small molecules

2026-08-20 | Epidemiology of Clonorchis sinensis infection in Guangdong, China, 2006–2024: a retrospective analysis of over 0.17 million reported cases from passive surveillance data

Clonorchiasis, caused by the ingestion of raw freshwater fish containing Clonorchis sinensis metacercariae, lacks national notifiable status in China, resulting in insufficiently characterized epidemiology. However, as an important endemic disease in Guangdong province, it is reported in the National Notifiable Disease Reporting System (NNDRS). We conducted an observational study to characterize clonorchiasis epidemiology using NNDRS data from Guangdong. Cases included in this study were patients diagnosed with clonorchiasis in Guangdong province (2006–2024) and reported via NNDRS. Proportion and reported incidence of cases by gender, ages, and occupations were analyzed and compared, when spatial analysis was employed to identify high clusters at county level. There were 170,709 cases reported during the study period, of which 166,728 cases lived in Guangdong. A steady increase in reported incidence from 1.77/100,000 in 2006 to 37.27/100,000 in 2024 was observed (average annual percent change (AAPC):17.22%, P <0.001). The median age of patients was 54.0 years (inter-quartile range, 44.0–64.0). Most cases were male (78.30%). House-hold or unemployed personnel (25.86%), retirees (20.43%) and farmers (16.66%) comprised the majority. The reported incidence was notably higher among males and individual aged 65–69 years. Although clonorchiasis was reported in all 21 prefecture-level cities, spatial clustering was observed, particularly in the developed Pearl River Delta region. Our findings revealed the importance of clonorchiasis as a public health problem in Guangdong province. Precise strategies for prevention and control of clonorchiasis should be employed for targeted populations, with an emphasis on middle-aged and elderly people, male individuals, and people living in Pearl River Delta region.

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2026-07-21 | Efficacy and safety of albendazole versus praziquantel in the treatment of Clonorchis sinensis infection: an infection intensity-stratified, open-label, phase 2, randomized controlled trial.

Praziquantel is the only drug recommended against Clonorchis sinensis, while adverse events are usually high. Albendazole is documented in treatment of clonorchiasis, but the evidence is low due to lack of high-quality randomized controlled trials. We did an exploratory randomized controlled trial in Binyang county, Guangxi, China. Eligible participants were adults aged 18-70 years with C. sinensis infection. Patients with light intensity (eggs per gram of feces (EPG): 1-999) and moderate plus heavy intensity (EPG: ≥ 1000) were randomly assigned to treatment groups respectively, with block sizes of two, four, and six, to receive praziquantel (25 mg/kg three times a day for 1 day) or albendazole (400 mg twice a day for 4 days) in a 1:1 ratio. Laboratory diagnosis technicians were blinded to group assignment. We assessed efficacy as cure rate and egg reduction rate in available-case analysis, while adverse events were also analyzed. Fifty-two patients with light intensity and 50 with moderate plus heavy intensity were assigned, when three were lost to follow-up. The cure rate was 84.0% (21/25, 95% confidential intervals: 63.9%-95.5%) using praziquantel and 30.8% (8/26, 14.3%-51.8%) using albendazole in light intensity stratum (χ²=14.7, p < 0.001). It was 60.9% (14/23, 38.5%-80.3%) using praziquantel and 8.0% (2/25, 1.0%-26.0%) using albendazole in moderate plus heavy intensity stratum (χ²=15.1, p < 0.001). The corresponding egg reduction rate using geometric mean of EPG was 99.9% (99.7%-100.0%), 97.1% (94.1%-98.8%), 100.0% (99.9%-100.0%), and 98.9% (97.8%-99.5%), respectively. The overall cure rate was 72.9% (35/48, 58.2%-84.7%) in praziquantel groups and 19.6% (10/51, 9.8%-33.1%) in albendazole groups (χ²=28.3, p < 0.001), while the corresponding egg reduction rate was 99.9% (99.8%-100.0%) and 98.3% (97.1%-99.1%). The proportion of adverse events was higher in praziquantel than albendazole in light intensity stratum (46.2% vs. 15.4%), while there was no difference in moderate plus heavy intensity stratum (20% vs. 20%). The overall proportion was 33.3% in praziquantel groups and 17.6% in albendazole groups. Albendazole (400 mg twice a day for 4 days) demonstrates high egg reduction rate and low adverse events, but low cure rate in C. sinensis infection. Its application in preventive chemotherapy for morbidity control deserves further exploration. Controlled-Trials.com, ISRCTN46268871.

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2026-07-09 | Recurrent Pyogenic Cholangitis with Probable Intercurrent Latent Clonorchiasis in a Filipino Migrant to Canada: A Review of Implications for Diagnosis and Management

Clonorchiasis is a parasitic infection caused by the trematode Clonorchis sinensis, which is endemic to East Asia but increasingly observed in non-endemic regions due to global migration. Chronic infection can lead to recurrent pyogenic cholangitis (RPC) and other hepatobiliary complications including cholangiocarcinoma. We herein review clonorchiasis and opisthorchiasis as potential contributory drivers of RPC in migrants from Southeast Asia through framing with a case of a Filipino woman residing in Canada who presented with a two-year history of intermittent right upper quadrant pain, liver enzyme derangement, and imaging features consistent with RPC. Despite negative stool ova and parasite (O&P) testing, empirical treatment with praziquantel (1800 mg three times daily for two days) led to clinical and biochemical improvement. Hepatobiliary trematodiasis, including clonorchiasis and opisthorchiasis, can be challenging to diagnose, and clonorchiasis and opisthorchiasis cannot be distinguished on clinical or radiographic grounds alone. Clonorchiasis can be challenging to diagnose in non-endemic areas due to low parasitologic test sensitivity and non-specific clinical features. Imaging findings and epidemiologic context are critical for raising clinical suspicion and guiding management. Healthcare providers in non-endemic regions should include clonorchiasis and opisthorchiasis in the differential diagnosis of unexplained cholestasis, particularly in migrants from areas endemic for clonorchiasis and opisthorchiasis. Negative stool tests do not exclude infection, and early empirical treatment may prevent irreversible hepatobiliary damage and reduce long-term malignancy risk.

