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Background: In areas where C. sinensis is endemic, early screening and diagnosis of C. sinensis infection is crucial to prevent complications and interrupt the chain of transmission. Testing for C. sinensis IgG antibodies is frequently employed as a screening method for detecting the disease. However, its effectiveness in populations with a high risk remains to be determined. This study aimed to evaluate the clinical value of IgG antibody testing for screening C. sinensis infection in high-risk populations., Methods: Between October 2020 and September 2023, 1,080 participants from Liuzhou Municipal Liutie Central Hospital patients were recruited. All participants underwent enzyme-linked immunosorbent assay (ELISA) to detect IgG antibodies and fecal examination for C. sinensis eggs using the Kato-Katz technique. The study examined the diagnostic concordance between two methods by using inter-rater agreement evaluation (Kappa). The diagnostic effectiveness of IgG antibodies was assessed comprehensively and across different gender and age categories, with the outcomes of the parasite egg test serving as the benchmark for diagnosis., Results: Out of the 1,080 participants, 48.0% (518/1,080) tested positive for C. sinensis eggs, and 46.9% (506/1,080) tested positive for IgG antibodies. The Kappa value of the two methods' diagnostic concordance was 0.599. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of IgG antibody detection were 78.0%, 81.9%, 79.8%, 80.1%, and 80.0%, respectively, using C. sinensis eggs as the diagnostic criterion. Gender and age subgroup analyses revealed that diagnostic specificity, negative predictive value (NPV), and accuracy were higher in females than males (p = 0.003, 0.001, and 0.049, respectively). Sensitivity tended to decrease, while specificity tended to increase with age (p = 0.007 and 0.010, respectively)., Conclusions: The technique for detecting Clonorchiasis IgG antibodies has a certain degree of accuracy in diagnosing C. sinensis, but its sensitivity is low, particularly in mild infections and in the elderly population. Diagnosis requires a combination of other assays or further optimization of the technique's performance.

Background & Aims: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer and is highly lethal. Clonorchis sinensis (C. sinensis) infection is an important risk factor for iCCA. Here we investigated the clinical impact and underlying molecular characteristics of C. sinensis infection-related iCCA., Methods: We performed single-cell RNA sequencing, whole-exome sequencing, RNA sequencing, metabolomics and spatial transcriptomics in 251 patients with iCCA from three medical centers. Alterations in metabolism and the immune microenvironment of C. sinensis-related iCCAs were validated through an in vitro co-culture system and in a mouse model of iCCA., Results: We revealed that C. sinensis infection was significantly associated with iCCA patients' overall survival and response to immunotherapy. Fatty acid biosynthesis and the expression of fatty acid synthase (FASN), a key enzyme catalyzing long-chain fatty acid synthesis, were significantly enriched in C. sinensis-related iCCAs. iCCA cell lines treated with excretory/secretory products of C. sinensis displayed elevated FASN and free fatty acids. The metabolic alteration of tumor cells was closely correlated with the enrichment of tumor-associated macrophage (TAM)-like macrophages and the impaired function of T cells, which led to formation of an immunosuppressive microenvironment and tumor progression. Spatial transcriptomics analysis revealed that malignant cells were in closer juxtaposition with TAM-like macrophages in C. sinensis-related iCCAs than non-C. sinensis-related iCCAs. Importantly, treatment with a FASN inhibitor significantly reversed the immunosuppressive microenvironment and enhanced anti-PD-1 efficacy in iCCA mouse models treated with excretory/secretory products from C. sinensis., Conclusions: We provide novel insights into metabolic alterations and the immune microenvironment in C. sinensis infection-related iCCAs. We also demonstrate that the combination of a FASN inhibitor with immunotherapy could be a promising strategy for the treatment of C. sinensis-related iCCAs., Impact and Implications: Clonorchis sinensis (C. sinensis)-infected patients with intrahepatic cholangiocarcinoma (iCCA) have a worse prognosis and response to immunotherapy than non-C. sinensis-infected patients with iCCA. The underlying molecular characteristics of C. sinensis infection-related iCCAs remain unclear. Herein, we demonstrate that upregulation of FASN (fatty acid synthase) and free fatty acids in C. sinensis-related iCCAs leads to formation of an immunosuppressive microenvironment and tumor progression. Thus, administration of FASN inhibitors could significantly reverse the immunosuppressive microenvironment and further enhance the efficacy of anti-PD-1 against C. sinensis-related iCCAs., (Copyright © 2024 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Clonorchis sinensis is one of the most important fish-borne zoonotic parasitic worms in humans, and is distributed in several countries with more than 15 million people infected globally. However, the lack of a point-of-care testing (POCT) method is still the critical barrier to effectively prevent clonorchiasis. With the application of novel fluorescent nanomaterials, the development of on-site testing methods with high signal enhancement can provide a simple, precise and inexpensive tool for disease detection. In this study, Eu-(III) nanoparticles (EuNPs) were used as indicative probes, combined with C. sinensis tandem repeat sequence 1 (CSTR1) antigen to capture specific antibodies. Afterward, the complex binds to mouse anti-human IgG immobilized on the test line (T-line) producing a fluorescent signal under UV light. The EuNPs-fluorescent immunoassay (EuNPs-FIA) was successfully constructed, allowing sample detection within 10 min. It enabled both qualitative determination with the naked eye under UV light and quantitative detection by scanning the fluorescence intensity on the test line and control line (C-line). A total of 133 clinical human sera (74 negative, 59 clonorchiasis, confirmed by conventional Kato-Katz (KK) methods and PCR via testing fecal samples corresponding to each serum sample) were used in this study. For qualitative analysis, the cut-off value of fluorescence for positive serum was 31.57 by testing 74 known negative human samples. The assay had no cross-reaction with other 9 parasite-infected sera, and could recognize the mixed infection sera of C. sinensis and other parasites. The sensitivity and specificity of EuNPs-FIA were both 100% compared with KK smear method. Taking advantage of its high precision and user-friendly procedure, the established EuNPs-FIA provides a powerful tool for the diagnosis and epidemiological survey of clonorchiasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fasciola spp., Opisthorchis spp. and Clonorchis sinensis are common liver flukes that can cause a variety of diseases, mainly cholangiocarcinoma induced by clonorchiasis and liver damage and associated pathology induced by fascioliasis. Because these trematodes are parasites of humans and domestic animals, they have greatly affected the economy of agricultural industries and public health worldwide. Due to the emergence of drug resistance and the living habits of flukes, among other reasons, a possibility of reinfection remains even when antiparasitic drugs are used. Therefore, developing a safe, efficient and cost-effective vaccine against trematodes is an important goal. Here, we briefly describe the progress in the development of vaccines against liver flukes. Related innovations may provide effective protection against these helminths and the diseases that they cause., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: DX5 + NKT cells' distribution and population change in BALB/c and FVB mice infected by C sinensis and their function in liver damage were investigated., Methods and Results: Mice were infected by Clonorchis sinensis metacercariae, and lymphocytes were isolated from the livers, spleens and peripheral blood. NK, DX5 + NKT, INF-γ + DX5 + NKT cells and liver fibrosis were analysed. The DX5 + NKT cells displayed the largest amount in normal BALB/c mice liver followed by peripheral blood and spleen. Although the hepatic DX5 + NKT cells of BALB/c mice were more than that of FVB mice, they did not show significant percentage change after C sinensis infection. The hepatic DX5 + NKT cells of FVB mice increased remarkably after infection accompanied with heavier liver injury and fibrosis than the BALB/c mice. And hydroxyproline content was also positively correlated with DX5 + NKT cells only in FVB mice. However, the increase of IFN-γ producing DX5 + NKT cells was lower in FVB mice than in BALB/c mice which showed sharp increase with mild liver damage after infection. The frequencies of anti-fibrotic NK cells were similar in both of the two mouse strains., Conclusions: C sinensis could induce different DX5 + NKT cells responses in different mouse strains which may play roles in liver injury and fibrosis in FVB mice., (© 2020 John Wiley & Sons Ltd.)

