloid C-type lectin receptors (CLRs) are PRRs and recognise carbohydrate structures of various pathogens. As Toxocara excretory-secretory products (TES) are predominantly composed of glycoconjugates, they represent suitable targets for CLRs. However, the range of host-derived CLRs recognising Toxocara spp. is still unknown. Using a CLR-hFc fusion protein library, T. canis and T. cati L3 somatic antigens (TSOM) were bound by a variety of CLRs in enzyme-linked immunosorbent assay (ELISA), while their TES products interacted with macrophage galactose-type lectin-1 (MGL-1). Two prominent candidate CLRs, MGL-1 and macrophage C-type lectin (MCL), were selected for further binding studies. Immunofluorescence microscopy revealed binding of MGL-1 to the oral aperture of L3. Immunoblot experiments identified distinct protein fractions representing potential ligands for MGL-1 and MCL. To evaluate how these interactions influence the host immune response, bone marrow-derived dendritic cell (BMDC) assays were performed, showing MCL-dependent T. cati-mediated cytokine production. In conclusion, MGL-1 and MCL are promising candidates for immune modulation during Toxocara infection, deserving further investigation in the future.

Effects of Trichinella spiralis and its excretory/secretory products on autophagy of host muscle cells in vivo and in vitro

Trichinella spiralis (T. spiralis) is a widely distributed pathogenic microorganism that causes trichinellosis, a disease that has the potential of causing severe harm to their host. Numerous studies have demonstrated that autophagy can be triggered by microbial infection, such as bacteria, viruses, protozoa, and parasitic helminths. However, it’s still unknown whether autophagy can facilitate host resistance to T. spiralis infection. The present study examined the role of autophagy in striated muscle cell transformation following infection with T. spiralis in BALB/c mice. Transmission electron microscopy (TEM) was used to detect the production of the host diaphragm autophagosome after T. spiralis infection, and changes in the protein and transcriptional levels of autophagic marker proteins were also detected. The significance of autophagy in T. spiralis infection, namely inhibition of T. spiralis growth, was preliminarily evaluated by conducting in vivo experiments using autophagy inhibitors. Besides, we studied the effect of excretory-secretory products (ES) of T. spiralis on autophagy of C2C12 myoblasts. The changes in protein and gene expression levels in autophagy-related pathways in vitro and in vivo were measured as further evidence. The results showed that T. spiralis infection induced autophagy in the host muscle cells. Meanwhile, ES inhibited autophagy of myoblasts in vitro, but this did not affect the cell viability. The upregulation and downregulation of autophagy-related factors in skeletal muscle cells may indicate an adaptive mechanism providing a comfortable niche for the parasite.

Regulation of human THP-1 macrophage polarization by Trichinella spiralis

Trichinella spiralis is a foodborne zoonotic nematode, which causes trichinellosis. During the infection, parasite evades the host immune responses by direct and indirect (through excretory-secretory products) contact with host immune cells. One of the main targets for immunomodulation induced by helminths are macrophages. In this study, we examined whether direct contact of different stages of T. spiralis can affect the polarization of human THP-1 macrophages. Co-culture of adult parasite stage and cells in direct contact without LPS addition had a significant impact on TNFα levels. Interestingly, in settings with the addition of LPS, the levels of IL-1β and TNFα significantly increased in adult parasite and newborn larvae (NBL) but not for muscle larvae (ML). While we tested muscle larvae ESP products to compare its effect with whole ML parasite, we detect an increase of pro-inflammatory cytokines like IL-1β and TNFα in no LPS conditions. Whereas, muscle larvae ESP significantly suppressed the inflammatory response measured by IL-1β, TNFα, and IL-6 levels and anti-inflammatory IL-10 compared to LPS control. Our findings indicate the anti-inflammatory potential of T. spiralis muscle larvae excretory-secretory products and propose signaling pathways which might be engaged in the mechanism of how muscle larvae ESP affect human macrophages.

Clone, expression and plasminogen binding property of three fructose-1,6-bisphosphate aldolases from Clonorchis sinensis.

