Excretory-secretory Products Research Articles

Comparative characterization of two galectins excreted-secreted from intestine-dwelling parasitic versus free-living females of the soil-transmitted nematode Strongyloides

Helminths are complex pathogens that ensure their long-term survival by influencing the immune responses of their host. Excretory/secretory products (ESP) can exert immunoregulatory effects which foster parasite survival. Galectins represent a widespread group of β-galactoside-binding proteins which are involved in a multitude of biological processes operative in parasite-host interaction. We had earlier identified seven galectins in Strongyloides ratti, four of them detected in the ESP of distinct developmental stages of the parasite. In the present report, we focused on the characterization of two of them, Sr-galectin-1 (Sr-Gal-1) and Sr-galectin-3 (Sr-Gal-3). While Sr-Gal-3 expression was strongest in parasitic females, Sr-Gal-1 was predominantly expressed in free-living females. Both proteins were cloned and recombinantly expressed in an E. coli expression system. Their glycan-binding activity was verified by haemagglutination and glycan array analysis. Furthermore, primary immunological activities of the Sr-galectins were initially investigated by the application of an in vitro mucosal 3D-culture model, comprising of mucosa-associated epithelial and dendritic cells. The Sr-galectins stimulated preferentially the release of the type 2 cytokines thymic stromal lymphopoietin and IL-22, a first indication for immunoregulatory activity. In addition, the Sr-galectins dose-dependently fostered cell migration. Our results confirm the importance of these carbohydrate-binding proteins in host-parasite-interaction by indicating possible interaction with the host mucosa-associated cells.

Vaccination with DNA encoding ES 43-kDa /45-kDa antigens significantly reduces Trichinella spiralis infection in mice

Trichinella spiralis is an intestinal nematode parasite that can cause trichinellosis in humans and animals worldwide. The most important known hosts of T. spiralis are pigs, horses, dogs and cats. Pork and its products are the main sources of infection in human trichinellosis. Vaccines against these infections are urgently needed. In this study, the genes encoding the 43-kDa or 45-kDa glycoprotein present in the excretory-secretory (ES) products from T. spiralis muscle larvae (ML) were cloned into the eukaryotic expression vector pVAX1, resulting plasmids pVAX1-Ts43 and pVAX1-Ts45, respectively. Then BALB/c mice were intramuscularly immunized with the DNA vaccine pVAX1-Ts43, pVAX1-Ts45, or both to evaluate their immunogenicity and host protective potential. After the third immunization, mice of each group were challenged with 300 T. spiralis ML. The results showed that the mice immunized with the DNA vaccine pVAX1-Ts43 or pVAX1-Ts45 developed significant numbers of FAS+PNA+ B220+ B cells indicating the formation of the germinal centers (GCs), IFN-γ-secreting (mesenteric lymph nodes, MLN) cells, and IL-4-, and IL-10-secreting splenocytes. Mice immunized with the pVAX1-Ts43 or pVAX1-Ts45 vaccine elicited partial protective immunity against challenge infections with T. spiralis as shown by significant reduction in ML. Most notably, the combined immunity of pVAX1-Ts43 and pVAX1-Ts45 induced better immune responses than either of the DNA vaccines given alone and provided as high as 75.9% reductions in muscle larval burden. These results suggest that the plasmid DNA encoding the 43-kDa or 45-kDa glycoprotein could be considered as a potential vaccine candidate against T. Spiraling infection.

Silent Witness: Dual-Species Transcriptomics Reveals Epithelial Immunological Quiescence to Helminth Larval Encounter and Fostered Larval Development.

