The crosstalk between cholangiocytes and hepatic stellate cells promotes the progression of epithelial-mesenchymal transition and periductal fibrosis during Clonorchis sinensis infection.

ABSTRACT: BACKGROUND: Clonorchis sinensis infection is one of the risk factors that provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis and even cholangiocarcinoma (CCA). Disrupted or aberrant intercellular communication among liver-constituting cells leads to pathological states that cause various hepatic diseases. This study was designed to investigate the pathological changes caused by C. sinensis excretory-secretory products (ESPs) in non-cancerous human cell lines (cholangiocytes [H69 cell line] and human hepatic stellate cells [LX2 cell line]) and their intercellular crosstalk, as well the pathological changes in infected mouse liver tissues. METHODS: The cells were treated with ESPs, following which transforming growth factor beta 1 (TGF-ß1) and interleukin-6 (IL-6) secretion levels and epithelial-mesenchymal transition (EMT)- and fibrosis-related protein expression were measured. The ESP-mediated cellular motility (migration/invasion) between two cells was assessed using the Transwell and three-dimensional microfluidic assay models. The livers of C. sinensis-infected mice were stained using EMT and fibrotic marker proteins. RESULTS: Treatment of cells with ESPs increased TGF-ß1 and IL-6 secretion and the expression of EMT- and fibrosis-related proteins. The ESP-mediated mutual cell interaction further affected the cytokine secretion and protein expression levels and promoted cellular motility. N-cadherin overexpression and collagen fiber deposition were observed in the livers of C. sinensis-infected mice. CONCLUSIONS: These findings suggest that EMT and biliary fibrosis occur through intercellular communication between cholangiocytes and hepatic stellate cells during C. sinensis infection, promoting malignant transformation and advanced hepatobiliary abnormalities.

Elucidation of pericholangitis and periductal fibrosis in cholestatic liver diseases via extracellular vesicles released by polarized biliary epithelial cells.

Cholestatic liver diseases causes inflammation and fibrosis around bile ducts. However, the pathological mechanism has not been elucidated. Extracellular vesicles (EVs) are released from both the basolateral and apical sides of polarized biliary epithelial cells. We aimed to investigate the possibility that EVs released from the basolateral sides of biliary epithelial cells by bile acid stimulation induce inflammatory cells and fibrosis around bile ducts, and they may be involved in the pathogenesis of cholestatic liver disease. Human biliary epithelial cells (H69) were grown on cell culture inserts and stimulated with chenodeoxycholic acid + IFN-γ. Human THP-1-derived M1-macrophages, LX-2 cells, and KMST-6 cells were treated with the extracted basolateral EVs, and inflammatory cytokines and fibrosis markers were detected by RT-PCR. Highly expressed proteins from stimulated EVs were identified, and M1-macrophages, LX-2, KMST-6 were treated with these recombinant proteins. Stimulated EVs increased the expression of TNF, IL-1ß, and IL-6 in M1-macrophages, TGF-ß in LX-2 and KMST-6 compared with the corresponding expression levels in unstimulated EVs. Nucleophosmin, nucleolin, and midkine levels were increased in EVs from stimulated cells compared with protein expression in EVs from unstimulated cells. Leukocyte cell-derived chemotaxin-2 (LECT2) is highly expressed only in EVs from stimulated cells. Stimulation of M1-macrophages with recombinant nucleophosmin, nucleolin, and midkine significantly increased the expression of inflammatory cytokines. Stimulation of LX-2 and KMST-6 with recombinant LECT2 significantly increased the expression of fibrotic markers. These results suggest that basolateral EVs are related to the development of pericholangitis and periductal fibrosis in cholestatic liver diseases.NEW & NOTEWORTHY Our research elucidated that the composition of basolateral EVs from the biliary epithelial cells changed under bile acid exposure and the basolateral EVs contained the novel inflammation-inducing proteins NPM, NCL, and MK and the fibrosis-inducing protein LECT2. We report that these new results are possible to lead to the potential therapeutic target of cholestatic liver diseases in the future.

