Anti-Opisthorchis felineus effects of artemisinin derivatives: An in vitro study.

BACKGROUND: The drug of choice for the treatment of opisthorchiasis caused by trematodes Opisthorchis viverrini and O. felineus is praziquantel (PZQ), but there is a constant search for new anthelmintics, including those of plant origin. Positive results on the use of artemisinin derivatives against O. viverrini opisthorchiasis have been shown previously, but the effect of these compounds on O. felineus has not been studied. Therefore, here, a comparative analysis of anthelmintic properties of artemisinin derivatives (artesunate [AS], artemether [AM], and dihydroartemisinin [DHA]) was carried out in vitro in relation to PZQ. Experiments were performed on newly excysted metacercariae (NEMs) and adult flukes of O. felineus. RESULTS: Dose- and time-dependent effects of artemisinin derivatives and of PZQ were assessed in terms of motility and mortality of both NEMs and adult flukes. The most pronounced anthelmintic action was exerted by DHA, whose half-maximal inhibitory concentrations (IC50) of 1.9 (NEMs) and 2.02 µg/mL (adult flukes) were lower than those of PZQ (0.56 and 0.25 µg/mL, respectively). In contrast to PZQ, the effects of DHA and AS were similar when we compared the two developmental stages of O. felineus (NEMs and adult flukes). In addition, AM, AS, and especially DHA at doses of 100 µg/mL disrupted tegument integrity in adult flukes, which was not observed with PZQ. CONCLUSIONS: Artemisinin derivatives (AS, AM, and DHA) have good anthelmintic efficacy against the trematode O. felineus, and the action of these substances is comparable to (and sometimes better than) the effects of PZQ.

Plastoquinone-Derivative SkQ1 Improved the Biliary Intraepithelial Neoplasia during Liver Fluke Infection.

Carcinogenic food-borne liver fluke infections are a serious epidemiological threat worldwide. The major complications of Opisthorchis felineus infection are chronic inflammation and biliary intraepithelial neoplasia. Although evidence has accumulated that increased reactive oxygen species production is observed in liver fluke infection, a direct relationship between the oxidative stress and biliary intraepithelial neoplasia has not been shown. Quinones and SkQ1, a derivative of plastoquinone, have been demonstrated to be cytoprotective in numerous liver injuries due to their potent antioxidant properties. This study is aimed to assess the level of biliary intraepithelial neoplasia in O. felineus-infected hamsters after treatment with mitochondria-targeted SkQ1. SkQ1 significantly reduced the biliary intraepithelial neoplasia, which was accompanied by a decrease in lipid and DNA oxidation byproducts, mRNA expression and level of proteins associated with inflammation (TNF-α, CD68) and fibrogenesis (CK7, αSMA), and was also associated with an activation of the Keap1-Nrf2 pathway. Thus, a direct relationship was found between oxidative stress and the severity of biliary intraepithelial neoplasia in O. felineus-infected hamsters. The hepatoprotective effect of plastoquinone-derivative SkQ1 was established; therefore, this compound is a promising agent in complex therapy in the treatment of opisthorchiasis.

Hepatic vascular changes associated with Opisthorchis felineus infection in Syrian hamsters and humans.

The liver fluke Opisthorchis felineus is a foodborne zoonotic pathogen endemic to Russia, Kazakhstan, and several European countries. The adult flukes affect the hepatobiliary system of piscivorous mammals and humans, thereby causing numerous complications, including liver fibrosis. Detailing the mechanisms of progression of the fibrotic complications is a hot topic in the field of research on opisthorchiasis pathogenesis. Pathologic angiogenesis appears to be associated with the fibrogenic progression due to active participation in the recruitment of inflammatory cells and many factors involved in the modulation of the extracellular matrix. The aim of the study was to evaluate neoangiogenesis and amyloid deposits in liver tissues of model animals and patients with confirmed chronic opisthorchiasis. In addition, we assessed a possible correlation of neoangiogenesis with liver fibrosis. We found a significant increase in the number of newly formed vessels and amyloid deposits in the liver of people with chronic opisthorchiasis compared to that of uninfected ones. Thus, for the first time we have demonstrated neoangiogenesis and amyloid deposits during O. felineus infection in a Mesocricetus auratus model. Regression analysis showed that CD34+ newly formed vessels correlate with fibrosis severity in the course of the infection. Our results indicate the potential contribution of angiogenesis to the progression of liver fibrosis, associated with O. felineus infection.