Gastrointestinal nematodes are among the most prevalent parasites infecting humans and livestock worldwide. Infective larvae of the soil-transmitted nematode Ascaris spp. enter the host and start tissue migration by crossing the intestinal epithelial barrier. The initial interaction of the intestinal epithelium with the parasite, however, has received little attention. In a time-resolved interaction model of porcine intestinal epithelial cells (IPEC-J2) and infective Ascaris suum larvae, we addressed the early transcriptional changes occurring simultaneously in both organisms using dual-species RNA-Seq. Functional analysis of the host response revealed an overall induction of metabolic activity, without induction of immune responsive genes or immune signaling pathways and showing suppression of chemotactic genes like CXCL8/IL-8 or CHI3L1. Ascaris larvae, when getting in contact with the epithelium, showed induction of genes that orchestrate motor activity and larval development, such as myosin, troponin, myoglobin, and protein disulfide isomerase 2 (PDI-2). In addition, excretory-secretory products that likely facilitate parasite invasion were increased, among them, aspartic protease 6 or hyaluronidase. Integration of host and pathogen data in an interspecies gene co-expression network indicated links between nematode fatty acid biosynthesis and host ribosome assembly/protein synthesis. In summary, our study provides new molecular insights into the early factors of parasite invasion, while at the same time revealing host immunological unresponsiveness. Reproducible software for dual RNA-Seq analysis of non-model organisms is available at https://gitlab.com/mkuhring/project_asuum and can be applied to similar studies.

Dectin-1 on macrophages modulates the immune response to Fasciola hepatica products through the ERK signaling pathway

Fasciolosis is a zoonotic disease of increasing importance due to its worldwide distribution and elevated economic losses. Previously, we demonstrated that Fasciola hepatica excretory–secretory products (FhESP) induce immunomodulatory effects on peritoneal macrophages in a Dectin-1 dependent manner.In this study, we observed that peritoneal macrophages from naive BALB/c mice stimulated in vitro with FhESP presented increased expression levels of phosphorylated extracellular-signal-regulated kinase (ERK), and this effect was dependent on Syk, protein kinase C (PKC) and Dectin-1. In this sense, we observed increased levels of arginase activity, IL-10 and TGF-β in macrophages stimulated with FhESP, which were dependent on PKC and ERK. Furthermore, we observed that the increased arginase activity, as well as in TGF-β and IL-10 levels, was partially dependent on IL-10 receptor signaling in macrophages that were pre-incubated with anti-IL10R before being stimulated with FhESP.Taken together, these results suggest the participation of Dectin-1 and Syk in FhESP interaction with peritoneal macrophages and the possible role of ERK and IL-10 in downstream signaling pathways involved in the immunomodulatory effects induced by Fasciola hepatica products.

The Intestinal Roundworm Ascaris suum Releases Antimicrobial Factors Which Interfere With Bacterial Growth and Biofilm Formation.

Ascariasis is a widespread soil-transmitted helminth infection caused by the intestinal roundworm Ascaris lumbricoides in humans, and the closely related Ascaris suum in pigs. Progress has been made in understanding interactions between helminths and host immune cells, but less is known concerning the interactions of parasitic nematodes and the host microbiota. As the host microbiota represents the direct environment for intestinal helminths and thus a considerable challenge, we studied nematode products, including excretory-secretory products (ESP) and body fluid (BF), of A. suum to determine their antimicrobial activities. Antimicrobial activities against gram-positive and gram-negative bacterial strains were assessed by the radial diffusion assay, while effects on biofilm formation were assessed using the crystal violet static biofilm and macrocolony assays. In addition, bacterial neutralizing activity was studied by an agglutination assay. ESP from different A. suum life stages (in vitro-hatched L3, lung-stage L3, L4, and adult) as well as BF from adult males were analyzed by mass spectrometry. Several proteins and peptides with known and predicted roles in nematode immune defense were detected in ESP and BF samples, including members of A. suum antibacterial factors (ASABF) and cecropin antimicrobial peptide families, glycosyl hydrolase enzymes such as lysozyme, as well as c-type lectin domain-containing proteins. Native, unconcentrated nematode products from intestine-dwelling L4-stage larvae and adults displayed broad-spectrum antibacterial activity. Additionally, adult A. suum ESP interfered with biofilm formation by Escherichia coli, and caused bacterial agglutination. These results indicate that A. suum uses a variety of factors with broad-spectrum antibacterial activity to affirm itself within its microbe-rich environment in the gut.