Association of Cholangiocarcinoma among People with Periductal Fibrosis.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly fatal tumor, and the most favorable chance for long-term survival lies in curative resection. Periductal fibrosis (PDF), a precancerous condition associated with chronic inflammation of the bile ducts, can serve as a screening marker for CCA using hepatobiliary ultrasonography (US). However, limited studies have explored the relationship between PDF and CCA. This study aimed to investigate the association between PDF and CCA in a population at risk in Northeast Thailand. METHODS: The study included participants enrolled in the Cholangiocarcinoma Screening and Care Program (CASCAP) between 2013 and 2021 who underwent US. Histological evaluations were conducted following the standard protocol of the tertiary hospital at Khon Kaen University, Thailand. PDF was defined as the presence of fibrosis in the peripheral (PDF1), segmental (PDF2), or main bile duct (PDF3), diagnosed by well-trained general practitioners or radiologists. The association between PDF and CCA was assessed using multiple logistic regression, calculating adjusted odds ratios (AORs) and 95% confidence intervals (CIs). RESULTS: Out of 751,061 participants, the overall prevalence of PDF was 115,267 (15.35%), with an overall CCA rate of 0.11%. The rates of CCA were 0.1%, 0.15%, and 0.27% in participants with PDF1, PDF2, and PDF3, respectively. After adjusting for gender, age at enrollment, education levels, history of O. viverrini infection, smoking, and alcohol consumption, the AORs for CCA were 0.94 (95% CI: 0.74 - 1.20), 1.4 (95% CI: 1.03 - 1.91), and 2.52 (95% CI: 1.38 - 4.58) for participants with PDF1, PDF2, and PDF3, respectively. CONCLUSION: Our findings demonstrate a significant association between fibrosis of the segmental and main bile ducts (PDF2 and PDF3) and CCA, with the strongest association observed in participants with PDF3. Hepatobiliary US screening could serve as a valuable tool for early detection of CCA, enabling timely curative treatment.

Elevated circulating TLR4+ monocytes in patients with liver fluke Opisthorchis viverrini is associated with advanced periductal fibrosis.

INTRODUCTION: Opisthorchis viverrini (Ov) infection can lead to several disease manifestations of the bile duct including advanced periductal fibrosis (APF) and the most severe complication, cholangiocarcinoma (CCA). Monocytes migrate to the infection site and differentiate into tissue macrophages to express and release molecules such as cytokines, reactive oxygen species, and growth factors. TLR4+ monocytes are classified as having a pro-tumor phenotype and secrete tumor-promoting factors. The aim of this study is to investigate the role of monocytes in the pathogenesis of opisthorchiasis. METHODOLOGY: We used flow cytometry to measure the number of TLR4+ monocytes in the circulating blood of Ov infected patients with or without APF compared to healthy, non-Ov-infected controls. RESULTS: We found, for the first time, that patients with AFP have elevated numbers of circulating TLR4+ monocytes when compared to patients without fibrosis and healthy individuals. Intriguingly, when we measured ROS from these monocytes, we found increased ROS production in patients with APF. CONCLUSIONS: We propose that excessive production of ROS from these TLR4+ monocytes may lead to excessive injury of surrounding tissue and hence contribute to the pathological processes that lead to the development of advanced periductal fibrosis.

Opisthorchis viverrini-Current Understanding of the Neglected Hepatobiliary Parasite.