Clonorchiasis caused by Clonorchis sinensis is endemic in East Asia; approximately 15 million people have been infected thus far. To diagnose the infection, serodiagnostic tests with excellent functionality should be performed. First, 607 expressed sequence tags encoding polypeptides with a secretory signal were expressed into recombinant proteins using an in vitro translation system. By protein array-based screening using C. sinensis-infected sera, 18 antigen candidate proteins were selected and assayed for cross-reactivity against Opisthorchis viverrini-infected sera. Of the six antigenic proteins selected, four were synthesized on large scale in vitro and evaluated for antigenicity against the flukes-infected human sera using ELISA. CsAg17 antigen showed the highest sensitivity (77.1%) and specificity (71.2%). The sensitivity and specificity of the bacterially produced CsAg17-28GST fusion antigen was similar to those of CsAg17 antigen. CsAg17 antigen can be used to develop point-of-care serodiagnostic tests for clonorchiasis., Competing Interests: The authors have declared that no competing interests exist.

Background: Clonorchis sinensis, a fluke dwelling in the intrahepatic bile ducts causes clonorchiasis, which affect about 15 million people wide-distributed in eastern Asia. During C. sinensis infection, worm-host interaction results in activation of patterns recognition receptors (PRRs) such as Toll-like receptors (TLRs) and further triggers immune responses, which determines the outcome of the infection. However, the mechanisms by which pathogen-associated molecules patterns from C. sinensis interact with TLRs were poorly understood. In the present study, we assumed that the molecules from C. sinensis may regulate host immune responses via TLR2 signaling pathway., Methodology/principal Findings: In the present study, we have identified a ~34 kDa CsHscB from C. sinensis which physically bound with TLR2 as demonstrated by molecular docking and pull-down assay. We also found that recombinant CsHscB (rCsHscB) potently activates macrophage to express various proteins including TLR2, CD80, MHCII, and cytokines like IL-6, TNF-α, and IL-10, but rCsHscB failed to induce IL-10 in macrophages from Tlr2-/- mice. Moreover, ERK1/2 activation was required for rCsHscB-induced IL-10 production in macrophages. In vivo study revealed that rCsHscB triggered a high production of IL-10 in the wild-type (WT) but not in Tlr2-/- mice. Consistently, the phosphorylation of ERK1/2 was also attenuated in Tlr2-/- mice compared to the WT mice, after the treatment with rCsHscB., Conclusions/significance: Our data thus demonstrate that rCsHscB from C. sinensis interacts with TLR2 to be endowed with immune regulatory activities, and may have some therapeutic implications in future beyond parasitology., Competing Interests: Author Hai-Liang Liu was employed by company CapitalBio Technology Inc. All other authors declare no competing interests.

Clonorchis sinensis (C. sinensis) is one of the most serious food-borne parasites, which can lead to liver fibrosis or cholangiocarcinoma. Effective measures for clonorchiasis prevention are still urgently needed. Bacillus subtilis (B. subtilis) is an effective antigen delivery platform for oral vaccines. Chonorchis sinensis serpin (CsSerpin) was proved to be potential vaccine candidates. In this study, CsSerpin3 was displayed on the surface of B. subtilis spore and recombinant spores were orally administrated to BALB/C mice. CsSerpin3-specific IgA levels in faecal, bile and intestinal mucous increased at 4-8 weeks after the first administration compared with those in control groups. The mucus production and the number of goblet cells in intestinal mucosa elevated in B.s-CotC-CsSerpin3 (CotC, coat protein of B. subtilis spore) spores treated group compared to those in blank control. No significant difference in the activities of glutamic-pyruvic transaminase/ alanine aminotransferase and glutamic oxalacetic transaminase/aspartate aminotransferase were observed between groups. There was no side effect inflammation and observable pathological damage in the liver tissue of mice after administration. Moreover, collagen deposition and Ishak score were statistically reduced in B.s-CotC-CsSerpin3 spores treated mice. In conclusion, B. subtilis spores displaying CsSerpin3 could be investigated further as an oral vaccine against clonorchiasis.

Chronic infections with the food-borne liver flukes, Opisthorchis viverrini or Clonorchis sinensis , associate with cholangiocarcinoma, bile duct cancer, which generally has a poor prognosis. We have produced a rapid and simple immunochromatographic test (ICT) kit for the diagnosis of opisthorchiasis and clonorchiasis by the detection of IgG antibodies in human infection sera. Sera from volunteers with proven opisthorchiasis and several other parasitic diseases and from healthy controls were evaluated for the presence of liver fluke infection-specific antibodies using a preparation of excretory-secretory antigen from adult stage O. viverrini absorbed onto ICT strips. Diagnostic values were compared with an ELISA. The diagnostic sensitivity, specificity, and positive and negative predictive values of the ELISA were 100%, 98.3%, 97.9%, and 100%, whereas those for the ICT were 94.6%, 91.2%, 89.7%, and 95.4%, respectively. There was 91.7% concordance between the ICT with ELISA, and differences in performance between the tests were not statistically significant ( P > 0.05). Twenty-seven of 30 (90%) of the clonorchiasis sera also were positive by ICT. This new ICT provides a facile, rapid test for point-of-care testing tool, which can be used at the bedside without the need for sophisticated equipment. Moreover, the ICT can be anticipated to supplement stool examination as a screening tool in the clinic for the diagnosis of opisthorchiasis and clonorchiasis, and in addition, it may be useful in screens of populations at risk of liver fluke infection-associated cholangiocarcinoma.

Chinese liver fluke Clonorchis sinensis changes the host's immune system. Recently, it has been reported that helminths including C. sinensis can ameliorate immune-related diseases such as allergy. In addition, recent studies showed that helminth infection can alleviate immune-mediated disorders by altering the gut microbiome. However, changes in the gut microbiome due to C. sinensis have not been reported yet. In this study, changes in the gut microbiome of C57BL/6 mice infected with C. sinensis metacercariae were evaluated over time. Stool was analyzed by 16S rRNA amplicon analysis using high-throughput sequencing technology. There was no apparent difference in species richness and diversity between the infected and control groups. However, the composition of the microbiome was different between the infected and control groups at 20 days and 30 days post-infection, and the difference disappeared at 50 days post-infection. In particular, this microbiome alteration was associated with a change in the relative abundance of genus Lactobacillus and the probiotic Lactobacillus species that are known to have an immune-modulation role in immune-mediated diseases.

Due to the lack of an effective prophylactic intervention and diagnosis, human liver fluke Clonorchis sinensis continues to afflict a large human population, causing a chronic inflammatory bile duct disease. With an aim to identify target antigens for sensitive serodiagnosis, adenylate kinase 3 of C. sinensis (CsAK3) was successfully expressed in soluble form in Escherichia coli by fusion to an RNA-interacting domain derived from human Lys-tRNA synthetase and purified by Ni2+-affinity chromatography. Anti-CsAK3 serum was raised by immunization of mice, and Western blotting confirmed that CsAK3 was expressed in adult-stage C. sinensis. Histochemical analysis showed that CsAK3 was localized to the subtegumental tissue of C. sinensis and was excreted into the bile duct of the host. When tested against sera from various parasite-infected patients by enzyme-linked immunosorbent assay, the recombinant CsAK3 elicited a specific response to C. sinensis-infected sera. The results suggest that CsAK3, either alone or in combination with other antigens, could be used for improving the clinical diagnosis of clonorchiasis.

Background: Clonorchiasis, 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 Findings: A 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/significance: We 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., Competing Interests: The authors have declared that no competing interests exist.