Fructose-1,6-bisphosphate aldolase (FbA), a well characterized glycometabolism enzyme, has been found to participate in other important processes besides the classic catalysis. To understand the important functions of three fructose-1,6-bisphosphate aldolases from Clonorchis sinensis (CsFbAs, CsFbA-1/2/3) in host-parasite interplay, the open reading frames of CsFbAs were cloned into pET30a (+) vector and the resulting recombinant plasmids were transformed into Escherichia coli BL21 (DE3) for expression of the proteins. Purified recombinant CsFbAs proteins (rCsFbAs) were approximately 45.0 kDa on 12% SDS-PAGE and could be probed with each rat anti-rCsFbAs sera by western blotting analysis. ELISA and ligand blot overlay indicated that rCsFbAs of 45.0 kDa as well as native CsFbAs of 39.5 kDa from total worm extracts and excretory-secretory products of Clonorchis sinensis (CsESPs) could bind to human plasminogen, and the binding could be efficiently inhibited by lysine analog ε-aminocaproic acid. Our results suggested that as both the components of CsESPs and the plasminogen binding proteins, three CsFbAs might be involved in preventing the formation of the blood clot so that Clonorchis sinensis could acquire enough nutrients from host tissue for their successful survival and colonization in the host. Our work will provide us with new information about the biological function of three CsFbAs and their roles in hostparasite interplay.

Phage display-based vaccine with cathepsin L and excretory-secretory products mimotopes of Fasciola hepatica induces protective cellular and humoral immune responses in sheep

Fasciolosis is a foodborne zoonotic disease that affects grazing animals and causes substantial economic losses worldwide. Excretory/secretory (E/S) products and cathepsin L mimotopes from Fasciola hepatica were used to immunise experimentally infected sheep against liver flukes. The level of protection was measured in terms of fluke burden, morphometric measurements and faecal egg counts, as well as the humoral and cellular immune responses elicited. Five groups of 5 sheep each were immunised with 1 × 1013 phage particles of cathepsin L1 (group 1: SGTFLFS), cathepsin L1 (group 2: WHVPRTWWVLPP) and immunodominant E/S product (group 3) mimotopes with Quil A adjuvant, and wild-type M13KE phage (group 4) at the beginning and as a booster two weeks later. The control group received phosphate-buff ;ered saline. All groups were challenged with 300 metacercariae at week four and slaughtered 18 weeks later. The mean fluke burdens after challenge were reduced by 52.39 % and 67.17 % in sheep vaccinated with E/S products (group 3) and cathepsin L1 (group 1: SGTFLFS), respectively; no eff ;ect was observed in animals inoculated with cathepsin L1 (group 2: WHVPRTWWVLPP). Animals vaccinated showed a significant reduction in fluke length and width, wet weights and egg output Sheep immunised with phage-displayed mimotopes induced the development of specific IgG1 and IgG2, indicating a mixed Th1/Th2 immune response. Measurement of cytokine levels revealed higher levels of IFN-γ as well as lower production of IL-4 in sheep vaccinated with the mimotope peptide of F. hepatica. Fluke-specific production of IFN-γ in immunised animals was significantly correlated with fluke burden (P < 0.01). As helminth infection progressed, increased levels of IL-4 were evident in the wild-type M13KE phage (group 4) and the control groups (group 5), accompanied by a downregulation of IFN-γ production. Vaccinated animals with cathepsin L1 (group 1: SGTFLFS) showed that amino acids located in the middle (64SG65) of the linear sequence and C-terminal end (314TFLFS318) were associated with significant protection.

Dysregulation of hepatic microRNA expression in C57BL/6 mice affected by excretory-secretory products of Fasciola gigantica.

