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The effects of co-infections with SARS-CoV-2 and parasitic diseases have been little investigated in terms of immune response, disease dynamics, and clinical outcomes. This study aimed to explore the impact of co-infection with Opisthorchis viverrini and SARS-CoV-2 on the immune response concerning clinical symptoms and the severity of pulmonary abnormalities. A cross-sectional study was conducted, including healthy participants as controls, participants with opisthorchiasis, SARS-CoV-2 infection, and a co-infection group with both diseases. Characteristics of SARS-CoV-2 infection were assessed based on clinical parameters and severity of pulmonary abnormalities, whereas opisthorchiasis burden was evaluated by eggs-per-gram (EPG) counts. Immune responses were assessed by measuring levels of interferon-γ (IFN-γ), SARS-CoV-2 anti-spike receptor binding domain (RBD) IgG, and neutralizing antibody against SARS-CoV-2. In the co-infected group, clinical parameters and hospitalization rates were lower than in the SARS-CoV-2 group. Pulmonary abnormalities, such as bronchial fibrosis, were commonly observed in the SARS-CoV-2 group, leading to hospitalization in some cases. Participants with opisthorchiasis had higher IFN-γ levels than healthy individuals. IFN-γ levels were significantly lower in the co-infection group compared with the SARS-CoV-2 group (P = 0.002). There was a significant (P = 0.044) positive correlation between RBD-specific IgG and percent neutralization levels in the SARS-CoV-2 group. Levels of both were somewhat lower (not statistically significant) in the co-infection group. A negative correlation was observed between opisthorchiasis burden (EPG counts) and IFN-γ and RBD-specific IgG levels in the co-infected group. Following vaccination, the increase in IgG levels against the RBD protein was significantly lower in the co-infected group than in the SARS-CoV-2 group. These results suggest that O. viverrini infection suppresses immune responses and may lead to a reduction in severity in cases of SARS-CoV-2 co-infection., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Opisthorchis viverrini infection is a pressing health issue in rural Southeast Asia and is associated with the risk of cholangiocarcinoma. Despite control efforts, high infection rates persist, including evidence of reinfection post-treatment. This study aimed to address this public health concern through an integrated One-Health approach in endemic areas in rural Thailand over a 3-year period. The study included data from 3600 participants from Udon Thani Province, Thailand, during the years 2020 to 2022 and involved integrated epidemiological data collection and risk factor analysis to understand the impact of various interventions on disease transmission in the community. The efficacy of interventions was assessed by monitoring the incidence of O. viverrini reinfection in 2021 and 2022. In 2020, 218 cases of O. viverrini infection (6.0%) were identified. Significant risk factors included proximity to water bodies and consumption of raw fish. Variables contributing to infection risk among participants (P < 0.001) were education level, engagement in traditional ceremonies, poor sanitation, absence of ducks in nearby water bodies, self-medication for parasitic conditions, and multiple infections within a household. Dogs, cats, and cyprinoid fish showed prevalence rates of 5.4%, 6.3%, and 11.5%, respectively. Geographic analysis revealed clusters of infected households around water bodies. Interventions, including in-depth interviews, focus-group discussions, health education, anthelminthic treatment, and biological control using local free-range ducks, were implemented, resulting in no human reinfections in the second year and a minimal 0.3% prevalence rate in the third year. This study offers valuable insights into the dynamic changes in infection prevalence, making a significant contribution to effective disease control and community health promotion. This integrated One-Health approach proved to be an effective strategy for the prevention and control of opisthorchiasis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Comorbidity of Opisthorchis viverrini (OV) infection and nonalcoholic fatty-liver disease (NAFLD) enhances NAFLD progression to nonalcoholic steatohepatitis (NASH) by promoting severe liver inflammation and fibrosis. Here, we investigated the effect of supplementation with curcumin-loaded nanocomplexes (CNCs) on the severity of NASH in hamsters., Methodology: Hamsters were placed in experimental groups as follows: fed standard chow diet (normal control, NC); fed only high-fat and high-fructose (HFF) diet; O. viverrini-infected and fed HFF diet (HFFOV); group fed with blank nanocomplexes (HFFOV+BNCs); groups fed different doses of CNCs (25, 50 and 100 mg/kg body weight: HFFOV+CNCs25; HFFOV+CNCs50; HFFOV+CNCs100, respectively) and a group given native curcumin (HFFOV+CUR). All treatment were for three months., Results: The HFF group revealed NAFLD as evidenced by hepatic fat accumulation, ballooning, mild inflammation and little or no fibrosis. These changes were more obvious in the HFFOV group, indicating development of NASH. In contrast, in the HFFOV+CNCs50 group, histopathological features indicated that hepatic fat accumulation, cell ballooning, cell inflammation and fibrosis were lower than in other treatment groups. Relevantly, the expression of lipid-uptake genes, including fatty-acid uptake (cluster of differentiation 36), was reduced, which was associated with the lowering of alanine aminotransferase, total cholesterol and triglyceride (TG) levels. Reduced expression of an inflammation marker (high-mobility group box protein 1) and a fibrosis marker (alpha smooth-muscle actin) were also observed in the HFFOV+CNCs50 group., Conclusion: CNCs treatment attenuates the severity of NASH by decreasing hepatic steatosis, inflammation, and fibrosis as well as TG synthesis. CNCs mitigate the severity of NASH in this preclinical study, which indicates promise for future use in patients., Competing Interests: The authors have declared that no competing interests exist.

A combination of Opisthorchis viverrini infection and high fat/high fructose diets (HFa/HFr) intake is likely to enhance fatty liver and kidney pathologies. Here we investigated the combined effects of chronic O. viverrini infection and HFa/HFr intake on liver and kidney pathologies, metabolism, and gut microbiome in hamsters. Animals were infected with O. viverrini and fed with either standard chow (OV group) or HFa/HFr diet (OH group) and non-infected hamsters were fed with either standard chow (NC) or HFa/HFr diet (HF) for 8 months. The OH group exhibited dyslipidemia and the highest severity of fatty liver. Tubular damage, inflammatory cell infiltration, and tubular fibrosis were the most prominently observed in this group, supported by increased expression of KIM-1, HMGB-1, and MCP-1. Urinary 1 H NMR metabolic profiles revealed that tauro-β-muricholic acid level was increased in the OV and OH groups, whereas metabolites involved in the TCA cycle and gut microbiota-associated metabolites (phenylacetylglycine, trimethylamine, and trimethylamine-N-oxide) were lower in OV, HF and OH groups compared to the NC group. Gut microbial profiles of the OH group were also different from other groups. In conclusion, O. viverrini infection and HFa/HFr diet-induced disturbance of metabolites and gut microbiota associated with concurrent liver and kidney pathologies in hamsters., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Opisthorchis viverrini (Ov) infection causes hepatobiliary diseases and is a major risk factor for cholangiocarcinoma. While several omics approaches have been employed to understand the pathogenesis of opisthorchiasis, effects of Ov infection on the host systemic metabolism and fecal microbiota have not been fully explored. Here, we used a 1 H NMR spectroscopy-based metabolic phenotyping approach to investigate Ov infection-induced metabolic disturbances at both the acute (1 month postinfection, 1 mpi) and chronic (4 mpi) stages in hamsters. A total of 22, 3, and 4 metabolites were found to be significantly different in the liver, serum, and urine, respectively, between Ov+ and Ov- groups. Elevated levels of hepatic amino acids and tricarboxylic acid (TCA)-cycle intermediates (fumarate and malate) were co-observed with liver injury in acute infection, whereas fibrosis-associated metabolites (e.g., glycine and glutamate) increased at the chronic infection stage. Lower levels of lipid signals ((C H 2 ) n and C H 2 CH 2 CO) and higher levels of lysine and scyllo -inositol were observed in serum from Ov+ hamsters at 1 mpi compared to Ov- controls. Urinary levels of phenylacetylglycine (a host-bacterial cometabolite) and tauro-β-muricholic acid were higher in the Ov+ group, which coexisted with hepatic and mild kidney fibrosis. Furthermore, Ov+ animals showed higher relative abundances of fecal Methanobrevibacter (Archaea), Akkermansia , and Burkholderia-Paraburkholderia compared to the noninfected controls. In conclusion, along with liver and kidney pathologies, O. viverrini infection resulted in hepatic and mild renal pathologies, disturbed hepatic amino acid metabolism and the TCA cycle, and induced changes in the fecal microbial composition and urinary host-microbial cometabolism. This study provides the initial step toward an understanding of local and systemic metabolic responses of the host to O. viverrini infection.