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2026-07-08 | Empirical therapy for hepatic lesions in suspected clonorchiasis: a comparative efficacy analysis of Albendazole and Praziquantel

Background Clonorchiasis frequently presents as intrahepatic space-occupying lesions, posing a diagnostic challenge due to the low sensitivity of fecal examination. While Praziquantel is the standard first-line therapy, Albendazole is often used empirically in suspected cases. However, the comparative efficacy of these two agents across different levels of diagnostic certainty (confirmed vs. suspected) remains poorly defined. Methods This retrospective cohort study included 151 patients with clonorchiasis-related hepatic lesions treated at our institution between January 2018 and December 2025. Patients were categorized into a Praziquantel group ( n = 78) and an Albendazole group ( n = 73). The primary outcome was clinical cure within 90 days, defined as symptom resolution, lesion reduction ≥50%, and normalization of eosinophil counts. Multivariable logistic regression and Generalized Estimating Equations (GEE) were employed to evaluate the interaction between treatment regimens and diagnostic certainty. Results The overall clinical cure rate was higher in the Praziquantel group (76.9%, 60/78) than in the Albendazole group (67.1%, 49/73), though the difference was not statistically significant ( χ 2 = 1.804, P = 0.179). Stratified analysis revealed that Praziquantel was significantly more effective in confirmed cases (90.0% vs. 62.5%, P = 0.047), whereas no significant difference was observed in suspected cases (68.8% vs. 68.4%, P = 0.971). The “Drug/times Diagnosis” interaction test showed marginal significance ( P = 0.064). After adjusting for confounders, Albendazole showed similar clinical outcomes to Praziquantel (Adjusted OR = 0.665, 95% CI: 0.298–1.482, P = 0.318). GEE analysis confirmed a significant “Drug × Time” interaction ( P &lt; 0.001), indicating distinct kinetic patterns of recovery, with Praziquantel demonstrating a superior cure advantage over time. Age was identified as an independent predictor of clinical cure ( P = 0.017). Conclusion The efficacy of Albendazole in treating clonorchiasis-related hepatic lesions is modulated by diagnostic certainty. While Praziquantel remains the superior choice for confirmed cases, Albendazole demonstrates comparable clinical efficacy in stool-negative suspected cases. These findings indicate that Albendazole demonstrates similar lesion resolution to Praziquantel in stool-negative suspected cases. However, given the retrospective design, these results should be interpreted as hypothesis-generating, necessitating prospective trials for confirmation.

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2026-04-13 | New combination therapy of praziquantel and Lactobacillus plantarum for enhancing the treatment of clonorchiasis in mice.

Clonorchis sinensis, a zoonotic trematode, parasitizes the bile ducts of humans and other mammals. It has been implicated in the development of liver fibrosis and cholangiocarcinoma. Currently, praziquantel is the sole drug for treating clonorchiasis, but it is not capable of reversing liver fibrosis. Therefore, it is critical to develop novel treatment strategies. Probiotics have garnered attention for their potential in treating a variety of intestinal infections, and they can influence the progression of liver diseases. In this study, a C. sinensis-induced liver fibrosis infection model was established in mice, with daily oral administration of Lactobacillus plantarum initiated immediately post-infection to investigate its anti-clonorchiasis effects. Body weight, survival time, liver/body weight ratio, parasite burden, liver lesions, and fibrosis were examined in C. sinensis-infected mice treated with or without L. plantarum. L. plantarum alleviated clonorchiasis in mice, as evidenced by mitigating weight loss, reducing the mortality rate by approximately 35.0%, decreasing the liver/body weight ratio, and reducing the parasite burden by 62.2%. L. plantarum also attenuated liver fibrosis in C. sinensis-infected mice, demonstrated by decreased collagen deposition and downregulated α-SMA expression. Furthermore, the synergistic effect of L. plantarum and praziquantel on treating clonorchiasis in mice was evaluated 35 d post-inoculation (dpi). When administered as monotherapy, praziquantel or L. plantarum reduced the hepatic parasite burden by 93.0% and 51.3%, respectively, resulting in a reduction of the liver fibrosis area from 21.8% to 12.2% and 9.0%, respectively. The combination of praziquantel and L. plantarum effectively eliminated almost all the hepatic parasites and further alleviated hepatic and biliary damage, resulting in an additional decrease in the liver fibrosis area to 6.0%. In summary, L. plantarum alleviated hepatic and biliary damage and liver fibrosis caused by C. sinensis in mice. The combination of L. plantarum and praziquantel showed enhanced efficacy in the treatment of clonorchiasis in mice, providing a potential novel strategy for a more effective treatment of C. sinensis-induced liver fibrosis.

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proteins

2025-04-18 | Characteristics and immunoprotective functions of three cysteine proteases from Clonorchis sinensis.