Venom allergen-like (VAL) proteins are important to host-parasite interactions. We previously demonstrated that a Clonorchis sinensis VAL (CsVAL) protein-derived synthetic peptide suppresses allergic and inflammatory responses. However, little is known regarding the physicochemical and antigenic properties of CsVAL proteins. Here, we identified a novel 194 amino acid VAL protein, named C. sinensis VAL 28 (CsVAL28), and characterized its functional motifs and structural details as a new member of the CAP superfamily. Unlike members of the Schistosoma mansoni VAL (SmVAL) family, CsVAL28 has a single CAP1 motif and six highly conserved disulfide bond-forming cysteines. Tertiary models of wild-type CsVAL28 and mutants were built using SmVAL4 as template via homology modeling. Normal mode analysis predicted that disulfide bond breaking by mutation of cysteine 124 to serine would greatly affect protein mobility. Four major immunoreactive linear epitopes were identified in the surface-exposed region or its vicinity via epitope mapping, using sera from clonorchiasis patients and healthy controls. Our findings provide in-depth knowledge on the structure-function properties of VAL proteins and may help determine highly antigenic regions for developing new diagnostic approaches.

Clonorchis sinensis and Capillaria hepatica are zoonotic parasites that mainly infect the liver and cause serious liver disorders. However, immunological parameters induced by co-infection with these parasites remain unknown. In this study, for the first time, we investigated immunological profiles induced by co-infection with C. hepatica (CH) in C. sinensis (CS)-infected rats (Sprague-Dawley). Rats were infected primarily with 50 metacercariae of C. sinensis; 4 weeks later, they were subsequently infected with 1000 infective C. hepatica eggs. Significantly higher levels of C. sinensis- or C. hepatica-specific IgG antibodies were found in the sera of rats. Interestingly, no cross-reacting antibody was observed between C. sinensis and C. hepatica infections. Significantly raised eosinophil levels were found in the blood of C. sinensis/C. hepatica co-infected rats (CS + CH) compared to the blood of rats infected singly with C. sinensis. Co-infected rats showed significantly higher levels of lymphocyte proliferation and cytokine production compared to a single C. sinensis infection. The worm burden of C. sinensis was significantly reduced in co-infected rats compared to the single C. sinensis infection. These results indicate that the eosinophils, lymphocyte proliferation and cytokine production induced by subsequent infection with C. hepatica in C. sinensis-infected rats might contribute to the observed C. sinensis worm reduction.

Background: Clonorchiasis caused by Clonorchis sinensis has become increasingly prevalent in recent years. Effective prevention strategies are urgently needed to control this food-borne infectious disease. Previous studies indicated that paramyosin of C. sinensis (CsPmy) is a potential vaccine candidate., Methods: We constructed a recombinant plasmid of PEB03-CotC-CsPmy, transformed it into Bacillus subtilis WB600 strain (B.s-CotC-CsPmy), and confirmed CsPmy expression on the spore surface by SDS-PAGE, Western blotting and immunofluorescence assay. The immune response and protective efficacy of the recombinant spore were investigated in BALB/c mice after intragastrical or intraperitoneal immunization. Additionally, biochemical enzyme activities in sera, the intestinal histopathology and gut microflora of spore-treated mice were investigated., Results: CsPmy was successfully expressed on the spore surface and the fusion protein on the spore surface with thermostability. Specific IgG in sera and intestinal mucus were increased after intraperitoneal and intragastrical immunization. The sIgA level in intestinal mucus, feces and bile of B.s-CotC-CsPmy orally treated mice were also significantly raised. Furthermore, numerous IgA-secreting cells were detected in intestinal mucosa of intragastrically immunized mice. No inflammatory injury was observed in the intestinal tissues and there was no significant difference in levels of enzyme-indicated liver function among the groups. Additionally, the diversity and abundance of gut microbiota were not changed after oral immunization. Intragastric and intraperitoneal immunization of B.s-CotC-CsPmy spores in mice resulted in egg reduction rates of 48.3 and 51.2% after challenge infection, respectively. Liver fibrosis degree in B.s-CotC-CsPmy spores treated groups was also significantly reduced., Conclusions: CsPmy expressed on the spore surface maintained its immunogenicity. Both intragastrical and intraperitoneal immunization with B.s-CotC-CsPmy spores induced systemic and local mucosal immune response in mice. Although both intragastric and intraperitoneal immunization elicited a similar protective effect, intragastric immunization induced stronger mucosal immune response without side effects to the liver, intestine and gut microbiota, compared with intraperitoneal immunization. Oral immunization with B. subtilis spore expressing CsPmy on the surface was a promising, safe and needle-free vaccination strategy against clonorchiasis.

Background: Clonorchis sinensis (C. sinensis) is the most widespread human liver fluke in East Asia including China and Korea. Clonorchiasis as a neglected tropical zoonosis, leads to serious economic and public health burden in China. There are considerable evidences for an etiological relation between chronic clonorchiasis and liver fibrosis in human beings. Liver fibrosis is a highly conserved and over-protected response to hepatic tissue injury. Immune cells including CD4+ T cell as well as dendritic cell (DC), and pro-fibrogenic cytokines like interleukin 4 (IL-4), IL-13 have been identified as vital manipulators in liver fibrogenesis. Our previous studies had a mere glimpse of T helper type 2 (Th2) dominant immune responses as key players in liver fibrosis induced by C. sinensis infection, but little is known about the involved mechanisms in this pathological process., Methodology/principal Findings: By flow cytometry (FACS), adult-derived total proteins of C. sinensis (CsTPs) down-regulated the expression of surface markers CD80, CD86 and major histocompatibility complex class II (MHC-II) on lipopolysaccharide (LPS) induced DC. ELISA results demonstrated that CsTPs inhibited IL-12p70 release from LPS-treated bone marrow-derived dendritic cells (BMDC). IL-10 level increased in a time-dependent manner in LPS-treated BMDCs after incubation with CsTPs. CD4+ T cells incubated with LPS-treated BMDCs plus CsTPs could significantly elevate IL-4 level by ELISA. Meanwhile, elevated expression of pro-fibrogenic mediators including IL-13 and IL-4 were detected in a co-culture system of LPS-activated BMDCs and naive T cells containing CsTPs. In vivo, CsTPs-immunized mice enhanced expression of type 2 cytokines IL-13, IL-10 and IL-4 in both splenocytes and hepatic tissue. Exposure of BMDCs to CsTPs activated expression of mannose receptor (MR) but not toll like receptor 2 (TLR2), TLR4, C-type lectin receptor DC-SIGN and Dectin-2 on the cell surface by RT-PCR and FACS. Blockade of MR almost completely reversed the capacity of CsTPs to suppress LPS-induced BMDCs surface markers CD80, CD86 and MHC-II expression, and further made these BMDCs fail to induce a Th2-skewed response as well as Th2 cell-associated cytokines IL-13 and IL-4 release in vitro., Conclusions/significance: Collectively, we validated that CsTPs could suppress the maturation of BMDCs in the presence of LPS via binding MR, and showed that the CsTPs-pulsed BMDCs actively polarized naive T helper cells to Th2 cells though the production of IL-10 instead of IL-12. CsTPs endowed host with the capacity to facilitate Th2 cytokines production including IL-13 and IL-4 in vitro and vivo. The study might provide useful information for developing potential therapeutic targets against the disease.