Extracellular vesicles of the liver fluke Opisthorchis felineus stimulate the angiogenesis of human umbilical vein endothelial cells.

The liver fluke Opisthorchis felineus is a clinically important food-borne parasite of humans. Infection with O. felineus in mammals is associated with liver morbidities such as periductal fibrosis, bile duct neoplasia, and chronic inflammation. Previously we have shown that excretory-secretory products (ESP) can stimulate the healing of skin wounds in mice, which may be due to stimulated angiogenesis and extracellular matrix remodeling. However, there are no studies analyzing the angiogenic character of O. felineus, and its effects on angiogenesis, vascularity, and vascular endothelium. The aim of this study was to evaluate the capacity of ESP and extracellular vesicles (EVs) of O. felineus to stimulate angiogenesis and the formation of pseudo-capillaries in vitro. We also aimed at the assessment of the angiogenesis during the infection in vivo, and estimation of the endothelial cell type abundances from heterogeneous bulk liver transcriptome between uninfected and infected animals with single-cell information. The study revealed significant alterations in vascularity in the hamster liver and significant involvement of portal endothelial cells at the transcriptome level. We also demonstrated that the ESP and EVs of O. felineus have the capacity to stimulate the formation of pseudo-capillaries in vitro. Both ESP and EVs appeared to have similar effects on all four parameters, increasing node formation and total master segments length, and significantly decreasing total isolated branches length and number of isolated segments of pseudo-capillaries. The liver flukes manipulate the host's angiogenic response, a fact that has been related to the pathogenesis caused by these parasites. Understanding these pathogenic mechanisms may uncover new therapeutic targets to relieve or prevent the most severe complications of opisthorchiasis.

Curcumin and Its Supramolecular Complex with Disodium Glycyrrhizinate as Potential Drugs for the Liver Fluke Infection Caused by Opisthorchis felineus.

Opisthorchiosis is a parasitic liver disease found in mammals that is widespread throughout the world and causes systemic inflammation. Praziquantel remains the drug of choice for the treatment of opisthorchiosis, despite its many adverse effects. An anthelmintic effect is attributed to the main curcuminoid of Curcuma longa L. roots-curcumin (Cur)-along with many other therapeutic properties. To overcome the poor solubility of curcumin in water, a micellar complex of curcumin with the disodium salt of glycyrrhizic acid (Cur:Na2GA, molar ratio 1:1) was prepared via solid-phase mechanical processing. In vitro experiments revealed a noticeable immobilizing effect of curcumin and of Cur:Na2GA on mature and juvenile Opisthorchis felineus individuals. In vivo experiments showed that curcumin (50 mg/kg) had an anthelmintic effect after 30 days of administration to O. felineus-infected hamsters, but the effect was weaker than that of a single administration of praziquantel (400 mg/kg). Cur:Na2GA (50 mg/kg, 30 days), which contains less free curcumin, did not exert this action. The complex, just as free curcumin or better, activated the expression of bile acid synthesis genes (Cyp7A1, Fxr, and Rxra), which was suppressed by O. felineus infection and by praziquantel. Curcumin reduced the rate of inflammatory infiltration, whereas Cur:Na2GA reduced periductal fibrosis. Immunohistochemically, a decrease in liver inflammation markers was found, which is determined by calculating the numbers of tumor-necrosis-factor-positive cells during the curcumin treatment and of kynurenine-3-monooxygenase-positive cells during the Cur:Na2GA treatment. A biochemical blood test revealed a normalizing effect of Cur:Na2GA (comparable to that of curcumin) on lipid metabolism. We believe that the further development and investigation of therapeutics based on curcuminoids in relation Opisthorchis felineus and other trematode infections will be useful for clinical practice and veterinary medicine.

Proteomic characterization of Opisthorchis felineus exosome-like vesicles and their uptake by human cholangiocytes.