Opisthorchiasis due to Opisthorchis viverrini infection continues to be a significant public healthcare concern in various subregions of Southeast Asia, particularly in Thailand, Laos, Cambodia, Myanmar, and Vietnam. The main mode of transmission is via consumption of raw or undercooked fish, which is deeply embedded in the culture and tradition of the people living near the Mekong River. After ingestion, the flukes migrate to the bile ducts, potentially causing many hepatobiliary complications, including cholangitis, cholecystitis, cholelithiasis, advanced periductal fibrosis and cholangiocarcinoma. Several mechanisms of opisthorchiasis-associated cholangiocarcinogenesis have been proposed and elucidated in the past decade, providing insight and potential drug targets to prevent the development of the sinister complication. The gold standard for diagnosing opisthorchiasis is still via stool microscopy, but the advent of novel serological, antigen, and molecular tests shows promise as more convenient, alternative diagnostic methods. The mainstay of treatment of opisthorchiasis is praziquantel, while treatment of opisthorchiasis-associated cholangiocarcinoma depends on its anatomic subtype and resectability. Thus far, the most successful fluke control programme is the Lawa model based in Thailand, which raised awareness, incorporated education, and frequent surveillance of intermediate hosts to reduce transmission of opisthorchiasis. Development of vaccines using tetraspanins shows promise and is currently ongoing.

1H NMR fecal metabolic phenotyping of periductal fibrosis- and cholangiocarcinoma-specific metabotypes defining perturbation in gut microbial-host co-metabolism.

Background: The liver fluke Opisthorchis viverrini (OV), which subsequently inhabits the biliary system and results in periductal fibrosis (PDF), is one of the primarily causes of cholangiocarcinoma (CCA), a bile duct cancer with an exceptionally high incidence in the northeast of Thailand and other Greater Mekong Subregion (GMS) countries. Insights in fecal metabolic changes associated with PDF and CCA are required for further molecular research related to gut health and potential diagnostic biological marker development. Methods: In this study, nuclear magnetic resonance (NMR) metabolomics was applied for fecal metabolic phenotyping from 55 fecal water samples across different study groups including normal bile duct, PDF and CCA groups. Results: By using NMR spectroscopy-based metabolomics, fecal metabolic profiles of patients with CCA or PDF and of individuals with normal bile duct have been established with a total of 40 identified metabolites. Further multivariate statistical analysis and hierarchical clustering heat map have demonstrated the PDF- and CCA-specific metabotypes through various altered metabolite groups including amino acids, alcohols, amines, anaerobic glycolytic metabolites, fatty acids, microbial metabolites, sugar, TCA cycle intermediates, tryptophan catabolism substrates, and pyrimidine metabolites. Compared to the normal bile duct group, PDF individuals showed the significantly elevated relative concentrations of fecal ethanol, glycine, tyrosine, and N-acetylglucosamine whereas CCA patients exhibited the remarkable fecal metabolic changes that can be evident through the increased relative concentrations of fecal uracil, succinate, and 5-aminopentanoate. The prominent fecal metabolic alterations between CCA and PDF were displayed by the reduction of relative concentration of methanol observed in CCA. The metabolic alterations associated with PDF and CCA progression have been proposed with the involvement of various metabolic pathways including TCA cycle, ethanol biogenesis, hexamine pathway, methanol biogenesis, pyrimidine metabolism, and lysine metabolism. Among them, ethanol, methanol, and lysine metabolism strongly reflect the association of gut-microbial host metabolic crosstalk in PDF and/or CCA patients. Conclusion: The PDF- and CCA-associated metabotypes have been investigated displaying their distinct fecal metabolic patterns compared to that of normal bile duct group. Our study also demonstrated that the perturbation in co-metabolism of host and gut bacteria has been involved from the early step since OV infection to CCA tumorigenesis.

Incidental follicular cholecystitis with periductal fibrosis on liver biopsy: Rare findings.