Cs1, a Clonorchis sinensis-derived serodiagnostic antigen containing tandem repeats and a signal peptide.

BackgroundClonorchiasis, caused by the liver fluke Clonorchis sinensis, remains a serious public health issue in Asia, especially in China, and its relationship with cholangiocarcinoma has highlighted the importance of C. sinensis infection. Proteins containing tandem repeats (TRs) are found in a variety of parasites and, as targets of B-cell responses, are valuable for the serodiagnosis of parasite infections. Here, we identified a novel C. sinensis-specific antigen, Cs1, containing TRs, and investigated its diagnostic value, other immunological properties, and tissue distribution.Methodology/Principal findingsA partial Cs1 cDNA sequence was cloned by screening an adult C. sinensis cDNA expression library. The full-length Cs1 cDNA was obtained by 5′ rapid amplification of cDNA ends. The deduced Cs1 protein consists of a signal peptide and five TRs of 21 amino acids. The recombinant Cs1 (rCs1) was constructed and purified. rCs1 showed higher sensitivity (94.3%) and specificity (94.4%) than the C. sinensis excretory–secretory products (ESPs) according to ELISA of 114 serum samples. Native Cs1 was identified in C. sinensis ESPs and crude antigens of adult C. sinensis by western blotting using an anti-rCs1 monoclonal antibody. ELISA of recombinant peptides of different Cs1 regions demonstrated that the TR region was immunodominant in Cs1. Immunohistochemistry and confocal microscopy revealed that Cs1 is located in a granule-like structure surrounding the acetabulum of C. sinensis adults that has not previously been described.Conclusions/SignificanceWe identified a novel C. sinensis-specific TR protein, Cs1, which is an antigen of high serological significance, compared with C. sinensis ESPs. The deduced features of Cs1 show a unique structure containing TRs and a signal peptide and the TR region is immunodominant in Cs1. This provides a basis for targeted screens of other antigens. The novel structure in which Cs1 is located also deserves further investigation.

Assessment of Fasciola hepatica glutathione S-transferase as an antigen for serodiagnosis of human chronic fascioliasis

Due to the unsatisfactory performance of parasitological diagnosis of human fascioliasis; the use of immunodiagnosis based on the detection of anti-Fasciola antibodies is traditionally used as a diagnostic alternative using total or purified parasite excretory-secretory products (ESPs). Glutathione S-transferase (GST) protein, one of the F. hepatica ESP components, possesses well-known roles in the detoxification of xenobiotic and endogenously derived toxins within the host bile environment. GST has shown to be a good target for vaccine or drug development against fascioliasis. The current study aimed to evaluate the potential of GST protein purified from a soluble crude extract of adult flukes as an antigen for serodiagnosis of chronic human fascioliasis by indirect ELISA. The study included a panel of 116 serum samples collected from individuals with confirmed fascioliasis, individuals carrying heterologous parasitic infections and healthy subjects. The parasitological examination was used as gold standard and a previously optimized ESP-ELISA was used to compare the performance of the GST-ELISA method. Results demonstrated that GST-ELISA is 94.3% sensitive, 80.2% specific and exhibits a moderate positive correlation (r = 0.555) and substantial agreement (k = 0.786) with the results obtained with the ESP-ELISA method. Moreover, because no sera from patients with early F. hepatica infection were available, GST-ELISA was then tested with sera from rabbits experimentally infected with F. hepatica metacercariae. The assay was able to detect anti-Fasciola antibodies as early as the 3rd week of infection (p < 0.0001) with peaks at 4th and 10th week post-infection.

The somatic proteins of Toxocara canis larvae and excretory-secretory products revealed by proteomics

Toxocariasis is a widespread helminth infection of dogs and cats, caused by Toxocara canis and Toxocara cati larvae, respectively. Toxocara spp. can cause zoonotic infections in humans by invading tissues and organs causing pathology. Toxocara spp. larvae release excretory-secretory molecules (TES) into the body of their host that are fundamental to the host-parasite interaction and could be used as targets for novel diagnostics and vaccines. In the present study, we identified 646 T. canis proteins from TES and larval extract using 1D-SDS PAGE followed by mass spectrometry. A wide range of proteins was identified that may play a role both in the induction of the host immune response and host pathology, and in parasite metabolism and survival. Among these proteins there are potential candidates for novel diagnostics and vaccines for dogs and cats toxocariases.