Cysteine proteases from Clonorchis sinensis, including various proteins, are essential for its pathogenicity and serve as potential vaccine candidates. This study assesses the protective effects of three C. sinensis cysteine proteases (CsCP1-3). Mice immunized with recombinant CsCP1-3 and adjuvants were subsequently infected with C. sinensis metacercariae after three immunization rounds. Liver damage was evaluated through hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical analyses. The levels of IgG1, IgG2a antibodies, and cytokines (IFN-g, IL-2, IL-4, and IL-10) were quantified by enzyme-linked immunosorbent assay (ELISA). RT-qPCR revealed that CsCP1-2 exhibited the highest expression in newly encysted larvae (NEL), while CsCP3 was predominantly expressed in adult stages. Immunohistochemical localization confirmed that CsCP1-3 are present in the eggshells, syncytial layers of metacercariae, NEL cuticle, and adult intestines. Histological and immunohistochemical analysis demonstrated that the rCsCP1-3-immunized group displayed reduced liver inflammation and biliary fibrosis compared to the control group. The rCsCP1-3 induced a progressive increase in specific IgG1 and IgG2a antibody titers by the second week post-immunization. In the CsCP1-2 group, cytokines IFN-g, IL-2, IL-4, and IL-10 were elevated relative to the control, with particularly high levels of IFN-g and IL-10 in CsCP1, indicating a strong mixed Th1/Th2 immune response. In contrast, the CsCP3 immunization group exhibited a transient increase in cytokines (IFN-g, IL-2, IL-4, and IL-10) three days postinfection, which subsided after one to two weeks. These findings suggest that CsCP1-3 elicit robust antibody and cellular immune responses, mitigating liver damage caused by C. sinensis infection. CsCP1, in particular, induces a potent mixed Th1/Th2 response, positioning it as a promising vaccine candidate.

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2023-11-10 | ASPSCR-1 and Sirt-5 alleviate Clonorchis liver fluke rCsNOSIP-induced oxidative stress, proliferation, and migration in cholangiocarcinoma cells

Clonorchiasis, caused by the infection of Clonorchis sinensis (C. sinensis), is a kind of neglected tropical disease, but it is highly related to cholangiocarcinoma. It has been well known that NO from chronic inflammation responses are thought to be a major component of the damage and ultimate carcinogenesis ESPs such as nitric oxide synthase interacting protein (NOSIP) are thought to enhance the damage. The objective of this study was to identify the protein candidates interact with recombinant CsNOSIP (rCsNOSIP) and explore their role involved in CCA development or progression.We applied HuProt microarray containing 21,000 probe sets for a systematic identification of rCsNOSIP-binding proteins and grouped binding hits by gene function. Pull-down assays were used to confirm the interaction of rCsNOSIP with alveolar soft part sarcoma (ASPSCR-1) and sirtuins 5 (Sirt-5). ASPSCR-1/Sirt-5 over-expression and siRNA knockdown experiments were employed for obtain of ASPSCR-1/Sirt-5 high or low expression (ASP-oe/Sirt5-oe or ASP-si/Sirt5-si) cholangiocarcinoma cell line (CCLP-1) cells. Nitric oxide (NO) and reactive oxygen species assay (ROS) as well as cell proliferation and wound-healing assays were performed to observe the effect of rCsNOSIP on ASP-oe/Sirt5-oe or ASP-si/Sirt5-si CCLP-1 cells.Seventy candidate proteins protein "hits" were detected as rCsNOSIP-binding proteins by HuProt microarray and bioinformatics analysis. Pull down assay showed that ASPSCR-1 and Sirt-5 could interact with rCsNOSIP. In addition, endotoxin-free-rCsNOSIP could increase the production of NO and ROS and promote the migration of CCLP-1 cells, while its effect on enhancing cell proliferation was not significant. Furthermore, ROS/NO production, proliferation, or migration were increased in ASP-si or Sirt5-si CCLP-1 cells but decreased in Asp-oe or Sirt5-oe CCLP-1 cells when stimulated with rCsNOSIP.Our findings suggest that CsNOSIP as a component of CsESPs might promote the development and invasion of CCA and Sirt5/ ASPSCR1 as host molecules might play a novel protective role against adverse stimulus during C. sinensis infection. This work supports the idea that CsESPs induce the occurrence and progression of CCA through ROS/RNS-induced oxidative and nitrative DNA damage.

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2023-07-21 | Recombinant protein EBI3 attenuates Clonorchis sinensis-induced liver fibrosis by inhibiting hepatic stellate cell activation in mice

Abstract Background Chronic infection with Clonorchis sinensis can cause hepatobiliary fibrosis and even lead to hepatobiliary carcinoma. Epstein-Barr virus-induced gene 3 protein (EBI3) is a subunit of interleukin 35, which can regulate inflammatory response and the occurrence of fibrotic diseases. Previous studies have reported that the expression of EBI3 in the serum of patients with liver cirrhosis is reduced. The present study aims to investigate the biological effects of EBI3 on liver fibrosis caused by C. sinensis and the underlying molecular mechanisms. Methods We first established a mouse model of liver fibrosis induced by C. sinensis infection and then measured the serum expression of EBI3 during the inflammatory and fibrotic phase. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed to investigate the potential role of EBI3 in liver fibrosis by regulating the extracellular matrix structural constituent and collagen catabolic process. Recombinant protein EBI3 (rEBI3) was added to hepatic stellate cells (HSCs) in vitro with C. sinensis antigen to explore its function. Finally, the therapeutic effect of rEBI3 was verified by intravenous injection into C. sinensis -infected mice. Results The results showed that the serum expression of EBI3 increased in the inflammatory response phase but decreased in the fibrotic phase. The excretory-secretory products of C. sinensis (Cs.ESP) were able to stimulate HSC activation, while rEBI3 reduced the activation of HSCs induced by Cs.ESP. Also, the protein expression of gp130 and downstream protein expressions of JAK1, p-JAK1, STAT3 and p-STAT3 in HSCs were increased after rEBI3 incubation. Finally, intravenously injected rEBI3 inhibited hepatic epithelial-mesenchymal transition in C. sinensis -infected mice by inhibiting HSC activation and reducing liver injury. Conclusion This study confirms that rEBI3 can attenuate C. sinensis- induced liver fibrosis by inhibiting HSC activation and may be one of the potential treatments for liver fibrosis. Graphical Abstract