Alteration of immunoglobulin glycosylation correlates with inflammatory diseases and infectious diseases including parasitic infections. Immunoglobulin glycosylation patterns may be implicated in disease development and have also been proposed as diagnostic tools for several diseases. Previous studies have reported the immunoglobulin profiles in experimental animals and in patients infected with the carcinogenic human liver fluke, Opisthorchis viverrini. However, the N-glycosylation profiles of immunoglobulins and their subclass-specific glycoforms in opisthorchiasis patients have never been elucidated. Here, N-glycosylation patterns of immunoglobulins and their subclass-specific glycoforms in sera of O. viverrini-infected patients were investigated using triple quadrupole mass spectrometry coupled with multiple reaction monitoring. Peptide fragmentation was utilized to quantify the immunoglobulin glycoforms normalized to the unique peptide of each subclass. Overall, serum levels of IgG and IgA in O. viverrini patients were significantly increased compared to uninfected controls. Twenty-seven glycoforms were detected based on analysis of detached glycans in all immunoglobulin subclasses. The abundance of immunoglobulin glycopeptides in serum of opisthorchiasis patients deviated significantly from controls. Immunoglobulin glycosylation patterns were associated with both pro- and anti-inflammatory properties. In conclusion, O. viverrini infection alters the serum immunoglobulin glycosylation profile and these changes could distinguish between O. viverrini-infected individuals and healthy controls. SIGNIFICANCE: We demonstrated that both quantities and glycoforms of serum immunoglobulin subclasses were altered in Opisthorchis viverrini-infected individuals as investigated by the QqQ-MS-MRM method. Patterns of immunoglobulin with a specific glycoform might contribute to immune responses to O. viverrini infection., (Copyright © 2020. Published by Elsevier B.V.)

The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide, including in regions where helminth infections such as the fish-borne liver fluke Opisthorchis viverrini ( Ov ) also occur. We investigated the effects of a high-fat and high-fructose (HFF) diet on the development and progression of NAFLD in experimental opisthorchiasis. Two groups of hamsters were infected with Ov for 4 months before the experiment to induce chronic inflammation. One of these groups ( Ov HFF) was fed with a HFF diet for up to further 4 months. One uninfected group of hamsters served as the normal control group, and another received the HFF diet (HFF group) for up to 4 months. Histopathology, biochemical parameters, and ultrastructural features of liver were investigated. In a short-term treatment, the Ov HFF group showed significantly better homeostatic model assessment for insulin resistance level and lower liver lipid than did the HFF group. By contrast, histopathological characteristics of severe NAFLD were prominent in the Ov HFF group after 4 months on the HFF diet, findings which were supported by confirmatory ultrastructural changes. In conclusion, opisthorchiasis induced the severe NAFLD in hamsters fed high-fat/high-fructose diets.

Opisthorchiasis, caused by Opisthorchis viverrini, remains the public health significance in Thailand, particularly in the northeastern region. Number of parasitological techniques is available for diagnosis. However, the detection the parasite's eggs in stool still referred as gold standard. Today, most people living in the endemic areas harbored the light infection. In this study, we compared the performance of formalin-ethyl acetate concentration technique (FECT), Kato-Katz technique, fecal parasite concentrator kit (FPCK) and direct simple smear technique for O. viverrini egg examination in stool. The results revealed that the FECT gave the best sensitivity (91.0%) followed by Kato-Katz technique, FPCK and direct simple smear techniques. Interestingly, the FECT showed the highest sensitivity in both groups of egg per gram (EPG) <50 (94.3%) and EPG ≥ 50 (100%). The FPCK and direct simple smear technique exhibited the higher sensitivity in EPG ≥ 50 group compared with EPG < 50 group (p <  0.05). However, the sensitivity of FPCK was not statistically differed from that direct simple smear in EPG <50 and EPG ≥50 group. In conclusion, the FECT showed the highest efficiency to detect O. viverrini egg in stool, followed by Kato-Katz technique, FPCK and direct simple smear technique. Substituting FPCK (Mini Parasep® Kit) for FECT and Kato-Katz method is not recommended for low intensity infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The combination of Opisthorchis viverrini (OV) infection and chemical carcinogen induces cholangiocarcinoma (CCA) in hamsters via inflammation-mediated mechanisms. Thus, suppression of inflammatory cells at the initial stages of CCA development would be of benefit. We aimed to investigate whether IL-17-producing CD4+ T cells (Th17) and CD4+ Foxp3+ T cells (Treg) are involved in the early stages of CCA genesis and can be targeted for suppression by melatonin., Methods: Inflammation, an initial stage of CCA development, was induced in hamsters by a combination of O. viverrini infection and N-nitrosodimethylamine (NDMA) administration. Melatonin (50mg/kg) was additionally administered to one group for the 30days of the experiment. Liver tissue-resident T cells were investigated using immunostaining, western blotting, and real-time PCR., Results: OV+NDMA-induced CCA tissues showed significantly higher numbers of inflammatory cells, especially eosinophils, bile duct proliferation and IL-17+ cell infiltration compared to normal livers. Expression of Foxp3 was localized in the bile duct epithelial cells, and especially in the bile duct hyperplasia. Accumulation of CD4+ and IL-17+ cells and intense staining of the Foxp3+ marker were consistent with their protein levels. Infiltration of IL-17+ inflammatory cells and Foxp3+ cells, as well as increases in their transcription expression levels, were significantly lower in the melatonin-treated group. In contrast, increased CD4+ cell infiltration and TNF-α expression were also observed through melatonin treatment., Conclusion: Melatonin exerts an immunomodulatory effect, suppressing eosinophils and Th17 cells and expression of Foxp3, but enhancing CD4+ cells and TNF-α. This suggests that melatonin may be used for CCA chemoprevention., (Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Persistent infection with Opisthorchis viverrini causes hepatobiliary abnormalities, predisposing infected individuals to cholangiocarcinoma (CCA). In addition, Helicobacter pylori is highly prevalent in most countries and is a possible risk factor for CCA; however, its role in enhancing hepatobiliary abnormality is unclear. Here, we investigated the effects of coinfection with H. pylori and O. viverrini on hepatobiliary abnormality. Hamsters were divided into four groups: (i) normal, (ii) H. pylori infected (HP), (iii) O. viverrini infected (OV), and (iv) O. viverrini and H. pylori infected (OV+HP). At 6 months postinfection, PCR and immunohistochemistry were used to test for the presence of H. pylori in the stomach, gallbladder, and liver. In the liver, H. pylori was detected in the following order: OV+HP, 5 of 8 (62.5%); HP, 2 of 5 (40%); OV, 2 of 8 (25%). H. pylori was not detected in normal (control) liver tissues. Coinfection induced the most severe hepatobiliary abnormalities, including periductal fibrosis, cholangitis, and bile duct hyperplasia, leading to a significantly decreased survival rate of experimental animals. The greatest thickness of periductal fibrosis was associated with a significant increase in fibrogenesis markers (expression of alpha smooth muscle actin and transforming growth factor beta). Quantitative reverse transcription-PCR revealed that the highest expression levels of genes for proinflammatory cytokines (interleukin-1 [ IL-1 ], IL-6 , and tumor necrosis factor alpha) were also observed in the OV+HP group. These results suggest that coinfection with H. pylori and O. viverrini increased the severity of hepatobiliary abnormalities to a greater extent than either single infection did., (Copyright © 2017 American Society for Microbiology.)