Protection Against Arthritis by the Parasitic Worm Product ES-62, and Its Drug-Like Small Molecule Analogues, Is Associated With Inhibition of Osteoclastogenesis.

The immunomodulatory actions of parasitic helminth excretory-secretory (ES) products that serendipitously protect against development of chronic inflammatory disorders are well established: however, knowledge of the interaction between ES products and the host musculoskeletal system in such diseases is limited. In this study, we have focused on ES-62, a glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae that is immunomodulatory by virtue of covalently attached phosphorylcholine (PC) moieties, and also two synthetic drug-like PC-based small molecule analogues (SMAs) that mimic ES-62’s immunomodulatory activity. We have previously shown that each of these molecules prevents development of pathology in collagen-induced arthritis (CIA), a model of the musculoskeletal disease rheumatoid arthritis (RA) and reflecting this, we now report that ES-62 and its SMAs, modify bone remodeling by altering bone marrow progenitors and thus impacting on osteoclastogenesis. Consistent with this, we find that these molecules inhibit functional osteoclast differentiation in vitro. Furthermore, this appears to be achieved by induction of anti-oxidant response gene expression, thereby resulting in reduction of the reactive oxygen species production that is necessary for the increased osteoclastogenesis witnessed in musculoskeletal diseases like RA.

Onchocerca - infected cattle produce strong antibody responses to excretory-secretory proteins released from adult male Onchocerca ochengi worms

BackgroundThe front line molecules from filarial worms and other nematodes or helminthes are their Excretory-Secretory (ES) products. Their interaction with the host cells, proteins and immune system accounts for the skin and eye pathology or hyposensitivity observed in human onchocerciasis. ES products and adult worms’ crude extracts from Onchocerca ochengi, a filarial nematode that infects the African zebu cattle, were utilized in the present study as a model for studying Onchocerca volvulus that causes river blindness in man.MethodsThe ES products were generated from adult male and female worms in vitro and analyzed with poly acrylamide gel electrophoresis (PAGE) and enzyme-linked immunosorbent assay (ELISA) using sera from Onchocerca-infected cattle and humans. The cattle sera were collected from a herd that had been exposed for six years to natural transmission of Onchocerca spp. The expressed reactivity was evaluated and differences analyzed statistically using Kruskal-Wallis rank and Chi-square tests.ResultsThe gel electrophoretic analyses of 156 ES products from O. ochengi female and male worms and of two somatic extracts from three females and 25 males revealed differences in the protein pattern showing pronounced bands at 15, 30–50 and 75 kDa for male ES proteins and 15, 25 and 40–75 kDa for somatic extracts, respectively and less than 100 kDa for female worms. Proteins in the ES products and somatic extracts from female and male Onchocerca ochengi worms were recognized by IgG in sera from both Onchocerca-exposed cattle and humans. Bovine serum antibodies reacted more strongly with proteins in the somatic extracts than with those in the ES products. Interestingly, the reaction was higher with male ES products than with ES products from female worms, suggesting that the males which migrate from one nodule to another are more exposed to the host immune system than the females which remain encapsulated in intradermal nodules.ConclusionsThis study demonstrates that O. ochengi ES products and, in particular, extracts from male filariae may represent a good source of immunogenic proteins and potential vaccine candidates.