INTRODUCTION AND IMPORTANCE: Follicular cholecystitis (FC) is a rare entity found, it is found in 0.1-1 % of patients with chronic cholecystitis. 1,2 This pathologic finding has been associated with extrahepatic biliary obstruction distal to the gallbladder, such as primary sclerosing cholangitis, choledocholithiasis, and distal biliary strictures. CASE PRESENTATION: Our patient is a 32-year-old female with a past medical history significant for obesity presented with symptoms of postprandial nausea and spasmodic abdominal pain. An abdominal ultrasound was performed with findings adenomyosis and possible gallbladder polyps or adherent stones. The patient was referred to surgery and a routine laparoscopic cholecystectomy with liver biopsy was performed. On pathology, the gallbladder was found to have chronic, active follicular cholecystitis with cholelithiasis. Percutaneous needle liver biopsy revealed the following: focal, mild periductal fibrosis, mild portal fibrosis with minimal mixed micro- and macrovesicular steatosis, and no significant steatohepatitis. CLINICAL DISCUSSION: To the best of our knowledge, this is the first documented case of follicular cholecystitis with associated hepatic findings on pathology. Follicular cholecystitis is strongly associated with extrahepatic biliary obstruction distal to the gallbladder, but it has not been previously associated with liver fibrosis. We hope to bring awareness to this rare but significant pathology. CONCLUSION: Our case is unusual due to the findings of hepatic periductal fibrosis with follicular cholecystitis. Follicular cholecystitis is strongly associated with extrahepatic biliary obstruction distal to the gallbladder but it has not been documented it to be associated with any hepatic findings or pathology.

Persistent advanced periductal fibrosis is associated with cagA-positive Helicobacter pylori infection in post-praziquantel treatment of opisthorchiasis.

BACKGROUND: Liver fluke infection caused by Opisthorchis viverrini is associated with several hepatobiliary diseases including advanced periductal fibrosis (APF) and cholangiocarcinoma. Recently, we demonstrated a persistent APF in over one-third of opisthorchiasis patients after worm removal by praziquantel (PZQ) treatment. However, the underlying mechanism(s) of this phenomena is unclear. Given a co-infection with Helicobacter pylori (H. pylori) especially cagA-positive strain enhances APF, we hypothesized that H. pylori with CagA virulent factor contributes to persistent APF. MATERIALS AND METHODS: Seventy-five opisthorchiasis patients who underwent ultrasonography and treatment with PZQ were recruited in the 2-year follow-up study. Helicobacter and its cagA in the feces were examined by conventional and qPCR. Correlations between prevalence or bacterial loads of Helicobacter spp., H. pylori, and cagA-positive H. pylori before and after PZQ treatment were analyzed among resolved, slowly resolved, relapsed, and persistent APF groups. RESULTS: Overall, prevalence of Helicobacter spp., H. pylori, and cagA-positive H. pylori declined after PZQ treatment. However, only the prevalence and bacterial loads of cagA-positive H. pylori detected at 2-year post-treatment were significantly lower than those before treatment (p < .05). In addition, both prevalence and bacterial loads of cagA-positive H. pylori were significantly lower in the resolved APF group after PZQ treatment, while there were no significant changes in the slowly resolved, relapsed, and persistent APF groups. Among the APF subgroups, cagA-positive H. pylori prevalence in both relapsed and persistent APF groups were significantly higher than the resolved APF group. CONCLUSION: The results support our hypothesis that H. pylori, especially cagA-positive strain, contributes to the relapsed and persistent APF. A supplementary antibiotic treatment for H. pylori to reduce persistent APF and eventually CCA is warranted.

The Overactivation of NADPH Oxidase during Clonorchis sinensis Infection and the Exposure to N-Nitroso Compounds Promote Periductal Fibrosis.

Clonorchis sinensis, a high-risk pathogenic human liver fluke, provokes various hepatobiliary complications, including epithelial hyperplasia, inflammation, periductal fibrosis, and even cholangiocarcinogenesis via direct contact with worms and their excretory-secretory products (ESPs). These pathological changes are strongly associated with persistent increases in free radical accumulation, leading to oxidative stress-mediated lesions. The present study investigated C. sinensis infection- and/or carcinogen N-nitrosodimethylamine (NDMA)-associated fibrosis in cell culture and animal models. The treatment of human cholangiocytes (H69 cells) with ESPs or/and NDMA increased reactive oxidative species (ROS) generation via the activation of NADPH oxidase (NOX), resulting in augmented expression of fibrosis-related proteins. These increased expressions were markedly attenuated by preincubation with a NOX inhibitor (diphenyleneiodonium chloride) or an antioxidant (N-acetylcysteine), indicating the involvement of excessive NOX-dependent ROS formation in periductal fibrosis. The immunoreactive NOX subunits, p47phox and p67phox, were observed in the livers of mice infected with C. sinensis and both infection plus NDMA, concomitant with collagen deposition and immunoreactive fibronectin elevation. Staining intensities are proportional to lesion severity and infection duration or/and NDMA administration. Thus, excessive ROS formation via NOX overactivation is a detrimental factor for fibrogenesis during liver fluke infection and exposure to N-nitroso compounds.