The excretory-secretory products released by the liver fluke Fasciola gigantica (FgESPs) play important roles in regulating the host immune response during the infection. Identification of hepatic miRNAs altered by FgESPs may improve our understanding of the pathogenesis of F. gigantica infection. In this study, we investigated the alterations in the hepatic microRNAs (miRNAs) in mice treated with FgESPs using high-throughput small RNA (sRNA) sequencing and bioinformatics analysis. The expression of seven miRNAs was confirmed by quantitative stem-loop reverse transcription quantitative PCR (qRT-PCR). A total of 1,313 miRNAs were identified in the liver of mice, and the differentially expressed (DE) miRNAs varied across the time lapsed post exposure to FgESPs. We identified 67, 154 and 53 dysregulated miRNAs at 1, 4 and 12 weeks post-exposure, respectively. 5 miRNAs (miR-126a-3p, miR-150-5p, miR-155-5p, miR-181a-5p and miR-362-3p) were commonly dysregulated at the three time points. We also found that most of the DE miRNAs were induced by FgESPs in the mouse liver after 4 weeks of exposure. These were subjected to Gene Ontology (GO) enrichment analysis, which showed that the predicted targets of the hepatic DE miRNAs of mice 4 weeks of FgESPs injection were enriched in GO terms, including cell membrane, ion binding, cellular communication, organelle and DNA damage. KEGG analysis indicated that the predicted targets of the most downregulated miRNAs were involved in 15 neural activity-related pathways, 6 digestion-related pathways, 20 immune response-related pathways and 17 cancer-related pathways. These data provide new insights into how FgESPs can dysregulate hepatic miRNAs, which play important roles in modulating several aspects of F. gigantica pathogenesis.

Necator americanus Ancylostoma Secreted Protein-2 (Na-ASP-2) Binds an Ascaroside (ascr#3) in Its Fatty Acid Binding Site.

During their infective stages, hookworms release excretory-secretory (E-S) products, small molecules, and proteins to help evade and suppress the host's immune system. Small molecules found in E-S products of mammalian hookworms include nematode derived metabolites like ascarosides, which are composed of the sugar ascarylose linked to a fatty acid side chain. The most abundant proteins found in hookworm E-S products are members of the protein family known as Ancylostoma secreted protein (ASP). In this study, two ascarosides and their fatty acid moieties were synthesized and tested for in vitro binding to Na-ASP-2 using both a ligand competition assay and microscale thermophoresis. The fatty acid moieties of both ascarosides tested and ascr#3, an ascaroside found in rat hookworm E-S products, bind to Na-ASP-2's palmitate binding cavity. These molecules were confirmed to bind to the palmitate but not the sterol binding sites. An ascaroside, oscr#10, which is not found in hookworm E-S products, does not bind to Na-ASP-2. More studies are required to determine the structural basis of ascarosides binding by Na-ASP-2 and to understand the physiological significance of these observations.

The Functional Parasitic Worm Secretome: Mapping the Place of Onchocerca volvulus Excretory Secretory Products.

Nematodes constitute a very successful phylum, especially in terms of parasitism. Inside their mammalian hosts, parasitic nematodes mainly dwell in the digestive tract (geohelminths) or in the vascular system (filariae). One of their main characteristics is their long sojourn inside the body where they are accessible to the immune system. Several strategies are used by parasites in order to counteract the immune attacks. One of them is the expression of molecules interfering with the function of the immune system. Excretory-secretory products (ESPs) pertain to this category. This is, however, not their only biological function, as they seem also involved in other mechanisms such as pathogenicity or parasitic cycle (molting, for example). We will mainly focus on filariae ESPs with an emphasis on data available regarding Onchocerca volvulus, but we will also refer to a few relevant/illustrative examples related to other worm categories when necessary (geohelminth nematodes, trematodes or cestodes). We first present Onchocerca volvulus, mainly focusing on the aspects of this organism that seem relevant when it comes to ESPs: life cycle, manifestations of the sickness, immunosuppression, diagnosis and treatment. We then elaborate on the function and use of ESPs in these aspects.

DC-SIGN signalling induced by Trichinella spiralis products contributes to the tolerogenic signatures of human dendritic cells

Tolerogenic dendritic cells (tolDCs) are central players in the maintenance of immune tolerance and thereby have been identified as the most favourable candidates for cell therapy of autoimmune diseases. We have recently shown that excretory-secretory products (ES L1) released by Trichinella spiralis larvae induce stable human tolDCs in vitro via Toll-like receptor 2 (TLR2) and TLR4. However, engagement of these receptors did not fully explain the tolerogenic profile of DCs. Here, we observed for the first time that dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) interacts with highly glycosylated ES L1 and contributes to the generation of ES L1-induced tolDCs. Blocking DC-SIGN interfered with the ES L1-induced higher expression of CD40 and CCR7 and the production of IL-10 and TGF-β by DCs. The cooperation of TLR2, TLR4 and DC-SIGN receptors is of importance for the capacity of DCs to prime T cell response toward Th2 and to induce expansion of CD4+CD25+Foxp3+ T cells, as well as for the production of IL-10 and TGF-β by these cells. Overall, these results indicate that induction of tolDCs by ES L1 involves engagement of multiple pattern recognition receptors namely, TLR2, TLR4 and DC-SIGN.

