Differential Protein Expression Marks the Transition From Infection With Opisthorchis viverrini to Cholangiocarcinoma.

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.

Social defeat stress exacerbates the blood abnormalities in Opisthorchis felineus-infected mice.

A model of chronic opisthorchiasis combined with social stress is examined; this situation is more likely for humans and animals than a separate impact of the infectious factor. For this purpose, we evaluated the effects of Opisthorchis felineus ("OP" group) and 30-day social stress (confrontations between males, "SS" group) alone and in combination ("OP + SS" group) in inbred C57BL/6 male mice and compared these effects according to the parameters listed below. The animals exposed to neither factor formed the control group ("CON"). All animals were assayed for blood biochemical parameters, changes in blood cell composition, and pattern of bone marrow hematopoiesis. By the end of the experiment, we have observed crucial effects of the two factors on the blood and liver of "OP" and "OP + SS". Eosinophil and basophil counts increased and relative segmented neutrophil and monocyte counts decreased in "OP + SS" mice on the background of activated myelopoiesis, mainly determined by social stress. Despite depressed erythropoiesis, "OP" mice displayed no changes in the relative peripheral erythrocyte counts. On the contrary, social stress, which stimulated erythropoiesis in "SS" and "OP + SS" mice, was accompanied by a decrease in the relative erythrocyte counts and hematocrit. Hepatosplenomegaly was observed on the background of these two impacts. Changes in transaminase (ALT and AST) and alkaline phosphatase activities as well as an increase in cholesterol and product of lipid peroxidation suggest a pronounced destruction of the liver. Altogether, social stress exacerbates many of the assayed blood parameters in the mice infected with the liver fluke.

Development of an ELISA using anti-idiotypic antibody for diagnosis of opisthorchiasis.

Monoclonal antibody specific for an epitope of cretory-secretory antigen protein of Opisthorchis felineus (Rivolta, 1884) (Trematoda: Opisthorchiidae) with a molecular weight of 28 kDa was used in a sandwich enzyme-linked immunosorbent assay (ELISA) for immobilisation of liver fluke specific antigen to the solid phase. Examination of human sera by this ELISA compared with commercial assays demonstrated that the monoclonal antibody epitope is located within this significant parasite protein. Anti-idiotypic antibody specific for the paratope of this monoclonal antibody was obtained by a hybridoma technique. Mimicking an epitope of excretory-secretory antigen of O. felineus, it had the capacity to bind specific antibody and elicit an antibody response. The value of anti-idiotypic antibody as a substitute for the liver fluke antigen was tested by ELISA using serum samples of infected dogs. Anti-idiotypic antibody proved to be of value in both an indirect-ELISA and a competitive-ELISA for diagnosis of opisthorchiasis. Mature trematodes were isolated from all infected animals. The faecal egg counts were negative in dogs with a relatively small number of parasites, despite finding antibodies in serum by ELISA. Substitution of parasite antigen with anti-idiotype avoids the use of experimental animals and also reduces time-consuming steps of antigen preparation.

Increase of exostosin 1 in plasma as a potential biomarker for opisthorchiasis-associated cholangiocarcinoma.

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.

microRNA profiling of exosomes derived from plasma and their potential as biomarkers for Opisthorchis viverrini-associated cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a life-threatening disease that impacts patients worldwide. In Southeast Asian countries, the liver fluke Opisthorchis viverrini plays a major role in inducing carcinogenesis of the bile ducts. Due to its asymptomatic nature, O. viverrini infections are rarely treated, consequently leading to the development of advanced stages of CCA before diagnosis. Despite the current use of exosomal microRNAs (miRNA) as diagnostic biomarkers for the early detection of many types of cancer, the applications for miRNA remain limited with CCA. Circulating exosomes, membranous vesicles essential for intercellular communication, were found to contain unique miRNA. In this study, we conducted next-generation sequencing (Ion Torrent PGM) and bioinformatics to characterize and compare the contents of exosomal miRNA derived from the plasma of CCA patients, O. viverrini-infected patients, and healthy individuals, as well as to identify and validate key molecules as markers for screening the diagnosis of CCA and O. viverrini infection. The obtained results showed the success of using NGS technology in discovering exosomal miRNAs, specifically miR-194-5p and miR-192-5p, both of which were upregulated in the O. viverrini-infected group. Interestingly, miR-192-5p was upregulated while miR-194-5p was downregulated in CCA, suggesting their potential use as biomarkers for screening CCA and O. viverrini infection, especially in O. viverrini-endemic areas.