Discovery of Serotransferrin Glycoforms: Novel Markers for Diagnosis of Liver Periductal Fibrosis and Prediction of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) caused by chronic liver fluke infection is a major public health problem in Northeast Thailand. Identification of CCA risk groups is urgently needed for the control of CCA in this region. Periductal fibrosis (PDF) induced by chronic inflammation of bile ducts is known as a pre-neoplastic lesion of CCA. We aimed to identify the serum CCA and PDF biomarkers using mass spectrometry (UPLC-ESI-QqQ) with multiple reaction mode (MRM) analysis. Here, serum levels of serotransferrin glycoforms at the glycopeptide level were measured in the sera of CCA (n = 100), PDF (n = 50), and healthy control (n = 100) subjects. The results indicated that serotransferrin peptide levels were generally the same between the control and PDF groups, whereas CCA patients had reduced levels. Moreover, 56 serotransferrin glycoforms were detected, with nine increased in CCA compared to control subjects. Among them, the serum levels of four glycoforms were increased in PDF and CCA patients compared to control subjects. In particular, highly sialylated multi-branched glycans of serotransferrin serum were significantly correlated with poor prognosis and tumor stage in CCA patients. Taken together, these glycoforms could be used as risk biomarkers and prognosis and diagnosis markers of CCA.

Association between periductal fibrosis and bile duct dilatation among a population at high risk of cholangiocarcinoma: a cross-sectional study of cholangiocarcinoma screening in Northeast Thailand.

OBJECTIVES: To assess associations between periductal fibrosis (PDF) and bile duct dilatation (BDD) in ultrasonography (US) screening of population at risk of cholangiocarcinoma (CCA) due to residence in an endemic area for Opisthorchis viverrini. CCA survival rates are low, and early identification of risk factors is essential. BDD is one symptom that can identify patients at risk of CCA. Detection of PDF by US can also identify at-risk patients, at an earlier stage of CCA development. Identification of association between PDF and BDD will inform screening practices for CCA risk, by increasing the viability of PDF screening for CCA risk. SETTING: Nine tertiary care hospitals in Northeast Thailand. DESIGN: Cross-sectional study. PARTICIPANTS: Study subjects in the Cholangiocarcinoma Screening and Care Program (CASCAP) in Northeast Thailand. CASCAP inclusion criteria are all residents of Northeast Thailand aged ≥40 years. Participants are recruited through CCA screening centres and through primary healthcare units. So far, 394 026 have been enrolled. METHODS: PDF and BDD were identified through US. PDF was categorised into three groups, PDF1, 2 and 3, depending on their high echo locality in the peripheral, segmental and main bile duct, respectively. Associations between PDF and BDD were determined by adjusted OR and 95% CI using multiple logistic regression. RESULTS: BDD was found in 6.6% of PDF3, 1.7% of PDF2 and 1.4% of PDF1 cases. Among PDF cases, especially in PDF3, BDD was found in men more than in women (8.9% and 4.6%, respectively). Compared with non-PDF, the association between PDF3 and BDD was highly significant (adjusted OR=5.74, 95% CI 4.57 to 7.21, p<0.001). CONCLUSIONS: Our findings reveal that there is a relationship between PDF and BDD, which is associated with CCA. Therefore, PDF can also be an indicator for suspected CCA diagnosis through US.