3-Hydroxybenzaldehyde and 4-Hydroxybenzaldehyde enhance survival of mouse astrocytes treated with Angiostrongylus cantonensis young adults excretory/secretory products

BackgroundHuman cerebral angiostrongyliasis, induced by Angiostrongylus cantonensis, is an emerging disease in many parts of the world. A. cantonensis is also an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. 3-Hydroxybenzaldehyde (3-HBA) and 4-Hydroxybenzaldehyde (4-HBA) have been shown to increase intracellular antioxidant activity, vasculoprotective potency, wound healing, and cell migration. However, the function of 3-HBA and 4-HBA in mouse astrocytes in response to A. cantonensis young adults excretory-secretory products (ESPs) treatment remains unclear. MethodsHere, we examined the effect of 3-HBA and 4-HBA by real-time qPCR, western blotting, and cell viability assay in astrocytes after A. cantonensis young adults ESPs treatment. The real-time qPCR, western blotting were employed to detect the expression of apoptosis- and Shh pathway-related molecule. The percentage of cell viability was monitored by CCK-8 assay. ResultsWe demonstrated that expression of apoptosis-related molecules was increased in response to A. cantonensis young adults ESPs treatment. However, the cell viability of astrocytes was elevated by treatment with 3-HBA and 4-HBA. Further investigation found that 3-HBA and 4-HBA activate the Shh signaling pathway and inhibit apoptosis-related molecule expression. ConclusionsThese findings were confirmed using A. cantonensis young adults ESPs to activate apoptosis-related pathways in astrocytes. Moreover, 3-HBA and 4-HBA induced a protective phenotype through regulation of apoptosis in response to A. cantonensis young adults ESPs treatment. Hence, 3-HBA and 4-HBA represent potential therapeutic drugs for the treatment of human angiostrongyliasis.

Differences in the antibody response to adult Fasciola hepatica excretory/secretory products in experimentally and naturally infected cattle and sheep

Fasciola hepatica (the liver fluke) is a common, global parasite of livestock. It can be highly pathogenic and has health and welfare implications for infected individuals. Typically, in ruminants, infections are sub-clinical, but if undiagnosed, they can lead to significant production losses. Accurate diagnosis is crucial to identify infection. Antibody detection ELISAs are commonly used to diagnose infection due to their high sensitivity and specificity and are typically based on native fluke excretory/secretory (ES) products or cathepsin L1 (CL1), the immunodominant antigen within ES products. These tests have been developed based on the antibody response of experimentally infected animals; however, this response has not been well characterised in naturally infected animals. We compared the antibody recognition of a recombinant CL1 (rCL1) antigen and native adult fluke ES products. Whilst samples from experimentally infected animals showed strong recognition of rCL1, serum antibodies from naturally infected animals did not. These results were confirmed by peptide array. Immunoblotting sera against ES products showed that experimentally infected animals had a strong, specific response to CL1/CL2 proteins whilst antibodies from naturally infected animals recognised multiple proteins and had a variable response to CL1/CL2. Mass spectrometry of proteins separated by 2D SDS PAGE, identified several antigens recognised by serum antibodies from a naturally infected cow, including cathepsins L1, L2 and L5, glutathione S-transferase and a dihydrolipoyl dehydrogenase. Overall, these results show that the antibody response in naturally infected animals to adult fluke ES products is qualitatively different to experimentally infected animals. This suggests that a diagnostic test based on CL1 alone may not be appropriate for diagnosis of natural F. hepatica infections in sheep and cattle.

Toxoplasma gondii excreted/secreted proteases disrupt intercellular junction proteins in epithelial cell monolayers to facilitate tachyzoites paracellular migration.