Contamination of a popular fermented fish dish, pla-som, by Opisthorchis viverrini metacercariae (OVMC) is a possible cause of carcinogenic liver fluke infection in Thailand. Affected individuals are at risk of bile duct cancer, which is a major health problem for people in the Greater Mekong Subregion. In order to investigate concerns about food safety, we studied the effects of fermentation time and low temperature on the viability and infectivity of OVMC during the pla-som production process. Pla-som was prepared at room temperature for up to 1 week in duplicate experiments using cyprinid freshwater fish obtained from an O. viverrini-endemic area. OVMC were then isolated and identified under a stereomicroscope. Complete and viable OVMC were found on days 1-4 of fermentation, while their morphology was degenerated thereafter. After OVMC were fed to hamsters, the percentage of the worm recovery after 1 to 2 months of infection was 52%, 44.7%, 11.3% and 1% for days 1, 2, 3 and 4, respectively. In order to measure the effect of low temperature on OVMC, fish were kept in a refrigerator (4 °C) for up to five days and then subsequently fermented for three days. In fish stored in a refrigerator for 1 and 2 days, viable OVMC were clearly observed and were able to infect hamsters, a worm-recovery percentage of 3.3% and 12.7%, respectively. By contrast, in pla-som prepared from fish stored for 3 to 5 days, OVMC were degenerated and could not infect the host. In conclusion, pla-som fermentation for more than four days and refrigerating fish for three days before pla-som processing can prevent O. viverrini infection. This study may increase awareness of fermented-fish dish preparation to prevent liver fluke infection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

3 T MRI and (1)H MRS were useful for quantitative investigation of the serial development of hepatobiliary changes in Opisthorchis viverrini infection in hamsters, and the differential diagnosis of cholangiocacinoma (CCA) development from bile duct changes and normal condition is unclear. In this study, we investigated the serial development of hepatobiliary changes and CCAgenesis in O. viverrini-infected and N-nitrosodimethylamine (NDMA) treated hamsters (ON group) using 3 T MRI and (1)H MRS and the results were compared with those either in the O. viverrini-infected group (OV group) and uninfected normal controls. In the ON group, CCAs were first found at 9 weeks post-infection, with sizes of ~2 mm. The typical MR signal characteristics of CCA were hypo- and occasionally isointensity signal on T1-weighted images, and mild-moderate to hyper-intensity signal on T2-weighted images compared to the liver parenchyma. T2-weighted images with fat suppression revealed dilatation of the intra- and extrahepatic bile ducts, and often defined the anatomical level of biliary obstruction, cystic lesions, liver abscesses, and CCA which was starting seen of these noticeable abnormalities at 5 weeks onwards. The results of fibrosis grading using MR images showed a positive correlation (r=0.90, P<0.038 by Spearman's rank correlation test) with those of the histopathological grading. In addition, 3.0 T (1)H MRS showed elevated choline and decreased lipids levels in the liver tissues of the ON group. In conclusion, MRI and (1)H MRS are useful for the quantitative investigation of the serial development of hepatobilary changes and CCA in hamsters, and are potentially useful as early diagnostic tools for CCA., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Infection with the liver fluke Opisthorchis viverrini (Digenea) (Poirier, 1886) causes bile duct injury and periductal fibrosis by chronic overproduction of inflammatory-mediators and eventually results in cholangiocarcinoma development. While extensive research works have been done on O. viverrini infection-associated changes of bile ducts and periductal fibrosis, little attention was paid on morphological and biochemical changes of the bile canaliculi (BC), the origin of bile flow. We aimed to investigate the morphological and functional alterations of BC in the liver of hamsters infected with O. viverrini at one and three months post-infection. Ultrastructural changes of BC showed dilatation of BC and significant reduction of the density of microvilli as early as at one month post-infection. Immunohistochemistry revealed that CD10, a BC marker, expression was reduced early as one month post-infection. The mRNA expression of the genes encoding molecules related to bile secretion including bile acid uptake transporters (slc10a1 and slco1a1), bile acid dependent (abcb11) and independent (abcc2) bile flow and bile acid biosynthesis (cyp7a1 and cyp27a1) were significantly decreased at one month post-infection in association with the reduction of bile volume. In contrast, the expression of the mRNA of bile acid regulatory genes (fxr and shp-1) was significantly increased. These changes essentially persisted up to three months post-infection. In conclusion, O. viverrini infection induces morphological and functional changes of BC in association with the decrease of bile volume.

The pharmacological activities of herbal extracts can be enhanced by complex formation. In this study, we manipulated cyanidin and delphinidin-rich extracts to form an anthocyanin complex (AC) with turmeric and evaluated activity against inflammation and periductal fibrosis in Opisthorchis viverrini-infected hamsters. The AC was prepared from anthocyanins extracted from cobs of purple waxy corn (70%), petals of blue butterfly pea (20%) and turmeric extract (10%), resulting in an enhanced free-radical scavenging capacity. Oral administration of AC (175 and 700 mg/kg body weight) every day for 1 month to O. viverrini-infected hamsters resulted in reduced inflammatory cells and periductal fibrosis. Fourier transform infrared spectroscopy and partial least square discriminant analysis suggested nucleic acid changes in the O. viverrini-infected liver samples, which were partially prevented by the AC treatment. AC reduced 8-oxodG formation, an oxidative DNA damage marker, significantly decreased levels of nitrite in the plasma and alanine aminotransferase activity and increased the ferric reducing ability of plasma. AC also decreased the expression of oxidant-related genes (NF-κB and iNOS) and increased the expression of antioxidant-related genes (CAT, SOD, and GPx). Thus, AC increases free-radical scavenging capacity, decreases inflammation, suppresses oxidative/nitrative stress, and reduces liver injury and periductal fibrosis in O. viverrini-infected hamsters.