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2022-10-13 | IL-10 regulates Th17 response to inhibit hepatobiliary injury caused by Clonorchis sinensis infection in C57BL/6J mice

Clonorchiasis caused by Clonorchis sinensis is a mainly foodborne parasitic disease. It can lead to hepatobiliary duct inflammation, fibrosis, obstructive jaundice, liver cirrhosis, and even cholangiocarcinoma. Interleukin (IL)-10 is an immune-regulatory cytokine which plays an immunosuppressive role during infection. Our previous study found that IL-10 was increased in mice with C. sinensis infection. However, the role and mechanism of IL-10 playing in hepatobiliary injury induced by C. sinensis infection remain unknown. Herein, Il10 +/+ mice and Il10 +/- C57BL/6J mice were infected with C. sinensis . It was found that IL-10 deficiency aggravated biliary hyperplasia and exacerbated periductal fibrosis induced by C. sinensis infection. Moreover, IL-10 deficiency increased CD4 + T cells and CD8 + T cells but not macrophages in the liver of mice with infection. There were no apparent differences in Th1 and Treg cells between Il10 +/+ and Il10 +/- mice infected with C. sinensis . However, the proportion of Th17 cells in CD4 + T cells in Il10 +/- infected mice was significantly higher than that in Il10 +/+ infected mice. IL-10 deficiency also enhanced the increase of Th17 cells induced by ESPs stimulation in vitro . Taken together, our results suggest that IL-10 plays a protective role in hepatobiliary injury in C57BL/6J mice induced by C. sinensis infection via inhibiting Th17 cells, which could deepen our understanding of the immunopathology of clonorchiasis.

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2022-08-24 | Mapping of the Complement C9 Binding Region on Clonorchis sinensis Paramyosin

Heliminthic paramyosin is a multifunctional protein that not only acts as a structural protein in muscle layers but as an immune-modulatory molecule interacting with the host immune system. Previously, we found that paramyosin from Clonorchis sinensis (CsPmy) is bound to human complement C9 protein (C9). To analyze the C9 binding region on CsPmy, overlapping recombinant fragments of CsPmy were produced and their binding activity to human C9 was investigated. The fragmental expression of CsPmy and C9 binding assays revealed that the C9 binding region was located at the C-terminus of CsPmy. Further analysis of the C-terminus of CsPmy to narrow the C9 binding region on CsPmy indicated that the region flanking731Leu-780 Leu was a potent C9 binding region. The CsPmy fragments corresponding to the region effectively inhibited human C9 polymerization. These results provide a precise molecular basis for CsPmy as a potent immunomodulator to evade host immune defenses by inhibiting complement attack.

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vaccines

2026-03-02 | [Epidemiology, carcinogenicity, prevention and control of clonorchiasis in China].

Clonorchiasis is one of the most prevalent foodborne zoonotic parasitic diseases, with Clonorchis (C.) sinensis being the primary causative agent in China. In 2020, approximately 9.46 million people in China were infected, and nearly 100 million were at risk. The complex life cycle of C. sinensis, involving human and animal hosts and two intermediate hosts, poses significant challenges for control. Recognized as a Group 1 human carcinogen, C. sinensis contributes to cholangiocarcinoma through mechanical obstruction of bile ducts, physical damage from worm migration, and chemical irritation from excretory-secretory products. Effective control requires an integrated "One Health" strategy, including chemotherapeutic interventions, health education, management of animal hosts and environments, and the development and application of vaccines as novel preventive measures.

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2025-10-28 | Bioinformatics-Driven Vaccine Design for Trematodes: Advances, Challenges, and Future Directions

Trematodes, or flukes, are parasitic flatworms that cause neglected tropical diseases such as schistosomiasis, fascioliasis, clonorchiasis, opisthorchiasis, and paragonimiasis, affecting millions worldwide and burdening public health and livestock industries. Control currently relies on chemotherapy with drugs such as praziquantel and triclabendazole, but reinfection, emerging resistance, and challenges in sustained elimination underscore the urgent need for vaccines. Vaccine development, however, remains difficult due to the parasites’ complex biology, multi-stage life cycles, and immune evasion strategies. Traditional empirical vaccine approaches have achieved limited success, but bioinformatics now offers transformative opportunities. Immunoinformatics enables systematic mining of genomic and proteomic data, prediction of immune-relevant antigens, cost-effective narrowing of candidates, and even cross-species applications. Key challenges remain, including stage-specific antigen diversity, parasite immune evasion, gaps between in silico predictions and in vivo validation, host genetic variability, and limited resources in endemic regions. Future prospects include integrating multi-omics data, applying artificial intelligence for improved predictions, and advancing novel platforms such as mRNA and nanoparticle vaccines. A One Health strategy targeting both humans and livestock could further enhance impact. Harnessing these approaches may finally enable effective trematode vaccines and sustainably reduce their global disease burden. Bangladesh Journal of Infectious Diseases, June 2025;12(1):174-180