Toxoplasma gondii shows high dissemination and migration properties across biological barriers infecting immunologically privileged organs. Toxoplasma uses different routes for dissemination; however, the mechanisms are not fully understood. Herein, we studied the effects of proteases present in excretion/secretion products (ESPs) of Toxoplasma on MDCK cell monolayers. Ultrastructural analysis showed that ESPs of Toxoplasma disrupt the intercellular junctions (IJ) of adjacent cells. The tight junction (TJ) proteins ZO-1, occludin, and claudin-1 suffered a progressive decrease in protein levels upon ESPs treatment. In addition, ESPs induced mislocalization of such TJ proteins, along with the adherent junction protein E-cadherin, and this was prevented by pre-treating the ESPs with protease inhibitors. Reorganisation of cytoskeleton proteins was also observed. Endocytosis inhibitors, Dyngo®-4a and Dynasore, impeded the modifications, suggesting that TJ proteins internalisation is triggered by the ESPs proteases hence contributing to the loss of IJ. The observed disruption in TJ proteins went in line with a decrease in the transepithelial electrical resistance of the monolayers, which was significantly blocked by pre-treating ESPs with metalloprotease and serine protease inhibitors. Moreover, exposure of cell monolayers to ESPs facilitated paracellular migration of tachyzoites. Our results demonstrate that Toxoplasma ESPs contain proteases that can disrupt the IJ of epithelial monolayers and this could facilitate the paracellular route for Toxoplasma tissue dissemination and migration.

Metabolomes and Lipidomes of the Infective Stages of the Gastrointestinal nematodes, Nippostrongylus brasiliensis and Trichuris muris.

Soil-transmitted helminths, including hookworms and whipworms, infect billions of people worldwide. Their capacity to penetrate and migrate through their hosts’ tissues is influenced by the suite of molecules produced by the infective developmental stages. To facilitate a better understanding of the immunobiology and pathogenicity of human hookworms and whipworms, we investigated the metabolomes of the infective stage of Nippostrongylus brasiliensis third-stage larvae (L3) which penetrate the skin and Trichuris muris eggs which are orally ingested, using untargeted liquid chromatography-mass spectrometry (LC-MS). We identified 55 polar metabolites through Metabolomics Standard Initiative level-1 (MSI-I) identification from N. brasiliensis and T. muris infective stages, out of which seven were unique to excretory/secretory products (ESPs) of N. brasiliensis L3. Amino acids were a principal constituent (33 amino acids). Additionally, we identified 350 putative lipids, out of which 28 (all known lipids) were unique to N. brasiliensis L3 somatic extract and four to T. muris embryonated egg somatic extract. Glycerophospholipids and glycerolipids were the major lipid groups. The catalogue of metabolites identified in this study shed light on the biology, and possible therapeutic and diagnostic targets for the treatment of these critical infectious pathogens. Moreover, with the growing body of literature on the therapeutic utility of helminth ESPs for treating inflammatory diseases, a role for metabolites is likely but has received little attention thus far.

Proteomic Analysis of Exosome-Like Vesicles Isolated From Saliva of the Tick Haemaphysalis longicornis.

Extracellular vesicles (EVs), are considered as vehicles of cellular communication. Parasites usually release EVs in their excretory-secretory products to modulate host environment. However, little is known about the secretion of EVs by ticks. In this study, we show for the first time that the tick Haemaphysalis longicornis secretes EVs in saliva that resembles exosomes. EVs were purified from pilocarpine induced saliva of partially engorged H. longicornis ticks. Electron microscopy analysis revealed the presence of exosome-like vesicles with a size of 100 nm. Proteomic analysis by LC-MS/MS identified a total of 356 proteins in tick-derived EVs. Proteome data of tick-derived EVs was validated by Western blot analysis. Immunodetection of Hsp70 and GAPDH proteins indicated that the proteomics data of tick-derived EVs were highly reliable. Bioinformatics analysis (Gene Ontology) indicated association of certain biological and molecular functions with proteins which may be helpful during tick development. Likewise, KEGG database revealed involvement of vesicular proteins in proton transport, detoxification, ECM-receptor interaction, ribosome, RNA transport, ABC transporters, and oxidative phosphorylation. The results of this study provide evidence that EVs are being secreted in tick saliva and suggest that tick saliva-derived EVs could play important roles in host-parasite relationships. Moreover, EVs could be a useful tool in development of vaccines or therapeutics against ticks.

Suppression of inflammatory genes expression in the injured host intestinal wall during Mesocestoides vogae tetrathyridium larvae migration.