Evaluation of a short term effect of praziquantel treatment in opisthorchiasis-induced hepatobiliary inflammation by urinary 8-oxodG.

Inflammation of the hepatobiliary system in chronic opisthorchiasis is associated with an elevated level of urinary 8-oxo-7,8 dihydro-2'deoxyguanosine (8-oxodG) during active as well as past exposure to Opisthorchis viverrini infection. In this study, we evaluated the short-term effect of praziquantel treatment on hepatobiliary disease (HBD) using urinary 8-oxodG as an inflammatory marker in a cohort of residents in endemic areas of opisthorchiasis in Khon Kaen, Thailand. The HBD status in terms of periductal fibrosis (PDF) was determined by abdominal ultrasonography and O. viverrini infection was monitored at baseline and 2-4 weeks after curative treatment by praziquantel. Analysis of O. viverrini-infected participants who were PDF-ve revealed that there was a significant reduction of urinary 8-oxodG after treatment compared with the baseline levels (p < 0.001). By contrast, in PDF+ve individuals, the levels of urinary 8-oxodG were similar between baseline and those post-treatment. Although confirmation by using a larger sample size is needed, the positive association between HBD and urinary 8-oxodG level after worm clearance suggests that chronic hepatobiliary inflammation is neither affected nor interrupted by short-term praziquantel treatment. Individuals with persistent PDF at pre- and post-treatment who have a high risk of cholangiocarcinoma, could be identified within 2-4 weeks after parasite removal by drug treatment. Thus, urinary 8-oxodG is a useful biomarker for predicting persistent PDF in individuals with a recent drug treatment history who require further clinical investigation, management and treatment.

Characterisation of the Urinary Metabolic Profile of Liver Fluke-Associated Cholangiocarcinoma.

BACKGROUND: Human infection with Opisthorchis viverrini, a carcinogenic liver fluke inhabiting the biliary tree, is endemic in Southeast Asia. Chronic infection is associated with a fatal complication, cholangiocarcinoma (CCA), a late-presenting and aggressive malignancy. Currently, annual mortality rates from CCA mirror trends in incidence, due in part to limited availability of efficient prognostic and early diagnostic biomarkers. With ability to detect thousands of urinary metabolites using metabonomics, the urine metabolome holds great potential in providing an insight into system-level alterations in carcinogenesis and in identifying metabolic markers altered in response to disturbed homoeostasis. METHODS: Global molecular profiling using reversed-phase ultraperformance liquid chromatography mass spectrometry was utilised to acquire the urinary spectral profile of 137 Thai subjects (48 at high risk of infection, 41 with O. viverrini infection, 34 periportal fibrosis and 14 CCA) from Khon Kaen, Thailand. RESULTS: Multivariate statistical analysis identified perturbation in several molecular classes related to purine metabolism and lipid metabolism in the CCA urine metabolome. These markers mainly reflect changes in energy metabolism to support proliferation (increased fatty acid oxidation and purine recycling), DNA methylation and hepatic injury. CONCLUSIONS: Several metabolites of biological interest were discovered from this proof-of-principle dataset. Augmenting these findings is essential to accelerate the development of urinary metabolic markers in CCA.

Subsets of Inflammatory Cytokine Gene Polymorphisms are Associated with Risk of Carcinogenic Liver Fluke Opisthorchis viverrini-Associated Advanced Periductal Fibrosis and Cholangiocarcinoma.