[Experience of using reamberin for detoxification therapy at the stage of deworming in children with chronic opisthorchiasis].

The study presents data on the reversible aggregation of erythrocytes in 50 children with chronic opisthorchiasis and 45 children with inflammatory diseases of the upper digestive tract without opisthorchiasis (chronic gastroduodenitis, duodenal ulcers), all patients aged from 7 to 17 years. The results of using reamberin for detoxification therapy at the stage of deworming in children with chronic opisthorchiasis are assessed.

[Thrombocytic and coagulation hemostasis in pregnant women living in the Ob-Irtysh river basin with chronic opisthorchiasis].

Opisthorchiasis that is widely spreading in Russia and CIS countries was and remains a serious social, medical, and scientific problem. Local or imported cases of opisthorchiasis have been notified in 87.6% of the administrative territories of Russia. The world's largest focus of opisthorchiasis is the Ob-Irtysh river basin with natural preconditions for its high infection in fish. The main clinical manifestations of Opisthorchis infestation are associated with liver damage caused by the obligate presence of Opisthorchis in the bile capillaries with evolving cholangitis, cholangiocholecystitis, and cholangiopancreatitis. Opisthorchis complicates the course of many diseases, affecting gestation, labor, and postpartum. The state of thrombocytic and coagulation hemostasis was analyzed in different period of pregnancy and postpartum (days 2-4), by using the course of pregnancy in the inhabitants of the hyperendemic focus of Opisthorchis infestation (the Ob-Irtysh basin) as an example. In the newcomers, pregnancy develops in the presence of platelet hyperaggregation and accelerated continuous blood clotting. In pregnant women who belong to native little peoples (Khanty, Mansi), opisthorchiasis induces no significant changes in the hemostatic potential.

[Continuous blood coagulation in chronic opisthorchiasis].

Animal experiments have shown that chronic Opisthorchis invasion results in accelerated continuous blood coagulation, by inducing hypercoagulation and platelet activation. The state of hemostasis depends on the degree of infection and undergoes the largest changes during maximum invasion. Irrespective of invasion rates, chronic opisthorchiasis is accompanied by hypofibrinogenemia.

[The efficiency of the enzyme immunoassay test system opisthorchiasis-CIC-EIA-best to detect circulating immune complexes containing opisthorchis antigens in the serum of patients with opisthorchiasis].

The efficacy of a kit of Opisthorchiasis-CIC-EIA-Best reagents was evaluated using 270 sera from patients in the study and control groups. The kit showed a sufficient sensitivity (not less than 87.2%) and a high specificity (not less than 97.9%). The use of the above kit of the reagents for enzyme immunoassay in practical healthcare enables one to increase detection rates among the infested subjects on comprehensive examination of those with suspected opisthorchiasis.

[The clinical and functional state of the liver in patients with psoriasis in the presence of chronic opisthorchiasis].

OBJECTIVE: to study the clinical and functional state of the liver in patients with psoriasis concurrent with chronic opisthorchiasis (CO). SUBJECTS AND METHODS: Twenty-eight patients with psoriasis concurrent with CO, 15 patients with CO, 15 patients with the enlarged liver, and 15 healthy individuals were examined. Serum biochemical study and ultrasonography of the liver and gallbladder were used. RESULTS AND DISCUSSION: The patients with comorbidity were found to have hepatomegaly with syndromes of cytolysis, cholestasis, and mesenchymal inflammation of more degrees than those from Groups 2 and 3. CONCLUSION: The found clinical and functional liver changes in patients with psoriasis concurrent with CO make it necessary to perform anthelmintic therapy in this category of patients.

Advanced periductal fibrosis from infection with the carcinogenic human liver fluke Opisthorchis viverrini correlates with elevated levels of interleukin-6.