The roles of TLR4 in mediation of innate immune response and in regulation of adaptive immune responses triggered by Clonorchis sinensis remain unknown. In the present study, splenocytes derived from C3H/HeN (TLR4 wild ) and C3H/Hej mice (TLR4 mut ) that were infected with 45 metacercariae of C. sinensis were harvested, then stimulated by C. sinensis excretory/secretory products (ESP) or medium (control) for 48 h, respectively. Meanwhile, bone marrow-derived dendritic cells (BMDCs) from normal C3H/HeN and C3H/Hej mice were prepared and stimulated with medium, ESP, LPS, or ESP+LPS for 24 h, respectively. The supernatants were collected, and the concentrations of type 1 and type 2 relative cytokines were determined by ELISA. The maturation of BMDCs indicated by surface markers of CD80, CD86, and MHC II was evaluated by flow cytometry. The results showed that the levels of IFN-γ, IL-6, TNF-α, and IL-10 in the splenocytes from C. sinensis-infected TLR4 mut mice were significantly lower than those from TLR4 wild mice when they were further exposed to ESP. For BMDCs, the productions of the cytokines IL-12p70 and IL-10, but not IL-4, in the BMDCs from TLR4 mutation mice were predominantly decreased compared with those from TLR4 wild mice when the BMDCs were co-stimulated by ESP combined with LPS. Flow cytometry analysis showed that ESP could significantly decrease the high levels of CD80, CD86, and MHC II which were elevated by LPS. In conclusion, these data suggest that TLR4 may play a regulatory role in type 1 immune responses during C. sinensis infection.

Background: Clonorchiasis ranks among the most important food-borne parasitic diseases in China. However, due to low compliance to traditional fecal examination techniques in the general population and medical personnel, immunodiagnosis is expected. This study evaluated, in parallel, the performance of four immunodiagnostic kits detecting clonorchiasis in China., Results: A bank with 475 sera was established in this study. Except for the low performance of the kit detecting IgM, the other three kits detecting IgG showed sensitivities ranging from 81.51% (194/238) to 99.16% (236/238). Higher sensitivity was presented in heavy infection intensity [89.47% (68/76) to 100% (76/76)]. Among the four kits, the overall specificity varied from 73.42% (174/237) to 87.34% (207/237). It was observed that the specificity was lower in the sera of the participants living in clonorchiasis-endemic areas but without any parasite infection [67.5% (81/120) to 90% (108/120)], as compared to those from the non-endemic area [94% (47/50) to 98% (49/50)]. The cross-reaction rate varied from 14.93% (10/67) to 31.34% (21/67). Youden's index was -0.022, 0.689, 0.726, and 0.802 for kits T1, T2, T3 and T4, respectively. Repeatability was high in all four kits., Conclusions: Three immunodiagnosis kits targeting IgG antibody had high performance on detecting chronic Clonorchis sinensis infection, but that detecting IgM antibody had not. The kits detecting IgG antibody also showed high sensitivity in heavy infection intensity. Research on immunological diagnosis of clonorchiasis is expected to be strengthened to improve the sensitivity in light infection and specificity.

Background: Human clonorchiasis, caused by the infection of Clonorchis sinensis, is one of the major health problems in Southeast Asia. However, vaccine efficacy against C. sinensis infection remains largely unknown., Methods: In this study, for the first time, we generated virus-like particles (VLPs) vaccine containing the C. sinensis tegumental protein 22.3 kDa (CsTP 22.3) and the influenza matrix protein (M1) as a core protein, and investigated the vaccine efficacy in Sprague-Dawley rats., Results: Intranasal immunization of VLPs vaccine induced C. sinensis-specific IgG, IgG2a and IgG2c in the sera and IgA responses in the feces and intestines. Notably, upon challenge infection with C. sinensis metacercariae, significantly lower adult worm loads (70.2%) were measured in the liver of rats immunized with VLPs, compared to those of naïve rats. Furthermore, VLPs immunization induced antibody secreting cells (ASC) responses and CD4+/CD8+ T cell responses in the spleen., Conclusions: Our results indicated that VLPs vaccine containing C. sinensis CsTP 22.3 kDa provided partial protection against C. sisnensis infection. Thus, VLPs could be a potential vaccine candidate against C. sinensis.

Background: Clonorchis sinensis, the causative agent of clonorchiasis, is classified as one of the most neglected tropical diseases and affects more than 15 million people globally. This hepatobiliary disease is highly associated with cholangiocarcinoma. As key molecules in the infectivity and subsistence of trematodes, glycolytic enzymes have been targets for drug and vaccine development. Clonorchis sinensis pyruvate kinase (CsPK), a crucial glycolytic enzyme, was characterized in this research., Results: Differences were observed in the sequences and spatial structures of CsPK and PKs from humans, rats, mice and rabbits. CsPK possessed a characteristic active site signature (IKLIAKIENHEGV) and some unique sites but lacked the N-terminal domain. The predicted subunit molecular mass (Mr) of CsPK was 53.1 kDa. Recombinant CsPK (rCsPK) was a homopentamer with a Mr. of approximately 290 kDa by both native PAGE and gel filtration chromatography. Significant differences in the protein and mRNA levels of CsPK were observed among four life stages of C. sinensis (egg, adult worm, excysted metacercaria and metacercaria), suggesting that these developmental stages may be associated with diverse energy demands. CsPK was widely distributed in adult worms. Moreover, an intense Th1-biased immune response was persistently elicited in rats immunized with rCsPK. Also, rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro., Conclusions: The sequences and spatial structures, molecular mass, and expression profile of CsPK have been characterized. rCsPK was indicated to be a homopentamer. Rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CsPK is worthy of further study as a promising target for drug and vaccine development.

Clonorchiasis remains a nonnegligible public health problem in endemic areas. Cysteine protease of Clonorchis sinensis (CsCP) plays indispensable roles in the parasitic physiology and pathology, and has been exploited as a promising drug and vaccine candidate. In recent years, development of spore-based vaccines against multiple pathogens has attracted many investigators' interest. In previous studies, the recombinant Escherichia coli (BL21) and Bacillus subtilis spores expressing CsCP have been successfully constructed, respectively. In this study, the immune effects of CsCP protein purified from recombinant BL21 (rCsCP) and B. subtilis spores presenting CsCP (B.s-CsCP) in Balb/c mice model were conducted with comparative analysis. Levels of specific IgG, IgG1 and IgG2a were significantly increased in sera from both rCsCP and B.s-CsCP intraperitoneally immunized mice. Additionally, recombinant spores expressing abundant fusion CsCP (0.03125 pg/spore) could strongly enhance the immunogenicity of CsCP with significantly higher levels of IgG and isotypes. Compared with rCsCP alone, intraperitoneal administration of mice with spores expressing CsCP achieved a better effect of fighting against C. sinensis infection by slowing down the process of fibrosis. Our results demonstrated that a combination of Th1/Th2 immune responses could be elicited by rCsCP, while spores displaying CsCP prominently induced Th1-biased specific immune responses, and the complex cytokine network maybe mediates protective immune responses against C. sinensis. This work further confirmed that the usage of B. subtilis spores displaying CsCP is an effective way to against C. sinensis.

Clonorchis sinensis infection elicits hepatic inflammation, which can lead to cholangitis, periductal hepatic fibrosis, liver cirrhosis, and even cholangiocarcinoma. Hepatic macrophages are an intrinsic element of both innate and acquired immunity. This study was conducted to demonstrate the dynamics of hepatic macrophage polarization during C. sinensis infection in mice and to identify factors regulating this polarization. Treatment of hepatic macrophages isolated from normal mice with C. sinensis excretory/secretory products (ESPs) resulted in the preferential generation of classically activated hepatic macrophages (M1 macrophages) and the production of pro-inflammatory cytokines. Additionally, cells stimulated with C. sinensis ESPs exhibited changes in cellular morphology. During the early stages of C. sinensis infection, hepatic macrophages preferentially differentiated into M1 macrophages; however, during the C. sinensis mature worm stage, when eggs are released, there were significant increases in the abundance of both M1 macrophages and alternatively activated hepatic macrophages (M2 macrophages). Moreover, there was a further increase in the M2 macrophage count during the fibrotic and cirrhotic stage of infection. Notably, this fibrotic and cirrhotic stage promoted a strong increase in the proportion of Arg-1-producing macrophages (M2 phenotype), which were associated with fibrosis and tissue repair in the liver. Our results suggest that the dynamic polarization of hepatic macrophages as C. sinensis infection progresses is related to the histological lesions present in liver tissue. Hepatic macrophages thus play an important role in local immunity during C. sinensis infection.