Opisthorchis viverrini infection induces chronic inflammation, and a minor proportion of infected individuals develop advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA). Inflammatory cytokines and/or their gene polymorphisms may link to these biliary pathologies. We therefore investigated associations among cytokine gene polymorphisms and cytokine production in 510 Thai cases infected with O. viverrini who presented with APF+ or APF-, as established by abdominal ultrasonography as well as in patients diagnosed with CCA. Levels of pro-inflammatory and anti-inflammatory cytokines were determined in culture supernatants after stimulation of peripheral blood mononuclear cells (PBMCs) with O. viverrini excretory-secretory (ES) products. Pro-inflammatory cytokines, IL-1ß, IL-6, IFN-γ, LT-α, and TNF-α were significantly increased in CCA patients compared with non-CCA (APF- and APF+) cases. Polymorphisms in genes encoding IL-1ß-511C/T, IL-6-174G/C, IFN-γ +874T/A, LT-α +252A/G, and TNF-α -308G/A were then investigated by using PCR-RFLP or allele specific-PCR (AS-PCR) analyses. In the CCA cases, LT-α +252A/G and TNF-α -308G/A heterozygous and homozygous variants showed significantly higher levels of these cytokines than the wild type. By contrast, levels of cytokines in wild type of IFN-γ +874T/A were significantly higher than the variants in CCA cases. IFN-γ +874T/A polymorphisms were associated with advanced periductal fibrosis, whereas IL-6 -174G/C polymorphisms were associated with CCA. To our knowledge, these findings provide the first demonstration that O. viverrini infected individuals carrying several specific cytokine gene polymorphisms are susceptible to develop fibrosis and CCA.

Coinfection with Helicobacter pylori and Opisthorchis viverrini Enhances the Severity of Hepatobiliary Abnormalities in Hamsters.

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.

Urinary microRNA-192 and microRNA-21 as potential indicators for liver fluke-associated cholangiocarcinoma risk group.

Opisthorchis viverrini infection induces chronic inflammation in the bile ducts, leading to periductal fibrosis (PDF), which possibly associates to cholangiocarcinoma (CCA). Patients with CCA have a poor prognosis, which is linked to asymptomatic disease and late diagnosis. Hence, detecting early stage CCA is essential. Secretory miRNAs have been promoted as biomarkers for pathological changes associated with parasitic infections, fibrosis and/or cancer. We aimed to determine levels of miR-192 and miR-21 in the urine of O. viverrini infected, periductal fibrosis (PDF) and CCA groups using qRT-PCR. We found that miR-192 was significantly higher in O. viverrini infected, PDF and also CCA groups (p<0.05) than in healthy controls. By utilizing the Receiver Operation Characteristics (ROC) analysis, miR-192 differentiated patients with opisthorchiasis (the area under the curve; AUC=0.766), PDF subjects (AUC=0.781) and CCA patients (AUC=0.682) from healthy controls. MiR-21 was significantly higher in PDF and CCA groups (p<0.05) than in healthy controls. MiR-21 discriminated PDF subjects (AUC=0.735) and CCA patients (AUC=0.682) from healthy controls. Combined levels of these two miRNAs revealed an increased AUC of 0.812 for separating opisthorchiasis, AUC of 0.815 in discriminating PDF subjects, and AUC of 0.849 in differentiating CCA from healthy controls. Odds ratios (OR) indicated high levels of miR-192/miR-21 as risk predictors for opisthorchiasis, PDF and CCA. Levels of these miRNAs declined significantly for patients following praziquantel treatment. In conclusion, urinary miR-192/miR-21 have potential as risk indicators for opisthorchiasis and PDF-associated CCA in the endemic region.

Subsets of Inflammatory Cytokine Gene Polymorphisms are Associated with Risk of Carcinogenic Liver Fluke Opisthorchis viverrini-Associated Advanced Periductal Fibrosis and Cholangiocarcinoma