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2022-01-18 | Molecular Mechanisms of Clonorchis sinensis-Host Interactions and Implications for Vaccine Development

Infections caused by Clonorchis sinensis remain a significant public health challenge for both humans and animals, causing pyogenic cholangitis, cholelithiasis, cholecystitis, biliary fibrosis, and even cholangiocarcinoma. However, the strategies used by the parasite and the immunological mechanisms used by the host have not yet been fully understood. With the advances in technologies and the accumulated knowledge of host-parasite interactions, many vaccine candidates against liver flukes have been investigated using different strategies. In this review, we explore and analyze in-depth the immunological mechanisms involved in the pathogenicity of C. sinensis. We highlight the different mechanisms by which the parasite interacts with its host to induce immune responses. All together, these data will allow us to have a better understanding of molecular mechansism of host-parasite interactions, which may shed lights on the development of an effective vaccine against C. sinensis.

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2021-05-14 | The storage stability of Bacillus subtilis spore displaying cysteine protease of Clonorchis sinensis and its effect on improving the gut microbiota of mice.

Bacillus subtilis (B. subtilis) spore can serve as an ideal vehicle for expressing heterologous antigens, and elicit specific immune responses by oral administration. In previous studies, we successfully constructed the recombinant B. subtilis spores expressing cysteine protease of Clonorchis sinensis (C. sinensis, B.s-CsCP), and confirmed that oral administration of B.s-CsCP could elicit good protective immune responses in mice. In this study, Gram staining was used to observe the morphology of B.s-CsCP in different form, and the storage of liquid spores and lyophilized spores at different temperatures was compared. The mice were orally immunized with three different doses of spores (2×108, 1×109, and 5×109 CFU/day) for three times in total at biweekly interval. Then, antibody levels of mice were measured, the safety of spores was evaluated, and the changes of gut microbiota after oral gavage of spores (1×109 dose) were investigated. Results showed that B. subtilis was a typical Gram-positive bacterium, and its spore had good resistance to chemical dye. Liquid B. subtilis spores resuspended in sterile water could be stored for a long time at 4 °C or below, while lyophilized spores could be well stored even at RT and better at lower temperatures. Oral administration of B. subtilis spores to mice could stimulate both local mucosal and systemic immune responses in a dose-dependent manner without toxic side effects. Besides, beneficial bacteria producing butyrate such as Odoribacter were increased, while potential pathogens such as Escherichia-Shigella were decreased in mice intestine. Therefore, our work further confirmed that B. subtilis spores expressing CsCP could be a promising oral vaccine against C. sinensis with the advantages of stability, safety, easy storage, and promotion of intestinal health.Key Points• Recombinant CsCP B. subtilis spores could be easily preserved in either liquid or freeze-dried state.• Oral immunization of recombinant spores in mice could increase both local and system immune levels in a dose-dependent manner.• Oral administration of recombinant spores increased the number of beneficial bacteria and reduced the number of harmful bacteria in the intestinal tract of mice.

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2018-03-07 | Bacillus subtilis spore with surface display of paramyosin from Clonorchis sinensis potentializes a promising oral vaccine candidate

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.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.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.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.

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oligonucleotides

2025-10-21 | Induction of TRIM22 during Clonorchis sinensis infection triggers intracellular ROS accumulation by suppressing the mTOR-mediated Nrf2 signaling pathway.

Clonorchis sinensis infection triggers various hepatobiliary complications, including epithelial hyperplasia, chronic inflammation, periductal fibrosis, and cholangiocarcinogenesis through direct contact with worms and their excretory-secretory products (ESPs). We previously profiled differential transcriptome expression from three-dimensional cholangiocyte (H69 cells) spheroids constitutively and repetitively exposed to ESPs using microarrays and RNA-seq analysis. TRIM22 was upregulated in response to ESP exposure. This study aimed to elucidate the pathophysiological mechanism of TRIM22-mediated hepatobiliary abnormalities during C. sinensis infection. Quantitative reverse transcription polymerase chain reaction and immunoblot analyses were used to examine TRIM22 transcript and protein expression in ESP-treated H69 spheroids and 2D cultured cells. Transfection with TRIM22-specific small interfering RNA was performed to examine changes in intracellular reactive oxygen species levels and the expression of the redox-active transcription factor signaling pathway (mTOR/Nrf2) and its target genes. Liver tissues from C. sinensis-infected mice were stained with TRIM22 and Nrf2 polyclonal antibodies. Treatment of H69 cells with ESPs increased TRIM22 expression and AKT/mTOR signaling pathway activation. TRIM22 knockdown abolished ESP-induced mTOR activation but elevated Nrf2 nuclear translocation with subsequent increased expression of antioxidant and phase II detoxifying enzymes. The immunoreactivity of TRIM22 and Nrf2 showed distinctly different patterns in the liver of mouse infected with C. sinensis for three months. TRIM22 upregulation during C. sinensis infection contributes to hepatobiliary abnormalities by disrupting redox homeostasis.

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2025-07-01 | GalNAc-siRNA conjugates mediate the silencing of the parasite-derived lncRNA: A novel therapeutic approach for liver fluke-induced liver fibrosis.