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite-related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell-free protein synthesis system for large-scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high-throughput expression and identification of individual genes comprising the library, a cell-free synthesis reaction was designed such that both the template DNA and the expressed proteins were co-immobilized on the same microbeads, leading to microbead-based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832-837, 2017., (© 2017 American Institute of Chemical Engineers.)

Background: Heat shock proteins (HSPs) are found in all prokaryotes and most compartments of eukaryotic cells. Members of the HSP family mediate immune responses to tissue damage or cellular stress. However, little is known about the immune response induced by the oriental liver fluke, Clonorchis sinensis, even though this organism is carcinogenic to humans. We address this issue in the present study in mouse bone marrow dendritic cells (mBMDCs), using recombinant HSP70 and 90 from C. sinensis (rCsHSP70 and rCsHSP90)., Methods: rCsHSP70 and rCsHSP90 were produced in an E. coli system. Purified recombinant proteins were treated in BMDCs isolated from C57BL/6 mice. T cells were isolated from Balb/c mice and co-cultured with activated mBMDCs. Expression of surface molecules was measured by flow cytometry and cytokine secretion was quantified using ELISA. C57BL/6 mice were divided into four groups, including peptide alone, peptide/Freund's adjuvant, peptide/CsHSP70, peptide/CsHSP90, and were immunized intraperitoneally three times. Two weeks after final immunization, antibodies against peptide were measured using ELISA., Results: Both proteins induced a dose-dependent upregulation in major histocompatibility complex and co-stimulatory molecule expression and increased secretion of pro-inflammatory cytokines including interleukin (IL)-1β, -6, and -12p70 and tumor necrosis factor-α in mBMDCs. Furthermore, when allogenic T cells were incubated with mBMDCs activated by rCsHSP70 and rCsHSP90, the helper T cell (Th)1 cytokine interferon-γ was up-regulated whereas the level of the Th2 cytokine IL-4 was unchanged. These results indicate that rCsHSPs predominantly induce a Th1 response. Over and above these results, we also demonstrated that the production of peptide-specific antibodies can be activated after immunization via in vitro peptide binding with rCsHSP70 or rCsHSP90., Conclusion: This study showed for the first time that the HSP or HSP/peptide complexes of C. sinensis could be considered as a more effective vaccine against C. sinensis infection as results of the activator of host immune response as well as the adjuvant for antigenic peptide conjugate to induce peptide-specific antibody response in mice.

Clonorchiasis, caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensisis (C.sinensis), remains a common public health problem. New effective prevention strategies are still urgent to control this food-borne infectious disease. The previous studies suggested Bacillus subtilis (B. subtilis) spores was an ideal vaccines delivery system, and the C.sinensis enolase (CsENO) was a potential vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgM levels by ELISA in sera, intestinal mucus and skin mucus in grass carps (Ctenopharyngodon idella) through oral administration with B. subtilis spores surface expressing CsENO. In addition, immune-related genes expression was also measured by qRT-PCR. Grass carps orally treated with B. subtilis spores or normal forages were used as controls. The results of ELISA manifested that specific IgM levels of grass carps in CsENO group in sera, intestine mucus and skin mucus almost significantly increased from week 4 post the first oral administration when compared to the two control groups. The levels of specific IgM reached its peak in intestine mucus firstly, then in sera, and last in skin mucus. qRT-PCR results showed that 5 immune-related genes expression had different degree of rising trend in CsENO group when compared to the two control groups. Our study demonstrated that orally administrated with B. subtilis spores expressing CsENO induced innate and adaptive immunity, systemic and local mucosal immunity, and humoral and cellular immunity. Our work may pave the way to clarify the exact mechanisms of protective efficacy elicited by B. subtilis spores expressing CsENO and provide new ideas for vaccine development against C. sinensis infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma., (Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Background: Clonorchiasis, a food-borne zoonosis, is caused by Clonorchis sinensis. The intestinal tract and bile ducts are crucial places for C. sinensis metacercariae to develop into adult worms. The endospore of Bacillus subtilis is an ideal oral immunization vehicle for delivery of heterologous antigens to intestine. Cysteine protease of C. sinensis (CsCP) is an endogenous key component in the excystment of metacercariae and other physiological or pathological processes., Methods: We constructed a fusion gene of CotC (a coat protein)-CsCP and obtained B. subtilis spores with recombinant plasmid of pEB03-CotC-CsCP (B.s-CotC-CsCP). CotC-CsCP expressed on spores' surface was detected by Western blotting and immunofluorescence. Immunological characteristics of recombinant spore coat protein were evaluated in a mouse model. The levels of CsCP-specific antibodies were detected by ELISA. Effects of recombinant spores on mouse intestine were evaluated by histological staining. The activities of biochemical enzymes in serum were assayed by microplate. Liver sections of infected mice were evaluated by Ishak score after Masson's trichrome., Results: The B.s-CotC-CsCP spores displayed CsCP on their coat. Specific IgG and isotypes were significantly induced by coat proteins of B.s-CotC-CsCP spores after subcutaneous immunization. IgA levels in intestinal mucus and bile of B.s-CotC-CsCP orally treated mice significantly increased. Additionally, more IgA-secreting cells were observed in enteraden and lamina propria regions of the mouse jejunum, and an increased amount of acidic mucins in intestines were also observed. There were no significant differences in enzyme levels of serum among groups. No inflammatory injury was observed in the intestinal tissues of each group. The degree of liver fibrosis was significantly reduced after oral immunization with B.s-CotC-CsCP spores., Conclusions: Bacillus subtilis spores maintained the original excellent immunogenicity of CsCP expressed on their surface. Both local and systemic specific immune responses were elicited by oral administration of B.s-CotC-CsCP spores. The spores effectively promoted intestinal health by inducing secretion of acidic mucins, with no other side effects to the liver or intestine. Oral administration of spores expressing CsCP could provide effective protection against C. sinensis. This study may be a cornerstone for development of antiparasitic agents or vaccines against clonorchiasis based on B. subtilis spore expressing CsCP on the surface.

Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.

Clonorchiasis, caused by Clonorchis sinensis (C. sinensis), is an important food-borne parasitic disease and one of the most common zoonoses. Currently, it is estimated that more than 200 million people are at risk of C. sinensis infection, and over 15 million are infected worldwide. C. sinensis infection is closely related to cholangiocarcinoma (CCA), fibrosis and other human hepatobiliary diseases; thus, clonorchiasis is a serious public health problem in endemic areas. This article reviews the current knowledge regarding the epidemiology, disease burden and treatment of clonorchiasis as well as summarizes the techniques for detecting C. sinensis infection in humans and intermediate hosts and vaccine development against clonorchiasis. Newer data regarding the pathogenesis of clonorchiasis and the genome, transcriptome and secretome of C. sinensis are collected, thus providing perspectives for future studies. These advances in research will aid the development of innovative strategies for the prevention and control of clonorchiasis.