The epidemiologically important food-borne trematode Opisthorchis felineus infests the liver biliary tract of fish-eating mammals and causes disorders, including bile duct neoplasia. Many parasitic species release extracellular vesicles (EVs) that mediate host-parasite interaction. Currently, there is no information on O. felineus EVs. Using gel electrophoresis followed by liquid chromatography coupled with tandem mass spectrometry, we aimed to characterize the proteome of EVs released by the adult O. felineus liver fluke. Differential abundance of proteins between whole adult worms and EVs was assessed by semiquantitative iBAQ (intensity-based absolute quantification). Imaging, flow cytometry, inhibitor assays, and colocalization assays were performed to monitor the uptake of the EVs by H69 human cholangiocytes. The proteomic analysis reliably identified 168 proteins (at least two peptides matched a protein). Among major proteins of EVs were ferritin, tetraspanin CD63, helminth defense molecule 1, globin 3, saposin B type domain-containing protein, 60S ribosomal protein, glutathione S-transferase GST28, tubulin, and thioredoxin peroxidase. Moreover, as compared to the whole adult worm, EVs proved to be enriched with tetraspanin CD63, saposin B, helminth defense molecule 1, and Golgi-associated plant pathogenesis-related protein 1 (GAPR1). We showed that EVs are internalized by human H69 cholangiocytes via clathrin-dependent endocytosis, whereas phagocytosis and caveolin-dependent endocytosis do not play a substantial role in this process. Our study describes for the first time proteomes and differential abundance of proteins in whole adult O. felineus worms and EVs released by this food-borne trematode. Studies elucidating the regulatory role of individual components of EVs of liver flukes should be continued to determine which components of EV cargo play the most important part in the pathogenesis of fluke infection and in a closely linked pathology: bile duct neoplasia. SIGNIFICANCE: The food-borne trematode Opisthorchis felineus is a pathogen that causes hepatobiliary disorders in humans and animals. Our study describes for the first time the release of EVs by the liver fluke O. felineus, their microscopic and proteomic characterization, and internalization pathways by human cholangiocytes. Differential abundance of proteins between whole adult worms and EVs was assessed. EVs are enriched with canonical EV markers as well as parasite specific proteins, i.e. tetraspanin CD63, saposin B, helminth defense molecule 1, and others. Our findings will form the basis of the search for potential immunomodulatory candidates with therapeutic potential in the context of inflammatory diseases, as well as novel vaccine candidates.

Opisthorchis viverrini, Clonorchis sinensis and Opisthorchis felineus liver flukes affect mammalian host microbiome in a species-specific manner.

BACKGROUND: Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis are epidemiologically significant food-borne trematodes endemic to diverse climatic areas. O. viverrini and C. sinensis are both recognized to be 1A group of biological carcinogens to human, whereas O. felineus is not. The mechanisms of carcinogenesis by the liver flukes are studied fragmentarily, the role of host and parasite microbiome is an unexplored aspect. METHODOLOGY/PRINCIPAL FINDINGS: Specific pathogen free Mesocricetus auratus hamsters were infected with C. sinensis, O. viverrini and O. felineus. The microbiota of the adult worms, colon feces and bile from the hamsters was investigated using Illumina-based sequencing targeting the prokaryotic 16S rRNA gene. The analysis of 43 libraries revealed 18,830,015 sequences, the bacterial super-kingdom, 16 different phyla, 39 classes, 63 orders, 107 families, 187 genera-level phylotypes. O. viverrini, a fluke with the most pronounced carcinogenic potential, has the strongest impact on the host bile microbiome, changing the abundance of 92 features, including Bifidobacteriaceae, Erysipelotrichaceae, [Paraprevotellaceae], Acetobacteraceae, Coriobacteraceae and Corynebacteriaceae bacterial species. All three infections significantly increased Enterobacteriaceae abundance in host bile, reduced the level of commensal bacteria in the gut microbiome (Parabacteroides, Roseburia, and AF12). CONCLUSIONS/SIGNIFICANCE: O. felineus, O. viverrini, and C. sinensis infections cause both general and species-specific qualitative and quantitative changes in the composition of microbiota of bile and colon feces of experimental animals infected with these trematodes. The alterations primarily concern the abundance of individual features and the phylogenetic diversity of microbiomes of infected hamsters.