Opisthorchis viverrini infection induces chronic inflammation, and a minor proportion of infected individuals develop advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA). Inflammatory cytokines and/or their gene polymorphisms may link to these biliary pathologies. We therefore investigated associations among cytokine gene polymorphisms and cytokine production in 510 Thai cases infected with O. viverrini who presented with APF+ or APF−, as established by abdominal ultrasonography as well as in patients diagnosed with CCA. Levels of pro-inflammatory and anti-inflammatory cytokines were determined in culture supernatants after stimulation of peripheral blood mononuclear cells (PBMCs) with O. viverrini excretory-secretory (ES) products. Pro-inflammatory cytokines, IL-1β, IL-6, IFN-γ, LT-α, and TNF-α were significantly increased in CCA patients compared with non-CCA (APF− and APF+) cases. Polymorphisms in genes encoding IL-1β-511C/T, IL-6-174G/C, IFN-γ +874T/A, LT-α +252A/G, and TNF-α−308G/A were then investigated by using PCR-RFLP or allele specific-PCR (AS-PCR) analyses. In the CCA cases, LT-α +252A/G and TNF-α−308G/A heterozygous and homozygous variants showed significantly higher levels of these cytokines than the wild type. By contrast, levels of cytokines in wild type of IFN-γ +874T/A were significantly higher than the variants in CCA cases. IFN-γ +874T/A polymorphisms were associated with advanced periductal fibrosis, whereas IL-6 −174G/C polymorphisms were associated with CCA. To our knowledge, these findings provide the first demonstration that O. viverrini infected individuals carrying several specific cytokine gene polymorphisms are susceptible to develop fibrosis and CCA.

Nanoencapsulated curcumin and praziquantel treatment reduces periductal fibrosis and attenuates bile canalicular abnormalities in Opisthorchis viverrini-infected hamsters.

This study investigated the effects of nanoencapsulated curcumin (NEC) and praziquantel (PZQ) treatment on the resolution of periductal fibrosis (PDF) and bile canalicular (BC) abnormalities in Opisthorchis viverrini infected hamsters. Chronic O. viverrini infection (OV) was initially treated with either PZQ (OP) and subsequently treated with NEC (OP+NEC), curcumin (OP+Cur) or unloaded carriers (OP+carrier) daily for one month. OP+NEC treatment reduced the PDF by suppression of fibrotic markers (hydroxyproline content, α-SMA, CTGF, fibronectin, collagen I and III), cytokines (TGF-ß and TNF-α) and TIMP-1, 2, 3 expression and upregulation of MMP-7, 13 genes. Higher activity of NEC in reducing fibrosis compared to curcumin was also demonstrated in in vitro studies. Moreover, OP+NEC also prevented BC abnormalities and upregulated several genes involved in bile acid metabolism. These results demonstrate that NEC and PZQ treatment reduces PDF and attenuates BC defect in experimental opisthorchiasis. From the Clinical Editor: Infection by Opisthorchis viverrini leads to liver fibrosis and affects population in SE Asia. Currently, praziquantel (PZQ) is the drug of choice but this drug has significant side effects. In this study, the authors combined curcumin (NEC) and praziquantel in a nanocarrier to test the anti-oxidative effect of curcumin in an animal model. The encouraging results may pave a way for better treatment in the future.

Histological confirmation of periductal fibrosis from ultrasound diagnosis in cholangiocarcinoma patients.

Cholangiocarcinoma (CCA) has no specific clinical signs and symptoms and non-specific bio- and tumor-markers in the early disease stage. Usually patients present to tertiary care with advanced disease stage. In order to detect early cases of CCA that may present as a mass, dilatation of intrahepatic duct or combination, ultrasonography is accepted as a powerful imaging tool. A smaller mass or bile duct segmental dilatation requires further imaging for characterization, including computerized tomography (CT) or magnetic resonance imaging (MRI). We examined whether liver echo pattern was correlated with high risk for CCA in an endemic area of Opisthorchis viverrini (Ov). Ov infestation caused chronic inflammation of the biliary tree by periductal fibrosis (PDF), which may subsequently lead to CCA development. In our study, a World Health Organization classification of pattern of increased periportal echo (IPE) for schistosomiasis was applied. Two CCA patients gave consent for operation. Histopathological diagnosis showed both had cholangiocarcinoma with periductal fibrosis of the non-tumorous area of the liver. Ultrasonography was used to compare the non-tumorous area with parenchymal echo pattern and was shown to have an early CCA detection role and a surveillance role in an endemic area of Ov by detection of PDF.

Risk biomarkers for assessment and chemoprevention of liver fluke-associated cholangiocarcinoma.

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.