Standardization of recombinant Ancylostoma caninum cysteine protease 2 (rAcCP2) based indirect ELISA for serodiagnosis of hookworm infection in dogs

Diagnosis of hookworm infection in dogs during pre-patentency or in pregnant bitches harbouring encysted larvae in tissues can be achieved by employing serological tests using proteins derived from somatic or excretorysecretory products of adult or larvae of Ancylostoma caninum. In the present study, cathepsin-B protease (AcCP2) of A. caninum, which helps in development of parasitism and nutrient digestion, was used to develop an indirect ELISA for detection of specific antibodies to A. caninum in dogs. The rAcCP2 (approx. 59.0 kDa) was cloned, expressed and purified under denaturing conditions. Serum samples of 20 known A. caninum positive and 15 known negative dogs were used for the standardization of indirect ELISA. The sensitivity and specificity of the assay was determined by using sera samples from 123 dogs (48 positive for A. caninum eggs in faeces and 75 faecal negative). Out of the 48 A. caninum faecal positive sera, 46 were tested positive (OD &gt; 0.306) by indirect ELISA; whereas, 14 out of 75 faecal negative samples were also tested positive (OD &gt; 0.306) by indirect ELISA. Based on the results, the sensitivity and specificity of rAcCP2 based indirect ELISA was calculated to be 95.8% and 84.3%, respectively. No cross-reactions were observed with serum from dogs naturally infected with B. canis vogeli, B. gibsoni, E. canis, Dirofilaria immitis and Toxocara canis. The results of the present study indicate that indirect ELISA with rAcCP2 protein might be appropriate in large scale epidemiological screening and for serological diagnosis of A. caninum infection in dogs.

Fasciola hepatica Glutathione S-tranferase suppresses inflammatory cytokines and chemokines in vivo against lethal doses of lipopolysaccharide on C57Bl/6 mice.

Abstract The world-wide helminth parasite, Fasciola hepatica, is an excellent immunomodulator. It survives in the host by preventing immune responses against itself by using excretory-secretory products (ESPs). One of the ESPs well-known for its anti-oxidant properties is Glutathione S-transferase (GST). Although it has been studied as a vaccine, the function GST plays in immunomodulation has scarcely been studied. Previously, we reported how the use of GST prior to TLR ligands significantly decreased TLR activation in vitro and boosted the survival rate of mice injected intraperitoneally with GST before a lethal dose of LPS by 80%. In this study, we investigate whether these C57BL/6 mice that survived after a lethal dose of LPS experienced a reduction in inflammatory cytokines and/or chemokines. For this, C57BL/6 6–8 week-old mice were allotted into two groups of 12 animals each. The control group (n=12) was injected intraperitoneally with 0.1M endotoxin free PBS and allowed to rest 1 hr, while the experimental group (n=12) was injected intraperitoneally with 15 ug of GST and allowed to rest 1 hour. Afterwards, both groups were injected with a lethal dose (1 mg) of E.coli LPS (serotype 0111:B4; Sigma) and monitored during a 48 hour period. Sera from mice in the experimental group were compared to the control group by using a Cytokine 20-Plex Mouse Panel kit (Invitrogen) for a wide array of cytokines and chemokines. Our results demonstrate that injecting 15ug of GST intraperitoneally 1 hour before a lethal dose of LPS gives the surviving 80% of C57BL/6 mice significantly reduced amounts of pro-inflammatory cytokines and chemokines, such as IL-1β (p = 0.0004), IL-12p70 (p = 0.0001) and MIP-1α (p = 0.0024), after 48 hrs.

Helminth Infections: Recognition and Modulation of the Immune Response by Innate Immune Cells.

The survival of helminths in the host over long periods of time is the result of a process of adaptation or dynamic co-evolution between the host and the parasite. However, infection with helminth parasites causes damage to the host tissues producing the release of danger signals that induce the recruitment of various cells, including innate immune cells such as macrophages (Mo), dendritic cells (DCs), eosinophils, basophils, and mast cells. In this scenario, these cells are able to secrete soluble factors, which orchestrate immune effector mechanisms that depend on the different niches these parasites inhabit. Here, we focus on recent advances in the knowledge of excretory-secretory products (ESP), resulting from helminth recognition by DCs and Mo. Phagocytes and other cells types such as innate lymphocyte T cells 2 (ILC2), when activated by ESP, participate in an intricate cytokine network to generate innate and adaptive Th2 responses. In this review, we also discuss the mechanisms of innate immune cell-induced parasite killing and the tissue repair necessary to assure helminth survival over long periods of time.