Human liver fluke, Opisthorchis viverrini (Ov), is the major risk factor of cholangiocarcinoma (CCA) in northeastern Thailand. Our approach focuses on genetic progression and molecular changes in the carcinogenic pathway of liver fluke-associated CCA aimed at assessing patients at risk of CCA and using chemoprevention as the secondary cancer prevention to reduce the incidence of CCA. This review summarizes altered gene expressions, biomolecules and their modification, i.e. DNA adducts, oxidized proteins, oxysterols and fibrotic markers in hamster- and human-CCA. Potential risk biomarker(s) and chemopreventive agent(s) criteria and selection were based on results from experimental and epidemiological studies identifying hepatobiliary disease, including CCA. Laboratory results reveal that oxidative stress induced by Ov infection leads to bimolecular damage, tissue remodeling especially periductal fibrosis and alteration of gene expressions, which could be involved in all steps of CCA carcinogenesis. Some of these molecules are reported to change their levels in opisthorchiasis, periductal fibrosis diagnosed by ultrasonography and CCA. Chemoprevention in experimental CCA tumorigenesis is discussed. These multiple risk biomarkers could now be explored for screening including chemopreventive intervention of subjects living in endemic areas where the prevalence of opisthorchiasis remains high., (© 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery.)

We investigated the cytokine/chemokine secretions and alteration of protein expression from peripheral blood mononuclear cells (PBMCs) cocultured with adult liver flukes (Opisthorchis viverrini) for 6 to 24 h. PBMC-derived proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the cytokines/chemokines in the supernatant were assessed using a cytokine array. Exposure to O. viverrini induced increases in secretion of proinflammatory cytokines, costimulating protein, adhesion molecules, and chemotactic chemokines relative to untreated controls. In contrast, secretion of the CD40 ligand, interleukin 16, and macrophage inflammatory protein 1β decreased. Proteomic analysis revealed that expression of 48 proteins was significantly altered in PBMCs stimulated with O. viverrini. Annexin A1 (ANXA1) was selected for further study, and immunoblotting showed upregulation of ANXA1 expression in PBMCs after 12 and 24 h coculture with liver flukes. In an in vivo study, transcription and translation of ANXA1 significantly increased in livers of hamsters infected with O. viverrini at 21 days and from 3 months onwards compared to normal controls. Interestingly, immunohistochemistry revealed that ANXA1 was present not only in the cytoplasm of inflammatory cells but also in the cytoplasm of cholangiocytes, which are in close contact with the parasite and its excretory/secretory products in the biliary system. Expression of ANXA1 increased with time concomitant with bile duct enlargement, bile duct formation, and epithelial cell proliferation. In conclusion, several cytokines/chemokines secreted by PBMCs and upregulation of ANXA1 in PBMCs and biliary epithelial cells might have a role in host defense against O. viverrini infection and tissue resolution of inflammation.

A proteomic-based approach was used to search for potential markers in the plasma of hamsters in which cholangiocarcinoma (CCA) was induced by Opisthorchis viverrini infection and N-nitrosodimethylamine treatment. The plasma proteins of CCA-induced hamsters were resolved by 1-D PAGE, digested by trypsin, and analyzed by LC-MS/MS. From the criteria of protein ID scores >15 and an overexpression of at least three times across all time points, 37 proteins were selected. These overexpressed proteins largely consisted of signal transduction, structural, transport, and transcriptional proteins in the order. Among the most frequently upregulated proteins, exostosin 1 (EXT1) was selected for further validation. By western blot analysis, the EXT1 expression level in the plasma of hamster CCA was significantly higher than that of controls at 1 month and thereafter. Immunohistochemistry revealed that EXT1 was expressed at vascular walls and fibroblasts at 21 days (before tumor onset) and at 2 months (early CCA) posttreatment. Its expression was also observed in bile duct cancer cells during tumor progression at 6 months posttreatment. In the human CCA tissue microarray, EXT1 immunoreactivity was found not only in vascular walls and fibroblasts but also in bile duct cancer cells and was positive in 89.7 % (61/68) of the cases. By ELISA and immunoblotting, plasma EXT1 level was significantly higher in human CCA compared to healthy controls. In conclusion, these results suggest that increased expression of EXT1 level in the plasma might be involved in CCA genesis and might be a potential biomarker of CCA.

Opisthorchis viverrini infection causes inflammation and liver injury leading to periductal fibrosis. Little is known about the pathological alterations in bile canaliculi in opisthorchiasis. This study aimed to investigate bile canalicular alterations in O. viverrini-infected hamsters and to examine the chemopreventive effects of curcumin on such changes. Hamsters were infected with O. viverrini and one group of animals was fed with 1% dietary curcumin supplement. Animals were examined during the acute infection phase, days 21 and 30 post-infection (PI) and chronic infection phase (day 90 PI). Scanning electron microscopy revealed that in the infected group fed with a normal diet, bile canaliculi became slightly tortuous by 30 day PI and more tortuous at day 90 PI. Transmission electron microscopy showed a reduction in microvilli density of canaliculi starting at day 30 PI, with a marked loss of microvilli at day 90 PI. These ultrastructral changes were slightly seen at day 21 PI, which was similar to that found in infected animals fed with 1% curcumin-supplemented diet. Notably, curcumin treatment prevented the reduction of microvilli density, reduced the dilation of bile canaliculi, and decreased the tortuosity of the bile canaliculi relative to non-infected animals on a normal diet at days 30 and 90 PI. These results suggest that curcumin reduces alteration of bile canaliculi and may be a promising agent to prevent the onset of bile duct abnormalities induced by O. viverrini infection.

To increase public health awareness for prevention of opisthorchiasis caused by eating raw freshwater fish, the distribution and abundance of Opisthorchis viverrini metacercariae (OV MC) was investigated in freshwater fish obtained from 20 provinces in northeastern Thailand between April 2011 and February 2012. A cross-sectional survey was conducted on 12,890 fish consisting of 13 species randomly caught from 26 rivers, 10 dams, and 38 ponds/lakes. Fish, were collected in each of the rainy and winter seasons from each province. Fish were identified, counted, weighed, and digested using pepsin-HCl. Samples were examined for OV MC by a sedimentation method, and metacercariae were identified under a stereomicroscope. OV MC were found in 6 species of fish; i.e., Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti, and Puntioplites proctozysron from localities in 13 provinces. Among the sites where OV MC-infected fish were found, 70.0% were dams, 23.7% were ponds/lakes, and 7.7% were rivers. The mean intensity of OV MC ranged from 0.01 to 6.5 cysts per fish (or 1.3-287.5 cysts per kg of fish). A high mean intensity of OV MC per fish (>3 cysts) was found in 5 provinces: Amnat Charoen (6.5 cysts), Nakhon Phanom (4.3), Mukdahan (4.1), Khon Kaen, (3.5) and Si Sa Ket (3.4). In conclusion, OV MC are prevalent in natural cyprinid fish, with the infection rate varying according to fish species and habitats.