UNLABELLED: More than 750 million people are at risk of infection with foodborne liver flukes. Opisthorchis viverrini is considered among the most important of these parasites, due to its strong association with cholangiocarcinoma (CCA). O. viverrini infection results in a chronic inflammatory challenge to the host, which can lead to advanced, pathogen-specific disease sequelae including obstructive jaundice, hepatomegaly, cholecystitis, as well as CCA. However, before disease sequelae are apparent, important inflammatory changes to the liver can be detected early during O. viverrini infection. In a case-control study involving 328 men and women with O. viverrini infection, we determined the presence of advanced periductal fibrosis in asymptomatic, O. viverrini-infected individuals and then measured cytokine responses to O. viverrini excretory/secretory products (ES). In the 200 participants with advanced periductal fibrosis (cases), levels of interleukin-6 (IL-6) to O. viverrini ES were 8 times higher than levels of the 128 O. viverrini-infected individuals without advanced periductal fibrosis (controls). Moreover, elevated IL-6 to parasite ES was associated with increased risk of advanced periductal fibrosis by 63% in a model adjusted for sex and age. The risk of advanced periductal fibrosis was also found to increase with higher levels of IL-6: individuals in the third quartile of IL-6-ES production had a 127% higher risk of developing advanced periductal fibrosis than individuals in the first quartile of IL-6 production. O. viverrini-infected individuals with advanced periductal fibrosis showed other hepatobiliary abnormalities, including reduced gallbladder contractility and the presence of gallbladder sludge. CONCLUSION: These data strongly implicate a role for parasite-specific IL-6 in the pathogenesis of advanced periductal fibrosis in opisthorchiasis, with possible links to other hepatobiliary abnormalities, including CCA.

Plasma metabolomics of the time resolved response to Opisthorchis felineus infection in an animal model (golden hamster, Mesocricetus auratus).

BACKGROUND: Opisthorchiasis is a hepatobiliary disease caused by flukes of the trematode family Opisthorchiidae. Opisthorchiasis can lead to severe hepatobiliary morbidity and is classified as a carcinogenic agent. Here we investigate the time-resolved metabolic response to Opisthorchis felineus infection in an animal model. METHODOLOGY: Thirty golden hamsters were divided in three groups: severe infection (50 metacercariae/hamster), mild infection (15 metacercariae/hamster) and uninfected (vehicle-PBS) groups. Each group consisted of equal number of male and female animals. Plasma samples were collected one day before the infection and then every two weeks up to week 22 after infection. The samples were subjected to 1H Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate statistical modelling. PRINCIPAL FINDINGS: The time-resolved study of the metabolic response to Opisthorchis infection in plasma in the main lines agrees with our previous report on urine data. The response reaches its peak around the 4th week of infection and stabilizes after the 10th week. Yet, unlike the urinary data there is no strong effect of the gender in the data and the intensity of infection is presented in the first two principal components of the PCA model. The main trends of the metabolic response to the infection in blood plasma are the transient depletion of essential amino acids and an increase in lipoprotein and cholesterol concentrations. CONCLUSIONS: The time resolved metabolic signature of Opisthorchis infection in the hamster's plasma shows a coherent shift in amino acids and lipid metabolism. Our work provides insight into the metabolic basis of the host response on the helminth infection.

[Comparative assessment of bile and blood cholesterol, pigment, and protein metabolic parameters in patients with cholelithiasis in the presence and absence of opisthorchiasis].

AIM: to study the regularities of lithogenesis in patients with cholelithiasis (CL) and opisthorchiasis. SUBJECTS AND METHODS: Fifty-one patients with CL and opisthorchiasis were examined. Comparison groups comprised 32 with CL without opisthorchiasis and 14 patients with opisthorchiasis without CL. All the patients underwent a comprehensive examination involving laboratory and ultrasound studies. RESULTS: Opisthorchis invasion leads to increases in vesicular and hepatic bile cholesterol saturation and bile albumin levels due to intrahepatic ductal cholestasis, to a rise in bile unconjugated bilirubin concentration, which is caused by the activation of proliferative inflammation in the bile ducts and by the increased deposits of bilirubin complexes resulting from higher bile globulin levels. CONCLUSION: In opisthorchiasis, bile lithogenic factors mostly activate in the development of CH in patients with significant manifestations of cholestasis, angiocholitis with the most preserved concentration function of the gallbladder.