Clonorchiasis is a chronic infection disease often accompanied by formation of liver fibrosis. Previous study has identified that Clonorchis sinensis (C. sinensis, Cs) infection and CsRNASET2 (a member of CsESPs) immunization can drive Th2 immune response. IL-13, a multifunctional Th2 cytokine, has been widely confirmed to be profibrotic mediator. We want to determine whether IL-13 is involved in the generation of liver fibrosis during C. sinensis infection. A part of mice were infected with C. sinensis or immunized with CsRNASET2, respectively. Another part of mice were intravenously injected with rIL-13. Liver tissues of C. sinensis-infected mice were stained with hematoxylin-eosin and Masson's trichrome, respectively. The transcriptional levels of collagen-I, collagen-III, α-SMA, and TIMP-1 in the livers of infected mice and rIL-13-treated mice were measured by quantitative RT-PCR. Besides, splenocytes of C. sinensis-infected and CsRNASET2-immunized mice were isolated, respectively. The levels of IL-13 in splenocytes were detected by ELISA. Our results displayed that the livers of C. sinensis-infected mice had serious chronic inflammation and collagen deposition. The transcriptional levels of collagen-I, collagen-III, α-SMA, and TIMP-1 in the livers of C. sinensis-infected mice were obviously increased. Splenocytes from both C. sinensis-infected and CsRNASET2-immunized mice expressed high levels of IL-13. Moreover, rIL-13 treatment markedly promoted the transcriptional levels of collagen-I, collagen-III, α-SMA, and TIMP-1. These data implied that hepatic fibrosis was formed in the livers of C. sinensis-infected mice, and IL-13 induced by C. sinensis infection and CsRNASET2 immunization might favor this progression.

During clonorchiasis, immune responses of hosts are responsible for the removal of the worms and also are involved in the progress of the pathological damage caused by Clonorchis sinensis. Interleukin-33 (IL-33), a recently described cytokine signaling through the ST2 receptor, has emerged as a potent inducer to bile duct proliferation and fibrosis; however, little is known of this signaling in the pathogen-caused periductal inflammation and fibrosis. In the present study, using immunohistochemistry, real-time PCR, enzyme-linked immunosorbent assay (ELISA), and flow cytometry, we studied the expression of IL-33/ST2 during C. sinensis infection, as well as their potential roles in C. sinensis-induced host immune responses. The results showed that a higher level of IL-33 was detected in the sera of patients of clonorchiasis (n = 45), compared with in those of healthy donors (n = 16). Similarly, in FVB mice experimentally infected with C. sinensis, a higher level of IL-33 was detected at latent stage both in the serum and in the liver, as well as the up-regulated expression of ST2 receptor on the inflammatory cells, especially on CD4(+) T cells in the liver of infected mice. Our results, for the first time, indicated that the increased IL-33/ST2 may be involved in the regulation of immunopathology induced by C. sinensis.

Lactate dehydrogenase (LDH) is a terminal enzyme in anaerobic glycolytic pathway. It widely exists in various organisms and is in charge of converting the glycolysis product pyruvic acid to lactic acid. Most parasites, including Clonorchis sinensis, predominantly depend on glycolysis to provide energy. Bioinformatic analysis predicts that the LDHs from many species have more than one transmembrane region, suggesting that it may be a membrane protein. C. sinensis LDH (CsLDH) has been confirmed as a transmembrane protein mainly located in the tegument. The antibodies against CsLDH can inhibit the worm's energy metabolism, kill the worm, and may have the same effects on human cancer cells. In this study, we cloned and characterized human LDHA (HsLDHA), HsLDHB, and CsLDH. Semi-quantitative real-time RCP showed that HsLDHB only existed in hepatocarcinoma cell SMMC-7721. Confocal microscopy and Western blot experiments revealed that HsLDHB was localized in the plasma membrane of SMMC-7721 cells, and the antibodies against CsLDH could cross-react with it. This cross-reaction could inhibit the enzymatic activity of HsLDHB. The cancer cells co-cultured with anti-CsLDH sera showed a significant decrease in cell proliferation rate and increases in caspase 9 and reactive oxygen species (ROS) levels. Therefore, anti-CsLDH antibodies can induce the apoptosis of cancer cells SMMC-7721 and may serve as a new tool to inhibit tumor.

Schistosomiasis is a common zoonoses affecting humans. The atypical clinical symptoms, low morbidity, and low degree of infection impede diagnosis and assessment of epidemics. Detecting circulating antigens from adult worms in patients' body fluids should be diagnostically superior to examining eggs in feces. Herein, the excretory-secretory proteins of adult worms were analyzed by using 2-D protein electrophoresis and mass spectrometry. The Schistosoma japonicum enolase (Sj enolase) was identified as the most abundant excretory-secretory antigen. Purified recombinant Sj enolase was prepared, and specific monoclonal and polyclonal antibodies were raised against it. A sandwich enzyme-linked immunoassay (sandwich ELISA) was established that used the monoclonal antibody as a capture antibody and the polyclonal antibody as a detection antibody. The linear detection range was 0.7-1000 ng/ml (minimum 700 pg/ml). Sj enolase could be detected in the sera of infected rabbits and disappeared rapidly postpraziquantel treatment. The sensitivity and specificity of this sandwich ELISA to detect field serum samples of schistosomiasis were 84.61 and 95.83 %, respectively. The cross-reaction rates for clonorchiasis and paragonimiasis were 3.33 and 5 %, respectively. This ELISA assay was used to test 45 matching sera of schistosomiasis patients before treatment and at 3, 6, 9, and 12 months posttreatment. Among the sera, 88.89 % were positive before treatment. At 3, 6, 9, and 12 months postpraziquantel treatment, 93.33, 97.78, 100, and 100 % tested negative, respectively. Therefore, Sj enolase can be used to indicate active Schistosoma infection, and detecting serum Sj enolase is important for diagnosis and evaluating treatment effect.

Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.

Clonorchiasis, caused by the liver fluke Clonorchis sinensis, is a chronic parasitic infection regulated by T cell subsets. An imbalance of CD4+CD25+ Foxp3+regulatory T (Treg) and interleukin (IL)-17-secreting T cells (Th17) may control inflammation and play an important role in the pathogenesis of immune evasion. In the present study, we assessed the dynamics of Treg/Th17 and determined whether the Treg/Th17 ratio is altered in C. sinensis-infected mice. The results showed that the percentages of splenic Treg cells in CD4+ T cells were suppressed on day 14 post-infection (PI) but increased on day 56 PI, while Th17 cells were increased on day 56 PI compared with normal control (NC) mice. The Treg/Th17 ratio steadily increased from day 28 to day 56 PI. The hepatic levels of their specific transcription factors (Foxp3 for Treg and RORγt for Th17) were increased in C. sinensis-infected mice from day 14 to 56 PI, and significantly higher than those in NC mice. Meanwhile, serum levels of IL-2 and IL-17 were profoundly increased in C. sinensis-infected mice throughout the experiment; while the concentrations of IL-6 and transforming growth factor β1 (TGF-β1) peaked on day 14 PI, but then decreased on day 28 and 56 PI. Our results provide the first evidence of an increased Treg/Th17 ratio in C. sinensis-infected mice, suggesting that a Treg/Th17 imbalance may play a role in disease outcomes of clonorchiasis.

Introduction: Clonorchis sinensis is one of the most important foodborne pathogens in humans, and can cause biliary diseases such as gallstones, cholecystitis, cholangitis, and cholangiocarcinoma. Toll-like receptors (TLRs) as sensors are crucial to initiating both innate and adaptive immune defenses against pathogens. However, little is known about the hepatic expression of TLRs of hosts induced by C. sinensis infection., Methodology: In the present study, the expression and distribution of TLR2 and TLR4 were investigated in a mouse model of clonorchiasis on days 28, 56, 84, and 112 post-infection (PI) using real-time quantitative reverse transcription polymerase chain reaction (PCR) and immunohistochemically staining, respectively. The levels of cytokines that are mediated by TLR2 and TLR4 were also evaluated using a cytometric bead array., Results: Results showed that the transcripts of TLR2 and TLR4 were upregulated on day 28 PI in C. sinensis-infected mice compared with non-infected ones (p < 0.01). In addition, their proteins were strongly immunohistochemically positive in the cytoplasm and membrane of endothelial cells, fibroblasts, and biliary epithelium cells of C. sinensis-infected mice. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were increased with activation of TLR2 and TLR4., Conclusions: The expression of TLR2 and TLR4 is upregulated against C. sinensis infection, which suggests that TLR2 and TLR4 might be involved in immune responses during C. sinensis infection.

Caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensis, human clonorchiasis remains a major public health problem in China. In previous study, we had expressed enolase from C. sinensis (CsENO) on the surface of Bacillus subtilis spore and the recombinant spore induced a pronounced protection in terms of reduced worm burden and eggs per gram feces, suggesting B. subtilis spore as an ideal vehicle for antigen delivery by oral treatment and CsENO as a promising vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgG and IgA levels both in serum and in intestinal mucus from rats orally administrated with B. subtilis spore surface expressing CsENO by ELISA. Lysozyme levels in serum and in intestinal mucus were analyzed too. In addition, IgA-secreting cells in intestine epithelium of the rats were detected by immunohistochemistry assay. The intestinal villi lengths of duodenum, jejunum, and ileum were also measured. Rats orally treated with B. subtilis spore or normal saline were used as controls. Our results showed that, compared with the control groups, oral administration of B. subtilis spore expressing CsENO induced both systemic and local mucosal immune response. The recombinant spores also enhanced non-specific immune response in rats. The spores had no side effect on liver function. Moreover, it might facilitate food utilization and digestion of the rats. Our work will pave the way to clarify the involved mechanisms of protective efficacy elicited by B. subtilis spore expressing CsENO and encourage us to carry out more assessment trails of the oral treated spore to develop vaccine against clonorchiasis.

Paramyosin of Clonorchis sinensis (CsPmy) is a myofibrillar protein localized in subtegumental muscle, tegument, and the muscle layer surrounding the intestine of the parasite. Previously, we have identified that CsPmy reacted with sera of human clonorchiasis and this protein had a potential as a candidate antigen for serodiagnosis of clonorchiasis. However, we also found that CsPmy is able to bind to human immunoglobulin G (IgG) in non-specific manners, which can affect the diagnostic value of the protein. Here, we mapped CsPmy-specific IgG binding site on CsPmy to analyze the putative epitopes recognized by CsPmy-specific IgG in sera of human clonorchiasis. The fragmental expression of CsPmy followed by immunoblot analyses with sera from patients with clonorchiasis and non-specific human IgG revealed that the middle portion of CsPmy (CsPmyC: 301-600 amino acid residues) had epitopes responsible for CsPmy-specific IgG recognition. The precise CsPmy-specific IgG binding site was further narrowed down to a fragment (CsPmyC-2), which harbors 151 amino acid residues (375-525) of CsPmy. Specific antibodies for CsPmyC-2 were produced in rats after two-weeks of post-experimental infection. The CsPmyC-2 showed low levels of cross reactivity against the sera from patients with other helminth parasites. Our results suggested that CsPmyC-2 has real epitopes recognized by CsPmy-specific IgG in sera of human clonorchiasis and the fragment can be useful as a reliable serodiagnostic antigen to develop a serodiagnostic method for clonorchiasis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: To characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis., Methods: The full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein., Results: The recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1: 51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis., Conclusion: The pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis- associated hepatic fibrosis.

Background: Approximately 35 million people are infected with Clonorchis sinensis (C. sinensis) globally, of whom 15 million are in China. Glycolytic enzymes are recognized as crucial molecules for trematode survival and have been targeted for vaccine and drug development. Hexokinase of C. sinensis (CsHK), as the first key regulatory enzyme of the glycolytic pathway, was investigated in the current study., Principal Findings: There were differences in spatial structure and affinities for hexoses and phosphate donors between CsHK and HKs from humans or rats, the definitive hosts of C. sinensis. Effectors (AMP, PEP, and citrate) and a small molecular inhibitor regulated the enzymatic activity of rCsHK, and various allosteric systems were detected. CsHK was distributed in the worm extensively as well as in liver tissue and serum from C. sinensis infected rats. Furthermore, high-level specific IgG1 and IgG2a were induced in rats by immunization with rCsHK. The enzymatic activity of CsHK was suppressed by the antibody in vitro. Additionally, the survival of C. sinensis was inhibited by the antibody in vivo and in vitro., Conclusions/significance: Due to differences in putative spatial structure and enzymology between CsHK and HK from the host, its extensive distribution in adult worms, and its expression profile as a component of excretory/secretory products, together with its good immunogenicity and immunoreactivity, as a key glycolytic enzyme, CsHK shows potential as a vaccine and as a promising drug target for Clonorchiasis.

Clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis, is associated with hepatobiliary damage, inflammation, periductal fibrosis, and the development of cholangiocarcinoma. Hepatic stellate cells respond to liver injury through production of proinflammatory mediators which drive fibrogenesis; however, their endogenous sources and pathophysiological roles in host cells were not determined. C. sinensis ferritin heavy chain (CsFHC) was previously confirmed as a component of excretory/secretory products and exhibited a number of extrahepatic immunomodulatory properties in various diseases. In this study, we investigated the expression pattern and biological role of CsFHC in C. sinensis. CsFHC was expressed throughout life stages of C. sinensis. More importantly, we found that treatment of human hepatic stellate cell line LX-2 with CsFHC triggered the production of free radicals via time-dependent activation of NADPH oxidase, xanthine oxidase, and inducible nitric oxide synthase. The increase in free radicals substantially promoted the degradation of cytosolic IκBα and nuclear translocation of NF-κB subunits (p65 and p50). CsFHC-induced NF-κB activation was markedly attenuated by preincubation with specific inhibitors of corresponding free radical-producing enzyme or the antioxidant. In addition, CsFHC induced an increased expression level of proinflammatory cytokines, IL-1β and IL-6, in NF-κB-dependent manner. Our results indicate that CsFHC-triggered free radical-mediated NF-κB signaling is an important factor in the chronic inflammation caused by C. sinensis infection.

Background: Clonorchiasis, caused by Clonorchis sinensis, is widely distributed in Southeast Asia including China. Clonorchiasis is included in control programs of neglected tropical diseases by World Health Organization (WHO) because it is one of the major health problems in most endemic areas. Diagnosis of clonorchiasis plays a key role in the control programs. However, so far, there is no satisfactory method for clonorchiasis because of low sensitivity, poor practicality and high false positivity of available diagnostic tools., Methodology/principal Findings: We developed an immunomagnetic bead enzyme-linked immunosorbent assay (ELISA) based on IgY (egg yolk immunoglobulin) against cysteine proteinase of C. sinensis for detection of circulating antigen in serum samples of patients infected with C. sinensis. The polyclonal IgY, coated with magnetic beads, was used as a capture antibody and a monoclonal IgG labeled with horseradish peroxidase as a detection antibody in the IgY-based immunomagnetic bead ELISA system (IgY-IMB-ELISA). The results showed that the sensitivity of IgY-IMB-ELISA was 93.3% (14 of 15) in cases of heavy infection (5000 to 9999 eggs per gram feces, i.e, EPG 5000-9999), 86.7% (13 of 15) in cases of moderate infection (EPG 1000-4999) and 75.0% (9 of 12) in cases of light infection (EPG <1000) of clonorchiasis. Together 36 of total 42 (85.7%) serum samples of human clonorchiasis gave a positive reaction. There was a significant correlation between ELISA optical density and egg counts (EPG) with a correlation coefficient of 0.83 in total 42 patients. There were no positive results in patients with trichinosis (n = 10) or cysticercosis (n = 10). Cross-reactivity was 6.7% (2 of 30) with schistosomiasis japonica and 10.0% (3 of 30) with paragonimiasis, respectively. No positive reaction was found in 20 healthy persons., Conclusions: Our findings suggest that IgY-IMB-ELISA appears to be a sensitive and specific assay for detection of circulating antigen in human clonorchiasis.