The human liver fluke Opisthorchis viverrini infection and N-nitrosodimethylamine (NDMA) administration induce cholangiocarcinoma (CCA) and liver injury in hamsters. Melatonin protects against liver injury and reduces the alteration of mitochondrial structure, mitochondrial membrane potential, and mitochondrial pro- and anti-apoptotic pathways in various cancer types. To investigate the chemopreventive effect of melatonin on CCA genesis and liver injury, hamsters were treated with a combination of O. viverrini infection and NDMA concurrently administered with melatonin (10 mg/kg and 50 mg/kg) for 120 days. Melatonin treatment at 50 mg/kg caused a significant reduction in liver/body weight ratios and decreased tumor volumes leading to an increase in the survival of animals. In the tumorous tissues, the high-dose melatonin reduced DNA fragmentation and mitochondrial apoptosis by inducing anti-apoptotic protein (Bcl-2) in the mitochondrial fraction and down-regulating cytochrome c, pro-apoptotic protein (Bax), and caspase-3 in tumor cytosol. Moreover, a high-dose melatonin treatment significantly increased mitochondrial antioxidant enzymes and prevented mitochondrial ultrastructure changes in the tumor. Overall, melatonin has potent chemopreventive effects in inhibiting CCA genesis and also reduces liver injury in hamster CCA, which, in part, might involve in the suppression of CCA by reducing tumor mitochondria alteration., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The northeastern region of Thailand has long been known as an endemic area of the human liver fluke infection which is caused by Opisthorchis viverrini. Humans are infected by ingestion of uncooked cyprinoid fish in traditional dishes such as "koi-pla," "pla-som," "pla-jom," and "pla-ra." To date, the prevalence of this parasite infection remains high because of cultural behavior and local beliefs. The popular misunderstanding among people in this area is that alcohol, lemon juice, and fish sauce can kill the parasites. Thus, they believe that they can eat raw fish without the risk of infection. This study attempts to clarify the effects of ethyl alcohol and acidosis-alkalosis on O. viverrini metacercariae excystation. Metacercariae of O. viverrini were obtained from infected cyprinoid fish in a natural reservoir. Most metacercariae were obtained from small cyprinoid fish. Metacercariae were divided into three experimental groups and were treated with solutions containing four different concentrations of ethyl alcohol, four different concentrations of salt, and a range of acidic/basic pH. Metacercariae excystation was observed at the assigned times, and the data were then analyzed. Salt had no effect on excystation. Interestingly, the optimal conditions for O. viverrini excystation were pH 9 and 25 % ethyl alcohol. The present study suggests that raw fish should not be eaten while drinking alcohol or when consuming other ingredients with pH 9, because both alcohol and pH 9 could induce O. viverrini metacercariae excystation, leading to the early development of parasites in the hepatobiliary system.

Opisthorchis viverrini infection induces inflammation-mediated oxidative stress and liver injury, which may alter α-tocopherol and lipid metabolism. We investigated plasma α-tocopherol and lipid profiles in hamsters infected with O. viverrini. Levels of α-tocopherol, cholesterol, and low-density lipoprotein increased in the acute phase of infection. In the chronic phase, α-tocopherol decreased, while triglyceride and very low-density lipoprotein increased. Notably, high-density lipoprotein decreased both in the acute and chronic phases. In the liver, cholesteryl oleate, triolein, and oleic acid decreased in the acute phase, and increased in the chronic phase. Such chronological changes were negatively correlated with the plasma α-tocopherol level. The expression of α-tocopherol-related molecules, ATP-binding cassette transporter A1 (ABCA1) and α-tocopherol transfer protein, increased throughout the experiment. These results suggest that O. viverrini infection profoundly affects on lipid and α-tocopherol metabolism in due course of infection., (Crown Copyright © 2012. Published by Elsevier Ireland Ltd. All rights reserved.)

Among the various types of chronic helminthic infections, only schistosomiasis hematobium, opisthorchiasis, and clonorchiasis in a long-standing inflammation are found to be causative agents of cancer. Opisthorchiasis and Opisthorchis viverrini-associated bile duct cancer are prevalent among the residents in northeastern Thailand, who prefer consuming raw or improperly cooked cyprinid fish. Although numerous studies have reported on the presence of O. viverrini metacercariae in freshwater cyprinid fishes, none of the data has been derived directly from cyprinid fish dishes. Here, we have demonstrated the presence of viable metacercariae in a number of cyprinid fish dishes that are among the favorite foods of northeastern Thais. Traditional salt-fermented and grilled cyprinid fish were found to be safe to consume, but not raw fish salad or cyprinid fish fermented in sticky rice for 69 h. This information could be used as part of a rational and sustainable control program for O. viverrini-associated bile duct cancer.

Re-infection with liver fluke, Opisthorchis viverrini, increases proinflammatory molecules involved in inflammation-mediated disease and carcinogenesis in an animal model. To clarify whether these genes respond to parasite antigen in peripheral blood leukocytes (PBL) of opisthorchiasis patients, we examined the transcriptional level of oxidant-generating (toll-like receptor 2 (TLR-2), nuclear factor-kappa B (NF-KB), and cyclooxygenase 2 (COX-2)), anti-oxidant-generating (manganese superoxide dismutase 2 (SOD-2) and catalase (CAT)), proinflammatory cytokine (interleukin (IL)-1β), and anti-inflammatory cytokine (IL-10), in PBL exposed to parasite antigen in O. viverrini-infected patients compared with healthy individuals in an in vitro experiment. After O. viverrini antigen-treated PBL, quantitative RT-PCR analysis revealed that increased expression of cytokines and oxidant-generating genes in PBL was similar between O. viverrini-infected and healthy groups. Interestingly, compared with healthy subjects, increase of TLR-2, COX-2, and SOD-2 and decreased CAT mRNA expression levels were observed in O. viverrini-infected group. The results indicate that O. viverrini antigen induces upregulation of TLR-2, COX-2, and SOD-2 and downregulation of CAT genes in opisthorchiasis patients, suggesting that imbalance of oxidant/anti-oxidant transcripts during re-infection may be involved in the inflammatory-driven carcinogenesis. These molecules may be used as the chemopreventive target for intervention of opisthorchiasis patients in an endemic area.