Clonorchis sinensis, as a liver fluke, can cause chronic liver damage, liver fibrosis, and even cholangiocarcinoma (CCA). However, there is a lack of effective therapeutic approaches for the treatment of clonorchiasis. Thus, we aimed to investigate the role of long noncoding RNAs (lncRNAs) in liver fibrosis induced by C. sinensis and evaluate the therapeutic potential of GalNAc-modified siRNAs targeting these lncRNAs. In this study, we first isolated Clonorchis sinensis extracellular vesicles (CsEVs) and identified the fibrosis-related long noncoding RNA lnc28240.1 in the CsEVs. In vitro and in vivo experiments revealed that the CsEVs and lnc28240.1 could induce liver fibrosis. Thus, to inhibit the development of liver fibrosis, we designed and synthesized a short interfering RNA#1 (siRNA#1) modified with N-acetylgalactosamine (GalNAc) to silence the pathogenic gene lnc28240.1. The results demonstrated that the GalNAc-siRNA conjugates effectively silenced lnc28240.1, significantly reducing C. sinensis-induced liver fibrosis and normalizing liver function. Our findings suggest that GalNAc-siRNA for gene silencing of parasite-derived lncRNA is a promising therapeutic strategy for the treatment of liver fibrosis caused by C. sinensis.

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2025-05-13 | Clonorchis sinensis extracellular vesicles associated with Csi-let-7a-5p activate pro-inflammatory macrophages to induce biliary injury.

During Clonorchis sinensis (C. sinensis) infection, pro-inflammatory macrophages (M1 macrophages) are highly activated, yet their role in the disease remains poorly understood. Previous studies have demonstrated that extracellular vesicles from C. sinensis (CsEVs) can activate these macrophages, and inhibiting a specific miRNA (Csi-let-7a-5p) in CsEVs (InCsEVs) can reduce this activation. In the present study, liver macrophages in mice were removed using clodronate liposomes (Clodlip). Subsequently, different types of bone marrow-derived macrophages (BMDMs) were adoptively transferred into the mice lacking liver macrophages: untreated (PBS-BMDM), treated with CsEVs (CsEVs-BMDM), treated with a control (ScrCsEVs-BMDM), or treated with InCsEVs (InCsEVs-BMDM). Biliary damages were then evaluated. The results indicated that the transferred macrophages successfully repopulated the mice. CsEVs-BMDM led to significant inflammation and bile duct damage, accompanied by higher levels of inflammatory cytokines (TNF-α and IL-1β). However, when macrophages were treated with InCsEVs, the damage and inflammation were alleviated, and the levels of TNF-α and IL-1β decreased. These findings suggest that pro-inflammatory macrophages activated by CsEVs, especially through Csi-let-7a-5p, play a crucial role in biliary damage during C. sinensis infection. Although other immune cells may also be involved, this study emphasizes the significance of pro-inflammatory macrophages in clonorchiasis.

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2023-05-11 | TLR3 activation by Clonorchis sinensis infection alleviates the fluke-induced liver fibrosis

Clonorchis sinensis is a zoonotic parasite associated with liver fibrosis and cholangiocarcinoma development. The role of toll-like receptors (TLRs) in C. sinensis infection has not yet been fully elucidated. Here, the TLR3 signaling pathway, cytokine expression and liver fibrosis were examined in C. sinensis-infected wildtype (WT) and TLR3-/- mice. Polyinosinic-polycytidylic acid (Poly (I:C)) was used to treat C. sinensis infections. The results showed that TLR3 deficiency caused severe clonorchiasis with increased parasite burden, exacerbated proinflammatory cytokine expression and liver lesions, promoted the TGF-β1/Smad2/3 pathway and myofibroblast activation, exacerbated liver fibrosis (compared to WT mice). Poly (I:C) intervention increased the body weight, decreased mouse mortality and parasite burden, reduced liver inflammation, and alleviated C. sinensis-induced liver fibrosis. Furthermore, C. sinensis extracellular vesicles (CsEVs) promote the production of IL-6, TNF in WT biliary epithelial cells (BECs) via p38/ERK pathway, compared with control group, while TLR3 deletion induced much higher levels of IL-6 and TNF in TLR3-/- BECs than that in WT BECs. Taken together, TLR3 inhibit IL-6 and TNF production via p38/ERK signaling pathway, a phenomenon that resulted in the alleviation of C. sinensis-induced liver fibrosis. Poly (I:C) is a potential treatment for clonorchiasis.

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2022-01-03 | MicroRNA-497 induced by Clonorchis sinensis enhances the TGF-β/Smad signaling pathway to promote hepatic fibrosis by targeting Smad7.