The main causes of biliary obstruction are stones and cancers. Fascioliasis is a very rare case which causes biliary obstruction. Fascioliasis is a zoonosis caused by Fasciola hepatica which infects herbivores like sheep and cattle. F. hepatica lives in the biliary system or the liver parenchyma of a host. In Korea, the occurrence of this infection in human is very rare and only few cases have been reported. A 32-year-old male presented with upper abdominal pain and jaundice. His laboratory finding revealed elevated liver transaminases. Abdomen CT scan showed mild left intrahepatic bile duct dilatation. On ERCP, adult F. hepatica worms were found and were thus removed. Concurrently, clonorchiasis was diagnosed by stool exam and serologic enzyme-linked immunosorbent assay test. Clonorchiasis was treated with praziquantel. Herein, we report a case of intrahepatic bile duct dilatation due to F. hepatica infection with concurrent Clonorchis sinensis infestation.

Cathepsin C is an important exopeptidase of papain superfamily and plays a number of great important roles during the parasitic life cycle. The amino acid sequence of cathepsin C from Clonorchis sinensis (C. sinensis) showed 54, 53, and 49% identities to that of Schistosoma japonicum, Schistosoma mansoni, and Homo sapiens, respectively. Phylogenetic analysis utilizing the sequences of papain superfamily of C. sinensis demonstrated that cathepsin C and cathepsin Bs came from a common ancestry. Cathepsin C of C. sinensis (Cscathepsin C) was identified as an excretory/secretory product by Western blot analysis. The results of transcriptional level and translational level of Cscathepsin C at metacercaria stage were higher than that at adult worms. Immunolocalization analysis indicated that Cscathepsin C was specifically distributed in the suckers (oral sucker and ventral sucker), eggs, vitellarium, intestines, and testis of adult worms. In the metacercaria, it was mainly detected on the cyst wall and excretory bladder. Combining with the results mentioned above, it implies that Cscathepsin C may be an essential proteolytic enzyme for proteins digestion of hosts, nutrition assimilation, and immune invasion of C. sinensis. Furthermore, it may be a potential diagnostic antigen and drug target against C. sinensis infection.

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.

The vacuolar ATPase enzyme complex (V-ATPase) pumps protons across membranes, energized by hydrolysis of ATP. Extensive investigations on structural and biochemical features of these molecules have implied their importance in the physiological process. In this study, a full-length sequence encoding a vacuolar ATP synthase subunit ε-like protein of Clonorchis sinensis (CsATP-ε) was isolated from our cDNA library. The hypothetical 226 amino acid sequence shared 76% identity with ATP-ε proteins of Schistosoma japonicum and above 55% identity with ATP-ε proteins from human and other eukaryotes. Characteristic Asp₁₄₀ amino acid residues and seven B-cell epitopes were predicted in this sequence. The complete coding sequence of the gene was expressed in Escherichia coli. Recombinant CsATP-ε (rCsATP-ε) protein could be probed by anti-rCsATP-ε rat serum and C.sinensis-infected human serum in Western blotting experiment, indicating that it is an antigen of strong antigenicity. The high level of antibody titers (1:204,800) showed that CsATP-ε has a powerful immunogenicity. Both the increased level and the change trend of IgG1/IgG2a subtypes in serum showed that the rCsATP-ε can induce strong combined Th1/Th2 immune responses in rats and stimulate the immune response changes to the dominant Th2 from Th1 along with long time infection. The results of immunoblot and immunolocalization demonstrated that CsATP-ε was consecutively expressed at various developmental stages of the parasite, which was supported by real-time PCR analysis. In immunohistochemistry, CsATP-ε was localized on the intestine, vitellarium, and testicle of an adult worm and excretory bladder of metacercaria, implying that CsATP-ε may relate to energy intake and metabolism. This fundamental study would contribute to further researches that are related to growth and development and immunomodulation of C. sinensis.

Previous studies demonstrated that Clonochis sinensis-derived crude antigens suppress development of allergic responses. We investigated the effects of C. sinensis venom allergen-like (CsVAL) proteins on immune-modulating activities in allergic inflammatory response. Using RBL-2H3 rat mast cells, we demonstrated that CsVAL inhibits antigen-induced β-hexosaminidase release from immunoglobulin E-sensitized RBL-2H3 cells, and this inhibitory activity occurs by suppressing Lyn phosphorylation and intracellular reactive oxygen species production. In addition, CsVAL peptide treatment inhibits activation of protein kinase C-α and extracellular signal-regulated kinase 1/2, which are involved in degranulation of immunoglobulin E-sensitized mast cells. Furthermore, immunization with CsVAL suppressed development of skin inflammation by assessing ear thickness and cutaneous infiltration by eosinophils and mast cells in oxazolone-induced contact hypersensitivity in vivo mouse model. These results suggest that CsVAL is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Dendritic cells (DCs), which are regarded as the most potent antigen-presenting cells, are involved in innate and adaptive immunity. Upon uptake of pathogens, DCs express cell surface markers and secrete cytokines. In this study, we analyzed production of cytokines and found that interleukin (IL)-10 and transforming growth factor (TGF)-β production significantly increased in bone marrow-derived DCs and a mouse DC line, DC2.4, after treatment with crude antigen (CA) from liver fluke, Clonorchis sinensis. However, expression patterns of several activation molecules did not change. In addition, following treatment of DC2.4 cells with antigen from the lung fluke, Paragonimus westermani, production of IL-10 and TGF-β significantly increased compared with groups treated with other parasite antigens, Spirometra erinacei plerocercoid CA and Echinococcus granulosus hydatid cystic fluid. We also found that treatment of DC2.4 cells with C. sinensis CA resulted in rapid and significant phosphorylation of extracellular signal-regulated kinase 1/2, a mitogen-activated protein kinase. Following treatment of DC2.4 cells with C. sinensis CA, treatment with an inhibitor specific to an extracellular signal-regulated kinase inhibited production of IL-10 and TGF-β. Our results suggest that CA from C. sinensis has a role in the anti-inflammatory function of DC cells by inducing IL-10 and TGF-β through activation of extracellular signal-regulated kinase 1/2.

Concomitant infections of different species of parasites are common in the field. Infection with one parasite species likely triggers host responses that may influence the subsequent infection of another species and alter disease outcomes. So far, the majority of studies have focused on single species parasite infection, and the mechanisms of protection induced by the first parasite infection against the secondary infection remain poorly defined. In this study, we assess the impact of trematode Clonorchis sinensis infection on the course of another tissue nematode Trichinella spiralis challenge. We observed that mice with preexisting C. sinensis infection had lower worm burden of intestinal T. spiralis than those infected with T. spiralis alone; mice with preexisting C. sinensis also had severe enteric histopathological changes and higher counts of intestinal Paneth cells in responses to T. spiralis challenge. The mRNA levels of interleukin (IL)-4, IL-10, IL-13, and tumor necrosis factor (TNF)-α from the small intestine and spleen of the different groups were analyzed using quantitative real-time polymerase chain reaction. Compared with that in mice infected with T. spiralis alone, the mRNA expression of IL-13 was significantly increased in the small intestine tissues and IL-4, IL-13, and TNF-α were significantly increased in the spleen tissues in the dually infected mice. Our findings suggest that a "preexisting" trematode infection of C. sinensis is a factor which contributes to reducing the establishment of T. spiralis adult worms in the small intestine.