Mesocestoides vogae is a cestode parasite of the family Mesocestoididae (order Cyclophyllidea). Its larvae, tetrathyridium, are approximately 1 mm long and 300 μm wide and infect a wide range of host species including humans. Tetrathyridium migrate through the intestinal wall to invade the peritoneal cavity. Despite intestinal penetration by such a large-sized parasite, symptomatic intestinal disorders are not common during the migration period. In this study, the dynamics of tetrathyridia migration and their pathogenicity towards intestinal tissues were examined in mice infected orally with these parasites. Most tetrathyridia were found to migrate through the intestinal wall, moving into the peritoneal cavity or liver 24 to 48 hours after the oral infections. Next, the pathogenicity of tetrathyridium in the intestinal wall was histopathologically evaluated, and tissue injury from tetrathyridium migration was confirmed. Inflammatory foci were observed as tetrathyridium migration tracks from 48 hours after oral infection; however, the number of inflammatory foci had decreased by half more than 48 hours later. Therefore, we examined the gene expression levels of the macrophage driving cytokine, IL-1β, and the eosinophil recruiting chemokine, CCL11, by quantitative reverse-transcriptase PCR. The expression levels of these genes in the infected group were significantly lower than those of the non-infected group at 48 hours post-infection. Although the immunomodulating ability of the excretory-secretory products released from tetrathyridium has been previously shown by in vitro assays, the significance of this ability in their lifecycle has remained unclear. In this study, we discovered that tetrathyridium causes temporal inflammation in the intestinal wall during penetration and large-scale migration in this organ, but tetrathyridium simultaneously suppresses the host’s inflammatory gene expression, might to be a strategy that reduces inflammatory responses and increases survival of the parasite.

The prevalence and risk factors of liver fluke infection in Mexican horses, donkeys and mules in tropical and temperate regions

The objective of the present study was to determine the seroprevalence of Fasciola hepatica infection in horses, donkeys and mules from different climate regions in two states of Mexico. A total of 594 serum samples were analysed for immunoglobulin G (IgG) antibodies using enzyme-linked immunosorbent assay (ELISA), with excretory-secretory (E/S) products as the antigen. The diagnostic sensitivity and specificity of serum IgG ELISA were 100% and 97.2%, respectively. We collected data using a questionnaire. The overall prevalence of the parasite in equids between May 2018 and April 2019 was 13.1% (78 out of 594). The highest prevalence was found in mules (17.9%), followed by that in donkeys (13.9%) and horses (10.4%). In addition, the highest percentage of positive equines was detected in samples from the tropical climate (17.5%). The identification of risk factors was assessed by bivariate logistic regression analysis. Mules had 8.608 times higher risk for F. hepatica infections as compared with horses (odds ratio (OR) 8.608; 95% confidence interval (CI): 2.809-26.377), while the OR for 6-10-year-old equines was 93.375 compared with that of young equines (≤ 5years old) (OR 93.375; 95% CI: 11.075-787.239). Likewise, tropical climate equines had 83.7% increased odds for fasciolosis (OR 1.83).

Identification and Analysis of the Tegument Protein and Excretory-Secretory Products of the Carcinogenic Liver Fluke Clonorchis sinensis.

Liver fluke proteins, including excretory-secretory products (ESPs) and tegument proteins, are critical for the pathogenesis, nutrient metabolism, etiology and immune response of liver cancer. To understand the functions of various proteins in Clonorchis sinensis physiology and human clonorchiasis, the ESPs and tegument proteins of C. sinensis were identified. Supernatants containing ESPs from adult C. sinensis after culture for 6 h were harvested and concentrated. The tegument was detached using a freeze/thaw method and successively extracted using various extraction buffers. The outer surface proteins of C. sinensis were labeled with biotin, and the biotinylated proteins were purified. The ESP, tegument and labeled outer surface proteins were identified and analyzed by high-resolution LC-MS/MS. The identified proteins were compared with those of other flukes, and the protein functions associated with pathogenesis, carcinogenesis and potential vaccine antigens and drug targets were predicted and analyzed. A total of 175 proteins were identified after the 6-h culture of C. sinensis ESPs. A total of 352 tegument proteins were identified through sequential solubilization of the isolated teguments, and a subset of these proteins were localized to the surface membrane of the tegument by labeling with biotin. Thirty identified proteins, including annexins, actin and tetraspanins, were identified as potential immunomodulators and promising vaccine antigens. Interestingly, among the 352 tegument proteins, as many as 155 were enzymes, and most were oxidoreductases, hydrolases or transferases. A comparison of the outer surface proteins of C. sinensis with those of other flukes indicated that flukes have some common outer surface proteins, such as actin, tetraspanin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and annexin. Granulin, thioredoxin peroxiredoxin, carbonyl reductase 1 and cystatin were identified in the C. sinensis proteome and predicted to be related to liver disease and cancer. The analysis of the C. sinensis proteome could contribute to a more in-depth understanding of complex parasite-host relationships, improve the diagnosis of clonorchiasis and benefit research on the pathogenesis and development of novel interventions, drugs and vaccines to control C. sinensis infection.