Fasciola hepatica Glutathione S‐Transferase (FhGST) conformational studies and its effect on the suppression of toll‐like receptors stimulation

Fasciola hepatica is a parasite well known as a master of immunomodulation because of its ability to suppress the Th1 immune responses and elicit a strong Th2/Treg response. This occurs when the parasite releases excretory‐secretory products (ESPs), which are comprised of a myriad of glycoproteins that are believed to play an important role in the immune response. F. hepatica Glutathione S‐Transferase (FhGST) is one of the ESP components recognized for its anti‐oxidant properties and its capacity to provide part of the parasite's defense by detoxifying the secondary products of lipid peroxidation that are produced due to the immune free‐radical attack on the host or the parasite membrane. Our preliminary studies demonstrate that FhGST exerts a suppressive effect on the activation of TLR4 in macrophages which is evidenced by significant suppression of NF‐kB activation and down‐regulation of TNFα and IL1‐β in response to LPS‐stimuli. We speculated that the integrity of the tertiary structure, as well as the glycans that decorate the protein moiety of FhGST, could be essential for its capacity to suppress the NF‐kB activation. To test our hypothesis, we first subjected FhGST to a thermal denaturing process by boiling it at 90°C for 10 min followed by a treatment with PNGase F. Circular dichroism analysis (CD) comparing the FhGST spectra before and after heat denaturing served to confirm that FhGST was full and irreversibly denatured. Next, denatured or native FhGST were added alone or in the presence of the whole extract of 1 x 108 cells Klebsiella pneumonia (Kp) to a culture of human monocytes (THP1‐CD14) that express most TLRs as well as an NF‐kb inducible secreted embryonic alkaline phosphatase reporter gene. Cells stimulated with an attenuated whole extract of 1 × 108 cells Klebsiella pneumonia (Kp) were used as activation control. Our results demonstrated that native FhGST suppresses NF‐kB activation by 83%, whereas the heat‐denatured protein suppresses NF‐kB activation by 84%. These suppressions suggest that in contrast to expected, the capacity of FhGST to suppress the NF‐κB activation in THP1‐CD14 cells is independent of the integrity of its secondary/tertiary structure and is not associated to the glycans. Docking analysis in which the FhGST‐protein moiety was docked with the CD14 and MD2 co‐receptor structures suggested that FhGST could target these co‐receptors as part of its mechanisms of action.Support or Funding InformationThis study was supported by MBRS‐RISE R25GM061838‐13, 5R25GM061151‐16 and NIH grants G12MD007600 and 2P40OD012217This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparison of Secretome Profile of Pathogenic and Non-Pathogenic Entamoeba histolytica.

The obligatory intracellular protozoan parasite Entamoeba histolytica causes amebic dysentery and liver abscess. E. histolytica adheres to the host tissues in a contact-dependent manner. E. histolytica excretory-secretory products (ESP) might play critical roles during invasion. We comparatively analyzed the secretome profile of E. histolytica pathogenic HM-1:IMSS and non-pathogenic Rahman strains. The two ESP revealed similar but distinct spotting patterns. In both ESP, alcohol dehydrogenase, enolase 1, and transketolase, which control classical carbohydrate metabolism and other moonlighting effects, constituted the most abundant fractions. We recognized differently secreted molecules. Secretion of cytoskeletal organization proteins (actin, actin binding protein, and EHI_068510), protein remodeling amino peptidase, and multifunctional elongation factor 1-α were increased in Rahman. Conversely, carbohydrate metabolizing enolase 1, alcohol dehydrogenase, transketolase, calponin, phosphoglucose mutase, malic enzyme and EHI_156420, xenobiotic scavenging superoxide dismutase and EHI_140740, and pyruvate:ferredoxin oxidoreductase and coronin (carbohydrate metabolism/detoxification) showed reduced secretion. Transcription levels of some genes involved in these processes also decreased. Changes of secretory behavior, especially decreased secretion of multifunctional carbohydrate metabolizing enzymes and detoxifying proteins that importantly participated in amoeba pathogenesis might reflect avirulent nature of Rahman strain in the host.