Chronic inflammation induced by liver fluke (Opisthorchis viverrini) infection is the major risk factor for cholangiocarcinoma (CCA) in Northeastern Thailand. Increased levels of proinflammatory cytokines and nuclear factor kappa B that control cyclooxygenase-2 and inducible nitric oxide activities, disturb the homeostasis of oxidants/anti-oxidants and DNA repair enzymes, all of which appear to be involved in O. viverrini-associated inflammatory processes and CCA. Consequently oxidative and nitrative stress-related cellular damage occurs due to the over production of reactive oxygen and nitrogen species in inflamed target cells. This is supported by the detection of high levels of oxidized DNA and DNA bases modified by lipid peroxidation products in both animal and human tissues affected by O. viverrini-infection. Treatment of opisthorchiasis patients with praziquantel, an anti- trematode drug was shown to reduce inflammation-mediated tissue damage and carcinogenesis. The principal mechanisms that govern the effects of inflammation and immunity in liver fluke-associated cholangiocarcinogenesis are reviewed. The validity of inflammation-related biomolecules and DNA damage products to serve as predictive biomarkers for disease risk evaluation and intervention is discussed., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Opisthorchiasis caused by Opisthorchis viverrini infection induces hepatobiliary disease (HBD)-associated cholangiocarcinoma (CCA) via a chronic inflammatory immune response. Here, we evaluated specific IgG and IgA antibodies against different fractions of O. viverrini antigen in residents from an endemic community in Northeast Thailand with varying hepatobiliary abnormalities. Crude somatic O. viverrini antigen was purified into three fractions (viz., P1, P2 and P3) by gel infiltration chromatography and these served as antigens for detection of fluke-specific IgG and IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The results revealed fluke-specific IgG and IgA antibody levels-against these antigens from subjects with O. viverrini-positive HBD-higher than in subjects with O. viverrini-negative HBD. Interestingly, the rank of fluke-specific IgG (and not IgA) antibody levels against crude extract and P1 antigens was CCA>severe HBD>mild HBD>healthy individuals. Purified antigens reduced cross-reactivity with other parasites compared to the crude antigen. Multiple linear regression analysis showed that HBD status was significantly associated with the liver fluke-specific IgG antibody against purified antigens. These results suggest that purified O. viverrini-antigen improves serodiagnosis for the evaluation of opisthorchiasis-associated HBD, and may be useful in the screening of opisthorchiasis in subjects at risk of developing CCA., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Infection by the liver fluke (Opisthorchis viverrini) causes hepatobiliary disease and bile duct cancer (cholangiocarcinoma, CCA) in endemic areas in Southeast Asia. Measurements of humoral immune response particularly parasite-specific antibodies are useful not only for serodiagnosis but they have been implicated as risk factors of CCA. In this study, we used indirect Enzyme Immunosorbent Assay (ELISA) to measure O. viverrini-specific immunoglobulins in serum, urine and saliva and assessed efficacies in diagnosis of opisthorchiasis and evaluated the relationship of antibodies among clinical specimens in a sample population in endemic areas in Khon Kaen, Thailand. By employing the Receiver Operation Characteristics (ROC) analysis, diagnostic efficacy based upon the area under the curve (AUC) revealed that serum, salivary IgG and IgA performed better than urine for diagnosis of opisthorchiasis. Seropositive cases were found in both parasite egg-negative as well as O. viverrini egg-positive groups. The levels of serum IgG correlated with intensity of O. viverrini infection (P<0.05). Diagnostic sensitivities based on serum and salivary IgG, IgA also positively associated with the intensity of infection. Correlations between serum antibodies and those in saliva were found to be greater in egg-negative than egg-positive individuals for O. viverrini. Our findings indicated a complex interrelation between antibody responses in different clinical specimens triggered by liver fluke infection. More comprehensive examinations are needed to determine the potential utility of salivary antibody detection which, in combination with the conventional fecal examination method, may better assist in the identification of individuals with opisthorchiasis. Furthermore, it may provide a better indicator of the risk of disease, particularly CCA., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Host-parasite interaction during infection with the liver fluke Opisthorchis viverrini plays an important role in opisthorchiasis-associated cholangiocarcinoma via nitric oxide (NO) production. Host cells induce nitric oxide synthase (NOS)-dependent DNA damage and secrete Ras-related C3 botulinum toxin substrate (Rac)1, heme oxygenase (HO)-1, and gelatinases (matrix metalloproteinase (MMP)2 and MMP9). We evaluated whether these enzymes are expressed in O. viverrini. Colocalization of NOS and Rac1 was most prominently detected on day 30 post-infection (p.i.) in the gut, reproductive organ, eggs, acetabular and tegument. Expression of HO-1, an antioxidative enzyme, increased in a similar pattern to NOS, but was not present in the tegument. The levels of nitrate/nitrite, end products of NO, and ferric reducing antioxidant capacity, an indicator of antioxidant enzyme capacity, in parasite homogenates were highest on day 30 p.i. and then decreased on day 90 p.i. In contrast, zymography revealed that MMP2 and MMP9 were not present in parasite homogenates at all time points. In conclusion, O. viverrini induces NOS expression and NO production, but does not express gelatinases. The study may provide basic information and an insight into drug design for prevention and/or intervention approaches against O. viverrini infection., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Opisthorchiasis caused by Opisthorchis viverrini induces periductal fibrosis via host immune/inflammatory responses. Plasma protein alteration during host-parasite interaction-mediated inflammation may provide potential diagnostic and/or prognostic biomarkers. To search for target protein changes in O. viverrini-infected hamsters, a 1-D PAGE gel band was trypsin-digested and analyzed by a LC-MS/MS-based proteomics approach in the plasma profile of infected hamsters, and applied to humans. Sixty seven proteins were selected for further analysis based on at least two unique tryptic peptides with protein ID score >10 and increased expression at least two times across time points. These proteins have not been previously identified in O. viverrini-associated infection. Among those, proteins involved in structural (19%), immune response (13%), cell cycle (10%) and transcription (10%) were highly expressed. Western blots revealed an expression level of protein tyrosine phosphatase alpha (PTPα) which reached a peak at 1 month and subsequently tended to decrease. Fibronectin significantly increased at 1 month and tended to increase with time, supporting proteomic analysis. PTPα was expressed in the cytoplasm of inflammatory cells, while fibronectin was observed mainly in the cytoplasm of fibroblasts and the extracellular matrix at periductal fibrosis areas. In addition, these protein levels significantly increased in the plasma of O. viverrini-infected patients compared to healthy individuals, and significantly decreased at 2-months post-treatment, indicating their potential as disease markers. In conclusion, our results suggest that plasma PTPα and fibronectin may be associated with opisthorchiasis and the hamster model provides the basis for development of novel diagnostic markers in the future.

Opisthorchiasis-associated cholangiocarcinoma (CCA) is one of main public health problems in Opisthorchis viverrini endemic areas. Although the definite relationship between prevalence of CCA and the parasite infection has been demonstrated, the molecular mechanism of tumorigenesis is still unknown. In the present study, by using animal model of opisthorchiasis-associated CCA, a kinetic analysis of cDNA microarray was performed to screen the candidate genes that involve in the development of opisthorchiasis-associated CCA. Microarray analysis revealed that the expressions of 131 genes were up-regulated during the development of CCA, including the genes relative to cell proliferation, differentiation and transformation, cell growth and cycle regulation, apoptosis, DNA repair, and cytoskeletal structure. The expressions of 145 genes were down-regulated, including the genes relative to metabolic enzymes, tumor suppressor, apoptosis, and oxidative response and oxidation reduction. The present study listed up the candidate genes involving tumorigenesis, provided molecular information on the development of opisthorchiasis-associated CCA and the potential biomarkers for diagnosis and therapy, and suggested that the increased expression of cell differentiation, proliferation, transformation-related genes, and decreased expression of metabolic enzymes may play important roles in the tumorigenesis of CCA.