Immunomodulatory action of excretory-secretory products of Angiostrongylus cantonensis in a mouse tumour model.

Excretory-secretory products (ESPs) of parasitic helminths are well known to exert immunostimulation and immunomodulation in hosts. Immune regulation plays a key role in anti-tumour therapy. The present study explored the anti-tumour effect of ESPs released by Angiostrongylus cantonensis. In Hepa1-6 mouse tumour models, ESPs significantly reduced tumour growth. Tumour-bearing mice treated with ESPs had significantly higher CD3+, CD4+, and CD8+ T cell counts than those treated with Freund's adjuvant. In vitro, human hepatocarcinoma HepG2 cells, human lung cancer A549 cells, and normal human liver HL-7702 cells were co-incubated with ESPs for 24h and 48h. ESPs significantly accelerated HepG2 apoptosis but had no inhibitory effect on the proliferation of A549 and HL-7702 cells. Apoptotic HepG2 cells displayed condensed nuclei, apoptotic bodies, and swollen endoplasmic reticulum (ER). Expression of the endoplasmic reticulum stress (ERS)-related factors activating transcription factor 6 (ATF6) and C/EBP-homologous protein (CHOP) in HepG2 cells increased with increasing ESP concentration and treatment time. Calreticulin (CRT) is a key effector protein of ESPs, and recombinant calreticulin (rCRT) was produced in BL21 Escherichia coli (E. coli). In contrast to ESPs, rCRT markedly reduced the proliferation of HepG2 cells. The expression levels of ATF6 and CHOP in HepG2 cells treated with 30μg/mL rCRT significantly increased at 48h. Notably, these findings synergistically suggest that ESPs and rCRT are promising candidates for anti-tumour immunotherapy.

Fasciola hepatica Extracellular Vesicles isolated from excretory-secretory products using a gravity flow method modulate dendritic cell phenotype and activity.

Parasite-released extracellular vesicles (EVs) deliver signals to the host immune system that are critical to maintaining the long-term relationship between parasite and host. In the present study, total EVs (FhEVs) released in vitro by adults of the helminth parasite Fasciola hepatica were isolated using a recently described gravity flow method that protects their structural integrity. The FhEVs molecular cargo was defined using proteomic analysis and their surface topology characterised by glycan microarrays. The proteomic analysis identified 618 proteins, 121 of which contained putative N-linked glycosylation sites while 132 proteins contained putative O-linked glycosylation sites. Glycan arrays revealed surface-exposed glycans with a high affinity for mannose-binding lectins indicating the predominance of oligo mannose-rich glycoproteins, as well as other glycans with a high affinity for complex-type N-glycans. When added to bone-marrow derived dendritic cells isolated FhEV induced a novel phenotype that was categorised by the secretion of low levels of TNF, enhanced expression of cell surface markers (CD80, CD86, CD40, OX40L, and SIGNR1) and elevation of intracellular markers (SOCS1 and SOCS3). When FhEV-stimulated BMDCs were introduced into OT-II mice by adoptive transfer, IL-2 secretion from skin draining lymph nodes and spleen cells was inhibited in response to both specific and non-specific antigen stimulation. Immunisation of mice with a suspension of FhEV did not elicit significant immune responses; however, in the presence of alum, FhEVs induced a mixed Th1/Th2 immune response with high antigen specific antibody titres. Thus, we have demonstrated that FhEVs induce a unique phentotype in DC capable of suppressing IL-2 secretion from T-cells. Our studies add to the growing immuno-proteomic database that will be an important source for the discovery of future parasite vaccines and immunotherapeutic biologicals.