Identification, isolation, and cloning of the full-length sequence of a gene encoding trypsin inhibitor-like protein (TIL) secreted by the intestinal parasitic nematode Strongyloides ratti

BackgroundIntestinal nematodes may interact with the intestinal mucosal host cells through molecules they produced actively in the host environment. Many excretory/secretory products (ESP) of the intestinal nematodes are considered as interesting candidates to be examined in vitro on their tolerance-inducing activities. Nematode-derived protease inhibitors are believed to have multiple, specific abilities for the host immunomodulation, playing on various immune effector mechanisms. Among the previously identified 500 ESP from Strongyloides ratti, trypsin inhibitor-like (Sra-TIL) peptides were detected in high concentration in the ESP of the parasitic female (pF). The aim is to identify interesting tolerance-inducing proteins belonging to the longtime adapted human biome which may be potentially applied to moderate inflammatory immune reactions.ResultsIn the present study, the full-length cDNA (from the parasitic female) and gDNA of the ~ 1605 bp Sra-til is identified and isolated by polymerase chain reaction (PCR), subsequent to cluster/EST analysis via alignment and BLAST search. gDNA and RNA were extracted from infective third stage larvae (iL3) and pF respectively, and the cDNA was synthesized.Then, the Sra-til was cloned and sequenced. The genomic organization analysis revealed that introns were absent in the Sra-til gene structure. Sequence analyses of both nucleotide and predicted protein sequences were investigated using different bioinformatic tools. The predicted architecture of the translated amino acid sequence showed the presence of a signal peptide for secretion. The architecture of Sra-TIL was Sra-TIL = TIL, EGF (epidermal growth factor) × 6). Phylogenetic analysis of the deduced amino acid sequence showed how related this novel Strongyloides protein to TIL orthologs from other nematodes.ConclusionThe full-length Sra-til gene which encodes an interesting ESP candidate, secreted from pF, was identified, isolated, cloned, and then sequenced. Both nucleotide and amino acid sequences were analyzed by various bioinformatic tools. The predicted innovative structural advantages of Sra-TIL polypeptides secreted from a parasitic intestinal stage of S. ratti nematode is probably reflective of the abundance of different biological functions, including the protection against host proteases, host immunomodulation via proliferation induction of the damaged intestinal cells as well as wound healing properties.

Trichinella spiralis Excretory-Secretory Products Induce Tolerogenic Properties in Human Dendritic Cells via Toll-Like Receptors 2 and 4.

Trichinella spiralis, as well as its muscle larvae excretory–secretory products (ES L1), given either alone or via dendritic cells (DCs), induce a tolerogenic immune microenvironment in inbred rodents and successfully ameliorate experimental autoimmune encephalomyelitis. ES L1 directs the immunological balance away from T helper (Th)1, toward Th2 and regulatory responses by modulating DCs phenotype. The ultimate goal of our work is to find out if it is possible to translate knowledge obtained in animal model to humans and to generate human tolerogenic DCs suitable for therapy of autoimmune diseases through stimulation with ES L1. Here, the impact of ES L1 on the activation of human monocyte-derived DCs is explored for the first time. Under the influence of ES L1, DCs acquired tolerogenic (semi-matured) phenotype, characterized by low expression of HLA-DR, CD83, and CD86 as well as moderate expression of CD40, along with the unchanged production of interleukin (IL)-12 and elevated production of IL-10 and transforming growth factor (TGF)-β, compared to controls. The interaction with DCs involved toll-like receptors (TLR) 2 and 4, and this interaction was mainly responsible for the phenotypic and functional properties of ES L1-treated DCs. Importantly, ES L1 potentiated Th2 polarizing capacity of DCs, and impaired their allo-stimulatory and Th1/Th17 polarizing properties. Moreover, ES L1-treated DCs promoted the expansion of IL-10- and TGF-β- producing CD4+CD25hiFoxp3hi T cells in indolamine 2, 3 dioxygenase (IDO)-1-dependent manner and increased the suppressive potential of the primed T cell population. ES L1-treated DCs retained the tolerogenic properties, even after the challenge with different pro-inflammatory stimuli, including those acting via TLR3 and, especially TLR4. These results suggest that the induction of tolerogenic properties of DCs through stimulation with ES L1 could represent an innovative approach for the preparation of tolerogenic DC for treatment of inflammatory and autoimmune disorders.