Various stimuli, including Clonorchis sinensis infection, can cause liver fibrosis. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) with massive production of extracellular matrix (ECM). Our previous study showed that the TGF-β1-induced Smad signaling pathway played a critical role in the activation of HSCs during liver fibrosis induced by worm infection; however, the mechanisms that modulate the TGF-β/Smad signaling pathway are still poorly understood. Accumulating evidence demonstrates that miRNAs act as an important regulator of activation of HSCs during liver fibrosis. The target of miR-497 was determined by bioinformatics analysis combined with a dual-luciferase activity assay. LX-2 cells were transfected with miR-497 inhibitor and then stimulated with TGF-β1 or excretory/secretory products of C. sinensis (CsESPs), and activation of LX-2 was assessed using qPCR or western blot. In vivo, the mice treated with CCl4 were intravenously injected with a single dose of adeno-associated virus serotype 8 (AAV8) that overexpressed anti-miR-497 sequences or their scramble control for 6 weeks. Liver fibrosis and damage were assessed by hematoxylin and eosin (H&E) staining, Masson staining, and qPCR; the activation of the TGF-β/Smad signaling pathway was detected by qPCR or western blot. In the present study, the expression of miR-497 was increased in HSCs activated by TGF-β1 or ESPs of C. sinensis. We identified that Smad7 was the target of miR-497 using combined bioinformatics analysis with luciferase activity assays. Transfection of anti-miR-497 into HSCs upregulated the expression of Smad7, leading to a decrease in the level of p-Smad2/3 and subsequent suppression of the activation of HSCs induced by TGF-β1 or CsESPs. Furthermore, miR-497 inhibitor delivered by highly-hepatotropic (rAAV8) inhibited TGF-β/smads signaling pathway by targeting at Smad7 to ameliorate CCL4-induced liver fibrosis. The present study demonstrates that miR-497 promotes liver fibrogenesis by targeting Smad7 to promote TGF-β/Smad signaling pathway transduction both in vivo and in vitro, which provides a promising therapeutic strategy using anti-miR-497 against liver fibrosis.

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2026-08-31 | A pathogenic CD8⁺ TRM-chemokine axis orchestrates liver fibrosis and provides circulating biomarkers during chronic Clonorchis sinensis infection.

Chronic liver fluke infections represent a major global health burden, driving liver fibrosis and elevating cancer risk, yet the immunological mechanisms linking persistent infection to tissue damage remain poorly defined. Using Clonorchis sinensis, a carcinogenic liver fluke infecting millions worldwide, as a representative model, we map immune responses during chronic infection in mice. We show that intrahepatic CD8⁺ tissue-resident memory T cells (TRM) progressively accumulate and drive pathology by producing chemokines that promote immune recruitment and fibroblast activation. Depletion of CD8⁺ T cells during defined disease phases markedly alleviates liver inflammation and fibrosis, demonstrating their causal role. Strikingly, C-C motif chemokine ligand 4, predominantly secreted by CD8⁺ TRM cells, correlates with fibrosis severity in both experimental models and patient cohorts, establishing it as a circulating biomarker for noninvasive disease monitoring. These findings define a pathogenic CD8⁺ TRM-chemokine axis underlying helminth-induced liver injury and highlight TRM-derived chemokines as biomarkers and therapeutic targets for fibrosis across chronic parasitic infections.

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2025-08-20 | Clonorchis sinensis-driven hepatocarcinogenesis via E2F1-CD24 transcriptional axis: mechanistic and therapeutic implications.

Hepatocellular carcinoma (HCC) remains a global health burden, with disproportionately high mortality in China's Guangxi region, where endemic Clonorchis sinensis (C. sinensis) infection coincides with elevated HCC incidence. Preliminary single-cell sequencing revealed marked overexpression of cluster of differentiation 24 (CD24) in HCC tissues, suggesting its potential pathological role. This study aims to elucidate the oncogenic mechanisms of C. sinensis excretory-secretory products (CsESPs) and their link to CD24-mediated HCC progression. We employed an integrated clinical and experimental approach. First, clinical cohort analysis assessed CD24 expression in C. sinensis-associated HCC cases. Multiplatform bioinformatics validation (GEPIA/UALCAN/TIMER) evaluated CD24's prognostic significance and immune microenvironment modulation. Functional studies (quantitative polymerase chain reaction (qPCR), Western blotting, CCK-8 assays, flow cytometry) examined CsESPs' effects on CD24 expression, cell proliferation, and apoptosis. Mechanistic investigations (chromatin immunoprecipitation, dual-luciferase reporter assays) identified E2F1-mediated transcriptional activation of CD24. siRNA-mediated CD24 knockdown validated its role in CsESPs-driven oncogenesis. Additionally, the expression of immune checkpoint (CTLA-4, LAG-3) was assessed in the co-cultures of peripheral blood mononuclear cells (PBMCs)-HCC cells. Clinical cohort analysis confirmed significant CD24 upregulation in HCC, particularly in C. sinensis-infected cases. Bioinformatic analyses linked high CD24 expression to poor prognosis and immune microenvironment alterations. Functional assays demonstrated that CsESPs enhance CD24 expression, promoting proliferation and inhibiting apoptosis. Mechanistically, E2F1 directly binds to CD24 promoter, driving its transcription upon CsESPs exposure. CD24 silencing reversed CsESPs-induced oncogenic effects. Furthermore, CsESPs upregulated immune checkpoints (CTLA-4, LAG-3) in the co-cultures of PBMC-HCC cells, an effect reversed by CD24 knockdown. Our findings establish a novel parasitic carcinogenesis paradigm wherein C. sinensis promotes HCC development through E2F1-mediated transcriptional activation of CD24, simultaneously identifying prognostic biomarkers and therapeutic targets while suggesting combinatory immunotherapy strategies for parasite-associated HCC.