[Increased accumulation of some trace elements in peripheral blood and bile of patients infested with Opisthorchis felineus (Rivolta, 1884) and Metorchis bilis (Braun, 1890)].

The invasion with Opisthorchis felineus (Rivolta, 1884) is known to be common in the Ob River region, West Siberia. These trematodes parasitize biliary tract of devinitive host (man or some species of animals). Other opisthorchiid species occurring in West Siberia, Metorchis bilis (Braun, 1890), has also been recorded recently as human parasite. Life cycles of both these trematodes include fish-eating mammal hosts. Eggs of O. felineus and M. bilis are very similar morphologically and can hardly be indentified. Chronic invasion with the helminthes is found to be a cause of disbolism of trace elements or high accumulation of some essential and toxic elements in the organism of definitive host. The aim of the present study was to determine concentrations of some essential and toxic elements in samples of peripheral blood and bile obtained from patients infested with Opisthorchis and/or Metorchis using instrumental neuron-activation technique. At first, all patients with microscopically confirmed opisthorchiasis (by microscopic examination of faeces and bile for the helminth eggs) were examined with serological method (ELISA) for specific anti-Opisthorchis and anti-Metorchis antibodies. Among 139 examined patients, 56.1% had specific antibodies against both Opisthorchis and Metorchis, 41.7% showed anti-Opisthorchis antibodies only, and 2.1% turn out to be seropositive for anti-Metorchis antibodies only. Of 31 elements detected in the samples of peripheral blood and bile, the concentration of nine essential and toxic elements (mercury, chromium, cesium, rubidium, lanthanum, bromine, selenium, zinc, and cobalt) in the patients with the mixt-infection of Opisthorchis and Metorchis (78 individuals) and with the Opisthorchis mono-infection (58 individuals) were significantly higher than those in healthy uninfested individuals. Among these elements, mercury and chromium showed the highest concentrations in infested patients. We suggest that the significant increase in concentrations of some toxic elements in blood and bile of patients may be associated with increased bioaccumulation of these elements in environment, especially in the local river water and fish. Moreover, concentrations of such elements as chromium, mercury, cesium, lanthanum, selenium, and zinc in blood and/or bile of the patients with mixt-infection of Opisthorchis and Metorchis were significantly higher than those in the patients with mono-infection of Opisthorchis. Accumulation of these elements in the organisms of patients was positively correlated with the number of parasites and the duration of helminth infection.

[Nitric monoxide and other indicators of free radical processes and antioxidant system in patients with mixed or monoinvasion of Opisthorhis and Metorchis].

As compared with healthy individuals, patients with mixed Opisthorhis and Metorchis invasion and/or Opisthorhis monoinvasion were found to have a considerable increase in the serum levels of nitric monoxide and malondialdehyde with the concurrent suppression of catalase activity and lipid antioxidant activity, which correlated with the blood accumulation of some toxic trace elements. The highest accumulation levels of toxic trace elements, as well as the indicators of free radical processes were detected in patients with the mixed invasion as compared with the Opisthorhis monoinvasion group, which appears to be directly related to the intensity of invasion.

Development of an Immunochromatographic Point-of-Care Test for Serodiagnosis of Opisthorchiasis and Clonorchiasis.

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

Clonorchis sinensis omega-class glutathione transferases are reliable biomarkers for serodiagnosis of clonorchiasis and opisthorchiasis.