Identification and partial characterization of a novel serpin from Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea).

Background: Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation. Results: In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm’s excretory-secretory products (ESPs) was confirmed. Conclusion: EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin’s presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses.

Long Noncoding RNA Profiling from Fasciola Gigantica Excretory/Secretory Product-Induced M2 to M1 Macrophage Polarization

Background/Aims: Long noncoding RNAs (lncRNAs) are well known regulators of gene expression that play essential roles in macrophage activation and polarization. However, the role of lncRNA in Fasciola gigantica excretory/secretory products (ESP)-induced M2 polarization into M1 macrophages is unclear. Herein, we performed lncRNA profiling of lncRNAs and mRNAs during the ESP-induced macrophage polarization process. Methods: F. gigantica ESP was used to induce peritoneal cavity M2 macrophages in BALB/c mice (5-6 weeks old) in vivo, and these cells were subsequently isolated and stimulated with IFN-γ + LPS to induce M1 cells in vitro. LncRNA and mRNA profiling was performed via microarray at the end of both polarization stages. Results: In total, 2,844 lncRNAs (1,579 upregulated and 1,265 downregulated) and 1,782 mRNAs (789 upregulated and 993 downregulated) were differentially expressed in M2 macrophages compared to M1 macrophages, and six lncRNAs were identified during polarization. We selected 34 differentially expressed lncRNAs and mRNAs to validate the results of microarray analysis using quantitative real-time PCR (qPCR). Pathway and Gene Ontology (GO) analyses demonstrated that these altered transcripts were involved in multiple biological processes, particularly peptidase activity and carbohydrate metabolism. Furthermore, coding and non-coding gene (CNC) and mRNA-related ceRNA network analyses were conducted to predict lncRNA expression trends and the potential target genes of these lncRNAs and mRNAs. Moreover, we determined that four lncRNAs and four mRNAs might participate in F. gigantica ESP-induced M2 polarization into M1 macrophages. Conclusions: This study illustrates the basic profiling of lncRNAs and mRNAs during F. gigantica ESP-induced M2 polarization into M1 macrophages and deepens our understanding of the mechanism underlying this process.

Comparative Proteomic Study of the Antiproliferative Activity of Frog Host-Defence Peptide Caerin 1.9 and Its Additive Effect with Caerin 1.1 on TC-1 Cells Transformed with HPV16 E6 and E7.

Caerin is a family of peptides isolated from the glandular secretion of Australian tree frogs, the genus Litoria, and has been previously shown to have anticancer activity against several cancer cells. In this work, we used two host-defence peptides, caerin 1.1 and caerin 1.9, to investigate their ability to inhibit a murine derived TC-1 cell transformed with human papillomavirus 16 E6 and E7 growth in vitro. Caerin 1.9 inhibits TC-1 cell proliferation, although inhibition is more pronounced when applied in conjunction with caerin 1.1. To gain further insights into the antiproliferative mechanisms of caerin 1.9 and its additive effect with caerin 1.1, we used a proteomics strategy to quantitatively examine (i) the changes in the protein profiles of TC-1 cells and (ii) the excretory-secretory products of TC-1 cells following caerin peptides treatment. Caerin 1.9 treatment significantly altered the abundance of several immune-related proteins and related pathways, such as the Tec kinase and ILK signalling pathways, as well as the levels of proinflammatory cytokines and chemokines. In conclusion, caerin peptides inhibit TC-1 cell proliferation, associated with modification in signalling pathways that would change the tumour microenvironment which is normally immune suppressive.