Xenobiotic-Metabolizing Enzymes in Trematodes.

Trematode infections occur worldwide causing considerable deterioration of human health and placing a substantial financial burden on the livestock industry. The hundreds of millions of people afflicted with trematode infections rely entirely on only two drugs (praziquantel and triclabendazole) for treatment. An understanding of anthelmintic biotransformation pathways in parasites should clarify factors that can modulate therapeutic potency of anthelmintics currently in use and may lead to the discovery of synergistic compounds for combination treatments. Despite the pronounced epidemiological significance of trematodes, there is still no adequate understanding of the functionality of their metabolic systems, including xenobiotic-metabolizing enzymes. The review is focused on the structure and functional significance of the xenobiotic-metabolizing system in trematodes. Knowledge in this field can solve practical problems related to the search for new targets for antiparasitic therapy based on a focused action on certain elements of the parasite's metabolic system. Knowledge of the functionality of this system is required to understand the adaptation of the biochemical processes of parasites residing in the host and mechanisms of drug resistance development, as well as to select a promising molecular target for the discovery and development of new anthelmintic drugs.

Wound healing approach based on excretory-secretory product and lysate of liver flukes.

Exogenous bioactive peptides are considered promising for the wound healing therapy in humans. In this regard, parasitic trematodes proteins may potentially become a new perspective agents. Foodborne trematode Opisthorchis felineus is widespread in Europe and has the ability to stimulate proliferation of bile duct epithelium. In this study, we investigated skin wound healing potential of O. felineus proteins in mouse model. C57Bl/6 mice were inflicted with superficial wounds with 8 mm diameter. Experimental groups included several non-specific controls and specific treatment groups (excretory-secretory product and lysate). After 10 days of the experiment, the percentage of wound healing in the specific treatment groups significantly exceeded the control values. We also found that wound treatment with excretory-secretory product and worm lysate resulted in: (i) inflammation reducing, (ii) vascular response modulating, (iii) type 1 collagen deposition promoting dermal ECM remodeling. An additional proteomic analysis of excretory-secretory product and worm lysate samples was revealed 111 common proteins. The obtained data indicate a high wound-healing potential of liver fluke proteins and open prospects for further research as new therapeutic approaches.

Jagged-1/Notch Pathway and Key Transient Markers Involved in Biliary Fibrosis during Opisthorchis felineus Infection.

Chronic opisthorchiasis associated with Opisthorchis felineus infection is accompanied by severe fibrotic complications. It is of high practical significance to elucidate the mechanisms of hepatic fibrosis in chronic infection dynamics. The goal of the study is to investigate the temporal profile of key markers and the Jagged1/Notch signaling pathway in the implementation of fibrosis in a chronic O. felineus infection. For the first time, using histological methods and real-time PCR analysis, we demonstrated the activation of the Jagged1/Notch pathway in liver fibrogenesis, including the activation of the Hes1 and Hey1 target genes during experimental opisthorchiasis in Mesocricetus auratus. Cluster analysis followed by regression analysis of key markers during the infection showed that Jagged1 and Mmp9have the greatest contribution to the development of cholangiofibrosis and periductal fibrosis. Moreover, we detected a significant increase in the number of Jagged1-positive cells in the liver of chronic opisthorchiasis patients compared to that of the control group without infection. The results of the study are extremely informative both in terms of investigation both diverse fibrosis mechanisms as well as potential targets in complex antihelmintic therapy.

Similarities and differences among the Opisthorchiidae liver flukes: insights from Opisthorchis felineus.