OBJECTIVES: To determine the potential for immunodiagnostic application of two recombinant forms of Clonorchis sinensis omega-class glutathione transferases (rCsGSTo1 and rCsGSTo2) against human small liver-fluke C. sinensis and Opisthorchis viverrini infections. METHODS: Specific antibody levels against rCsGSTo1 and rCsGSTo2 in patients' sera of egg-positive opisthorchiasis (n = 87) and clonorchiasis (n = 120), as well as those in sera from patients with other helminthic infections (n = 252) and healthy controls (n = 40) were retrospectively analysed by ELISA. RESULTS: We observed highly positive correlation coefficients between specific antibody levels against rCsGSTo1 and rCsGSTo2 and egg counts per gramme of faeces (EPG) of patients with opisthorchiasis (n = 87; r = 0.88 for rCsGSTo1 and r = 0.90 for rCsGSTo2). Sera from opisthorchiasis patients whose EPG counts >100 (n = 43) revealed high antibody titres against both antigens. Patients' sera with low EPG counts (<100, n = 44) also exhibited reliable sensitivities of 93.2% and 97.7% for rCsGSTo1 and rCsGSTo2, respectively. Sera from clonorchiasis patients showed sensitivities of 90% (108/120 samples) and 89.2% (107/120 sera) for rCsGSTo1 and rCsGSTo2. Overall diagnostic sensitivities for liver-fluke infections were 92.3% for rCsGSTo1 (191/207 samples) and 93.2% for rCsGSTo2 (193/207 samples). Specificities were 89.7% (rCsGSTo1) and 97.6% (rCsGSTo2). CONCLUSIONS: Detection of specific antibody levels against rCsGSTo1 or rCsGSTo2 might be promising for the serodiagnosis of patients infected with these two phylogenetically close carcinogenic liver-flukes.

[Impact of Opisthorchis invasion on lipid peroxidation and hemostasis during pregnancy].

Blood cell lipid peroxidation processes and the state of thrombocytic and coagulative hemostasis were studied in 214 pregnant women and parturients with Opisthorchis invasion amongst the indigenous population and newcomers of the Khanty-Mansi Autonomic District-Yugra. Chronic opisthorchiasis was found to enhance lipid peroxidation in the blood cells, to accelerate the activation of thrombocytic and coagulative hemostasis during gestation, at labor, and in the postpartum period. Opisthorchis invasion variously affected the state of lipid peroxidation and hemostasis in the examined groups. In the indigenous women, the number of activated forms of platelets increased, without any obvious change in their functional potential, which was unaccompanied by changes in the parameters of coagulative hemostasis and lipid peroxidation. Increased activation of platelets, their hyperaggregability, and subsequent depletion of their functional potential were revealed in the newcomers. This was attended by hypercoagulation with signs of acceleration of continuous blood coagulation, by enhanced lipid peroxidation and reduced antioxidative potential in the blood cells.

[Change in the production of key cytokines for regulation eosinophilic granulocytes in Opisthorchis felineus invasion].

Opisthorchis invasion is accompanied by the imbalanced lymphocytic subpopulational composition manifested itself as induction of the B-link and, on the contrary, depression of T-lymphocytic populations (CD4+, CD8+), with their weaker helper-suppressor association. The immunocompetent cells were ascertained to show a higher production of TH2 cytokines that had an eosinophil-stumulating effect.

[Dysregulation of cooperative interactions of immunocytes and eosinophils in the mechanism of development of eosinophilia in Opisthorhis felineus invasion].

The authors studied the levels of mononuclear cell production of eosinophil-specific cytokines (IL-3, IL-5), the serum levels of eotaxin by enzyme immunoassay; the expression of the eosinophilic cell receptor apparatus by flow cytofluorometry in patients with acute and chronic Opisthorchis invasion. Eosinophilia-associated Opisthorchis invasion was found to be accompanied by a pronounced change in the serum production of the key cytokine regulators of eosinophilic homeostasis (elevated IL-3 and IL-5 levels) and eotaxin by peripheral blood mononuclear leukocytes. There was an increase in the number of receptor structures to eosinophil-specific cytokines (IL-5R-, IL-3R-, and CCR3-positive cells) in patients with opisthorchiasis. In vitro incubation of the eosinophils, obtained from patients with opisthorchiasis, with the recombinant forms of cytokines (IL-5, IL-3, and eotaxin) demonstrated the decreased expression of IL-5 and IL-3 receptors with the normal presentation of CCR3. With the developed acute helminthiasis, the revealed changes were more pronounced than those observed in chronic Opisthorchis invasion.