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2022-10-22 | Interleukin-33 deficiency prevents biliary injuries and repairments caused by Clonorchis sinensis via restraining type 2 cytokines

Clonorchiasis caused by Clonorchis sinensis is a zoonotic parasitic disease characterized by cholangitis, biliary proliferation, biliary fibrosis, and even cholangiocarcinoma. Our previous study showed that the expression of interleukin (IL)-33 is increased in both humans and mice infected by C. sinensis, suggesting that IL-33 is potentially involved in the pathogenesis of clonorchiasis. However, the roles and potential mechanism of IL-33 underlying remain unknown.Wild-type (WT) and IL-33 knockout (KO) mice (BALB/c female mice) were orally infected with 45 metacercariae of C. sinensis for 8 weeks. Biliary injuries and fibrosis were extensively evaluated. Hepatic type II cytokines (IL-4, IL-13, and IL-10) were detected by ELISA.For wild-type mice, we found that the mice infected with C. sinensis showed severe biliary injuries and fibrosis compared with the normal mice that were free from worm infection. In addition, the levels of type II cytokines such as IL-4, IL-13, and IL-10 in infected wild-type mice were significantly higher than in the control mice without infection (P < 0.05). However, IL-33 deficiency (IL-33 KO) prevents the augmentation of biliary injuries and fibrosis caused by C. sinensis infection. Furthermore, the increased levels of these type II cytokines induced by worm infection were also reversed in IL-33 KO mice.Our present study demonstrates that IL-33 contributes to the pathogenesis of C. sinensis-induced biliary injuries and repair, which can potentially orchestrate type 2 responses. These findings highlight the pathophysiological role of IL-33 in the progression of clonorchiasis.

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2015-10-01 | Prognosis of sepsis induced by cecal ligation and puncture in mice improved by anti-Clonorchis Sinensis cyclopholin a antibodies

Cyclophilin A (CyPA), a ubiquitously distributed intracellular protein, is thought to be one of the important inflammatory factors and plays a significant role in the development process of sepsis. In the form of cytokine, CyPA deteriorates sepsis by promoting intercellular communication, apoptosis of endothelial cells and chemotactic effect on inflammatory cells. In our previous study, cyclophilin A of Clonorchis sinensis (CsCyPA), a type of excretory-secretory antigen, could induce the patients infected with Clonorchis sinensis to produce specific anti-CsCyPA antibodies. In this study, we investigated whether anti-CsCyPA antibodies could cross-react with CyPA and then play a protective role against sepsis, just like other anti-cytokine antagonists. The mice model with sepsis was established with cecal ligation and puncture (CLP). Fifty mg/kg purified anti-CsCyPA antibodies were injected via the caudal vein 6 h after the CLP operation, and persistent observation was performed for 72 h. Blood samples and tissues were collected at 6 h, 12 h, 24 h, 48 h and 72 h after CLP. Cytokines in serum were measured by ELISA. Lung and mesentery tissues were stained with hematoxylin-eosin. Endothelial cells (ECs) isolated from murine aorta were co-cultured with CyPA of mice (MuCyPA) and anti-CsCyPAs for 24 h, then, viability was measured by Cell Counting Kit-8. Anti-CsCyPA antibodies could combine with MuCyPA and inhibite its peptidyl prolyl isomerase (PPIase) activity. In the antibodies treatment group, blood coagulation indicators including PT, aPTT, D-dimer and platelet count were obviously more ameliorative, the proinflammary factors like IL-6, TNF-α, IL-1β were significantly lower at 12 h and 24 h after surgery and the viability of ECs was significantly improved compared to those in the control group. Furthermore, the survival rate was elevated, ranging from 10.0 % to 45.0 % compared to the control group. These antibodies may have a favorable effect on sepsis via inhibition of enzymic activity or protection of endothelial cells.

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1992-03-26 | Analysis of Clonorchis sinensis antigens and diagnosis of clonorchiasis using monoclonal antibodies.

Clonorchis sinensis is a common parasite of man in Korea. Researches on the specific antigens of C. sinensis would be valuable not only because those elucidate the molecular characteristics of this fluke but also because it is applicable to immunodiagnosis. Although many monoclonal antibodies have been used in the field of parasite immunology, few articles on monoclonal antibodies against C. sinensis have been published so far. The aim of this study was to analyze C. sinensis antigens recognized by monoclonal antibodies, and to set up ELISA-inhibition test using C. sinensis specific monoclonal antibodies for improved specificity of immunodiagnostic tests. By fusion between spleen cells of the mice immunized with C. sinensis water-soluble crude adult worm antigens and plasmacytoma cells of mouse origin, 29 hybridoma clones secreting anti-C. sinensis monoclonal antibodies were made, and 8 clones among those were found specific. After cell cloning, isotypes of 6 selected specific monoclonal antibodies were determined to be IgG1, IgG2b and IgA. Four exposed antigenic determinants of natural infection were recognized by different specific monoclonal antibodies. By enzyme-immunoelectrotransfer blot, 10 KD, 34 KD antigenic determinants were found to be reacted with CsHyb 0714-20, CsHyb 0605-10 monoclonal antibodies, respectively. The antigenic determinant recognized by CsHyb 0714-20 monoclonal antibody was revealed to be located at the surface and parenchyme of a parasite by indirect immunofluorescent antibody technique, and those reacted with CsHyb 0605-10, CsHyb 0714-25 monoclonal antibodies were found at the parenchyme and intestine. The antigenic determinant reacted with CsHyb 0605-23 monoclonal antibody was found mainly around the uterine eggs. Four antigenic determinants recognized by specific monoclonal antibodies were all found to be present in the early eluted fractions of C. sinensis antigens separated by Sephadex G-200 gel filtration. By conventional ELISA, 75% of clonorchiasis cases were found positive, but 7.1% of normal controls and 37.5% of paragonimiasis cases showed false positives. However, by ELISA-inhibition test using C. sinensis specific monoclonal antibody (CsHyb 0605-23), 77.1% of clonorchiasis cases were found positive, and there were no false positives in normal controls or paragonimiasis cases, indicating 100% specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

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