Opisthorchiasis is the major public health problem in the endemic areas of Thailand and Laos because Opisthorchis viverrini infection causes serious hepatobiliary diseases including CCA. The molecular mechanism of the CCA carcinogenesis induced by the infection remains obscure. To reveal the potential genes and signaling pathways to involve in the carcinogenesis, the present study investigated the expression of c-Ski, an oncogene, and two TGF-β signaling pathway relative genes, TGF-β and Smad4, during the development of CCA induced by O. viverrini infection in hamster model, and in human opisthorchiasis associated CCA. The results showed that the expression of c-Ski gene was greatly up-regulated during the carcinogenesis of CCA in hamster model. The overexpression of c-Ski was confirmed by immunohistological staining result which showed the increased expression of c-Ski protein in cytoplasm of the epithelial lining of hepatic bile ducts. Moreover, the immunohistological staining of the specimens of human opisthorchiasis associated CCA revealed the up-regulated expression of c-Ski and Smad4 proteins in the cytoplasm of the epithelial lining of hepatic bile ducts and stomal fibrosis respectively. The expression of TGF-β and Smad4 were up-regulated, which expression kinetics was time-dependent of CCA development. These results suggest that c-Ski is likely involved in the carcinogenesis of CCA induced by O. viverrini infection through regulating TGF-β signaling pathway.

Opisthorchiasis has the significant relationship with the high prevalence of cholangiocarcinoma (CCA; a bile duct cancer) in the endemic areas in Southeast Asia. To reveal the molecular mechanism of the tumorigenesis induced by Opisthorchis viverrini infection, the present study investigated the kinetic expression of RB pathway genes, including RB1, p16(INK4), cyclin D1, and CDK4, during the development of opisthorchiasis-associated CCA in hamster model. The results of quantitative real-time polymerase chain reaction indicated that the expressions of RB1 and p16(INK4) were down-regulated during the development of CCA induced by infection plus N-nitrosodimethylamine treatment in a time-dependent manner. On the other hand, the expressions of cyclin D1 and CDK4 were up-regulated. The expression kinetics was corresponding to the pathological progression of the opisthorchiasis-associated CCA, revealed by histopathological observation. Moreover, the analysis of the expression of these genes in human opisthorchiasis-associated CCA cases showed the decreased expression of RB1 and p16(INK4) in 50% and 82.7% cases and overexpression of cyclin D1 and CDK4 in half cases, respectively. The results suggested that RB pathway is likely involved in the tumorigenesis of opisthorchiasis-induced CCA and proposed the potential application of some of these genes as biomarkers in predispose and molecular therapy of the parasite-associated cancer.

Praziquantel has been used for the treatment of diseases caused by infection with various parasites. Praziquantel treatment in Opisthorchis viverrini-infected patients reduces the hepatobiliary fibrosis due to tissue resorption, however, some fibroses are irreversible. To clarify the effect of praziquantel treatment on hepatobiliary fibrosis, we examined the expression of matrix metalloproteinases (MMPs) in relation to fibrolysis on praziquantel-treated hamsters after O. viverrini acute infection (AI) for 21 days and chronic infection (CI) for 4 months. The reduction rate of hydroxyproline content in the livers of the AI group with 6-month praziquantel treatment (71%) was higher than that of corresponding praziquantel-treated CI group (13%), similar to the decrease in the thickness of peribiliary fibrosis. Hepatic mRNA levels of collagen I significantly decreased compared with untreated control after praziquantel treatment both in AI and CI groups. Expression of collagen III significantly decreased in the AI group but unchanged in the CI group. MMP-9 and MMP-13 (except 3 months in CI group) levels significantly decreased in both groups. Notably, expression of MMP-7 level increased at 1 and 6 months in both AI and CI groups, respectively. MMP-2 level did not change in both groups. Gelatinase activity of MMPs-2 and -9 was associated with their transcriptional levels. Tissue inhibitors of MMP (TIMP)-1 and TIMP-2 significantly decreased in the AI group, whereas TIMP-1 levels did not change and TIMP-2 level was significantly increased in CI group. Praziquantel treatment increased the expression of TNF-alpha in both groups, suggesting an association with MMP-7 expression. TGF-beta expression was significantly increased only in the CI group indicating its may involve in TIMPs expression. These results suggest that in animals with chronic O. viverrini infection, praziquantel treatment induces the expression of TGF-beta, and TIMPs and resultant inhibition of MMP activity, leading to slow resorption of hepatic fibrosis.

Praziquantel causes adverse effects after short-term treatment. To examine the mechanism of these effects, we studied the distribution of Opisthorchis viverrini antigens and the expression of inducible nitric oxide synthase (iNOS), nuclear factor-kappaB (NF-kappaB), and antioxidant enzymes in O. viverrini-infected hamsters during short-term praziquantel treatment. Praziquantel-induced dispersion of parasite antigens produced a recruitment of inflammatory cells. NF-kappaB and iNOS mRNA expression was significantly elevated and associated with their immunoreactivity in the bile duct epithelium and inflammatory cells. Plasma nitrate, end products of nitric oxide, and malondialdehyde level increased significantly. Expression of mRNA for antioxidant enzymes (superoxide dismutases, catalase, and glutathione peroxidase) also increased significantly, which suggests host defense against oxidative stress. These results suggest that short-term praziquantel treatment induces inflammation and resulting oxidative and nitrative stress through O. viverrini antigen release. Data in this study can be used as a basis to understand potential side effects of praziquantel treatment in humans.

Inflammation-mediated DNA damage triggered by Opisthorchis viverrini (OV) infection is a major risk factor of cholangiocarcinoma (CCA). We have recently reported that nitrative and oxidative DNA damage participates in CCA development caused by repeated infection with OV [Pinlaor et al., Carcinogenesis 2004; 25:1535-42]. Therefore, to clarify the preventive effect of the antihelminthic drug praziquantel against cholangiocarcinogenesis, we assessed the effect of this drug on nitrative and oxidative DNA damage, including the formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and the expression of inducible nitric oxide synthase (iNOS) by immunohistochemistry in OV-infected hamsters. We also examined the expression of nuclear factor-kappaB (NF-kappaB), which functions as a tumor promoter in inflammation-associated cancer. Our results showed that although 1-week treatment with praziquantel did not kill parasites completely in hamsters on days 14 and 30, this drug dramatically reduced inflammatory cell infiltration. Double immunofluorescence staining showed that drug treatment almost completely diminished OV-induced 8-nitroguanine and 8-oxodG formation in bile duct epithelial cells. Quantitative analysis using an electrochemical detector coupled to HPLC revealed that 8-oxodG level in the liver of OV-infected hamsters was significantly decreased by drug treatment (p<0.05). Western blotting and immunohistochemistry revealed that the expression of NF-kappaB and iNOS in bile duct epithelium was reduced by drug treatment. The amount of nitrate plus nitrite in the liver and plasma was significantly decreased after drug treatment. It is concluded that praziquantel can exhibit a preventive effect against OV-induced cholangiocarcinoma by inhibiting iNOS-dependent DNA damage through not only elimination of parasites but also a potential antiinflammatory effect., (Copyright 2006 Wiley-Liss, Inc.)

We assessed the frequency and distribution of infection with opportunistic and non-opportunistic intestinal parasites and the liver fluke, Opisthorchis viverrini, in HIV-seropositive and HIV-seronegative subjects. Age- and sex-matched HIV-seropositive (n = 78) and HIV-seronegative patients (n = 78) from two hospitals in Khon Kaen Province, Thailand, participated in this study from November 1998 to August 2000. These subjects were divided according to the presence of diarrhea and CD4 counts. A single stool sample was obtained and analyzed by using specific techniques. Opisthorchis viverrini, was the most common parasite (19.2%) in each group. The prevalence rates of Cryptosporidium spp (11.5%) and Strongyloides stercoralis (17.9%) in the HIV-seropositive group were significantly (p < 0.05) higher than those in the HIV-seronegative group (1.0% for Cryptosporidium spp and 7.7% for S. stercoralis infections). The prevalences of these two parasites were 28% for Cryptosporidium spp and 20% for S. stercoralis in HIV-seropositives with diarrhea and CD4 counts lower than 100 cells/mm3, and were higher compared with patients without diarrhea or with high CD4 counts. These results suggest that infection with these parasites increases during HIV infection. The epidemiological distribution of Cryptosporidium and S. stercoralis may have implications for AIDS-related diseases.

Opisthorchis viverrini infection induces inflammation in and around the bile duct, leading to cholangiocarcinoma in humans. To examine the mechanism of O. viverrini-induced inflammatory response, we assessed the expression of Toll-like receptors (TLRs) in RAW 264.7 macrophage cell line treated with an extract of O. viverrini antigen. Flow cytometry and immunocytochemistry showed that O. viverrini antigen induced the expression of TLR2 but not TLR4. Western blotting and immunocytochemistry revealed that nuclear factor-kappaB (NF-kappaB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were expressed in RAW 264.7 cells treated with O. viverrini antigen in a dose-dependent manner. These results suggest that O. viverrini induces inflammatory response through TLR2-mediated pathway leading to NF-kappaB-mediated expression of iNOS and COX-2.

We investigated pathological changes, antibody response, and liver enzymes in hamsters re-infected with Opisthorchis viverrini. Group 1 received a single dose of 50 metacercariae; Groups 2 and 3 were first dosed with of 30 metacercariae and re-infected with 20 more once or twice at three month intervals. Inflammation and liver cell necrosis were observed on 3D (day 3) for Group 3 and 7D for Group 2 in comparison with 21D for Group 1. Pathological changes included peri-ductal fibrosis, bile duct dilation, and small bile duct formation. Increased O. viverrini-specific IgG levels ranked in the order Group 3>Group 2>Group 1. Liver enzyme activity was related to inflammatory cell infiltration. Re-infection induced faster inflammation and more severe pathological changes in association with parasite-specific antibody during chronic inflammation. This study emphasizes that there is an important relationship between the gradual decreases of inflammation with a concomitant increase in fibrosis after re-infection.

Inflammation mediated by infection is an important factor causing carcinogenesis. Opisthorchis viverrini (OV) infection is a risk factor of cholangiocarcinoma (CHCA), probably through chronic inflammation. Formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) were assessed in the liver of hamsters infected with OV. We newly produced specific anti-8-nitroguanine antibody without cross-reaction. Double immunofluorescence staining revealed that 8-oxodG and 8-nitroguanine were formed mainly in the same inflammatory cells and epithelium of bile ducts from day 7 and showed the strongest immunoreactivity on days 21 and 30, respectively. It is noteworthy that 8-oxodG and 8-nitroguanine still remained in epithelium of bile ducts on day 180, although amount of alanine aminotransferase activity returned to normal level. A time course of 8-nitroguanine was associated with iNOS expression. Furthermore, this study demonstrated that HO-1 expression and subsequent iron accumulation may be involved in enhancement of oxidative DNA damage in epithelium of small bile ducts. In conclusion, nitrative and oxidative DNA damage via iNOS expression in hamsters infected with OV may participate in CHCA carcinogenesis.

Chronic inflammation induced by repeated infection with Opisthorchis viverrini has been postulated to be a risk factor for cholangiocarcinoma. To clarify the mechanism of carcinogenesis induced by repeated O.viverrini infection, we investigated the timecourse of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, inducible nitric oxide synthase (iNOS) expression, nitric oxide production and pathological features in hamsters with two (2-IF) or three (3-IF) O.viverrini infections. Inflammatory cell infiltration triggered by repeated infection (3-IF > 2-IF > 1-IF) was earlier than by single infection (1-IF). HPLC coupled with an electrochemical detector revealed that 8-oxodG level in the liver was the highest on day 3 in 3-IF and day 7 in 2-IF, earlier than that on day 21 in 1-IF. Notably, a double immunofluorescence study revealed that formation of 8-nitroguanine and 8-oxodG appeared to increase in the epithelium of bile ducts in the order 3-IF > 2-IF > 1-IF after the decrease in inflammatory cells. This may be explained by the fact that repeated infection increased iNOS expression in the epithelium of bile ducts in the order 3-IF > 2-IF > 1-IF on day 90. Proliferating cell nuclear antigen accumulated in the epithelium of bile ducts on day 90 after repeated O.viverrini infection, supporting the hypothesis that cell proliferation was promoted by inflammation-mediated DNA damage. In conclusion, more frequent O.viverrini infection can induce the expression of iNOS not only in inflammatory cells but also in the epithelium of bile ducts and subsequently cause nitrosative and oxidative damage to nucleic acids, which may participate in the initiation and/or promotion steps of cholangiocarcinoma development.

Complications arising from infection with the carcinogenic liver fluke Opisthorchis viverrini cause substantial morbidity and mortality in Thailand and adjacent lower Mekong countries. In parallel, the incidence rate of diabetes mellitus (DM) is increasing in this same region, and indeed worldwide. Many residents in opisthorchiasis-endemic regions also exhibit DM, but the hepatobiliary disease arising during the co-occurrence of these two conditions remains to be characterized. Here, the histopathological profile during co-occurrence of opisthorchiasis and DM was investigated in a rodent model of human opisthorchiasis in which diabetes was induced with streptozotocin. The effects of excretory/secretory products from the liver fluke, O. viverrini (OVES) on hepatocyte and cholangiocyte responses during hyperglycemic conditions also were monitored. Both the liver fluke-infected hamsters (OV group) and hamsters with DM lost weight compared to control hamsters. Weight loss was even more marked in the hamsters with both opisthorchiasis and DM (OD group). Hypertrophy of hepatocytes, altered biliary canaliculi, and biliary hyperplasia were more prominent in the OD group, compared with OV and DM groups. Profound oxidative DNA damage, evidenced by 8-oxo-2'-deoxyguanosine, proliferating cell nuclear antigen, and periductal fibrosis characterized the OD compared to OV and DM hamsters. Upregulation of expression of cytokines in response to infection and impairment of the pathway for insulin receptor substrate (IRS)/phosphatidylinositol-3-kinases (PI3K)/protein kinase B (AKT) signaling attended these changes. In vitro, OVES and glucose provoked time- and dose-dependent effects on the proliferation of both hepatocytes and cholangiocytes. In overview, the co-occurrence of opisthorchiasis and diabetes exacerbated pathophysiological damage to the hepatobiliary tract. We speculate that opisthorchiasis and diabetes together aggravate hepatobiliary pathogenesis through an IRS/PI3K/AKT-independent pathway. [ABSTRACT FROM AUTHOR]