The foodborne liver trematode Opisthorchis felineus (Rivolta, 1884) is a member of the triad of phylogenetically related epidemiologically important Opisthorchiidae trematodes, which also includes O. viverrini (Poirier, 1886) and Clonorchis sinensis (Loos, 1907). Despite similarity in the life cycle, Opisthorchiidae liver flukes also have marked differences. Two species (O. viverrini and C. sinensis) are recognized as Group 1A biological carcinogens, whereas O. felineus belongs to Group 3A. In this review, we focus on these questions: Are there actual differences in carcinogenicity among these 3 liver fluke species? Is there an explanation for these differences? We provide a recent update of our knowledge on the liver fluke O. felineus and highlight its differences from O. viverrini and C. sinensis. In particular, we concentrate on differences in the climate of endemic areas, characteristics of the life cycle, the range of intermediate hosts, genomic and transcriptomic features of the pathogens, and clinical symptoms and morbidity of the infections in humans. The discussion of these questions can stimulate new developments in comparative studies on the pathogenicity of liver flukes and should help to identify species-specific features of opisthorchiasis and clonorchiasis pathogenesis.

Global changes in gene expression related to Opisthorchis felineus liver fluke infection reveal temporal heterogeneity of a mammalian host response.

The food-borne trematode Opisthorchis felineus colonizes bile ducts of the liver of fish-eating mammals including humans. Among chronically infected individuals, this opisthorchiasis involves hepatobiliary problems, including chronic inflammation, periductal fibrosis, biliary intraepithelial neoplasia, and even cholangiocarcinoma. Despite numerous studies at the pathomorphological level, the systemic response and cellular pathogenesis of these disorders are not well studied. To conduct in-depth research and to gain insights into the mechanism by which O. felineus infection causes precancerous liver lesions, we (i) applied a next-generation-sequencing-based technology (high-throughput mRNA sequencing) to identify differentially expressed genes in the liver of golden hamsters infected with O. felineus at 1 and 3 months postinfection and (ii) verified the most pronounced changes in gene expression by western blotting and immunohistochemistry. A total of 2151 genes were found to be differentially expressed between uninfected and infected hamsters ("infection" factor), whereas 371 genes were differentially expressed when we analyzed "time × infection" interaction. Cluster analysis revealed that sets of activated genes of cellular pathways were different between acute (1 month postinfection) and chronic (3 months postinfection) opisthorchiasis. This enriched KEGG pathways were "Cell adhesion molecules", "Hippo signaling", "ECM-receptor interaction", "Cell cycle", "TGF-beta", and "P53 signaling". Moreover, epithelial-mesenchymal transition was the most enriched (q-value = 2.2E-07) MSigDB hallmark in the set of differentially expressed genes of all O. felineus-infected animals. Transcriptomic data were supported by the results of western blotting and immunohistochemistry revealing the upregulation of vimentin, N-cadherin, and α-smooth muscle actin postinfection. Our data expand knowledge about global changes in gene expression in the O. felineus-infected host liver and contribute to understanding the biliary neoplasia associated with the liver fluke infection.

Population genetic structure of diphyllobothriid tapeworms (Cestoda: Diphyllobothriidea) parasitising fish in the Baikal Rift Zone.

Tapeworms of the genus Dibothriocephalus are widely distributed throughout the world, and some are agents of human diphyllobothriasis, one of the most important fish-borne zoonoses caused by a cestode parasite. Until now, the population genetic structure of diphyllobothriid tapeworms in the Baikal Rift Zone (BRZ) has remained unexplored. The major aim of this study was to analyse the population genetic structure of D. dendriticus and D. ditremus parasitising fish in the BRZ based on internal transcribed spacer 1 (ITS1) and mitochondrial gene cytochrome oxidase subunit I (cox1) sequences. We found that both species had complex population genetic structures. Each species formed 2 clades (D. dendriticus: Clade 1 & 2; D. ditremus Clade A & B) that differed in genetic diversity. D. dendriticus haplotypes in Clade 1 formed a star-like sub-network with a main haplotype, whereas the haplotypes in Clade 2 formed a diffuse network. We assumed that the complex population genetic structure of D. dendriticus was a consequence of populations evolving under different palaeoecological conditions during the Last Glacial Maximum. In contrast to D. dendriticus, both clades in the D. ditremus samples formed a diffuse network. Our findings revealed hypothetical pathways in the formation of the population genetic structure of diphyllobothriids in the BRZ. On one hand, isolation by distance played an important role; on the other hand, lake recolonisation from refugia and a genetic bottleneck after the end of the Last Glacial Maximum had a possible influence.

Time-dependent renal pathologies associated with the liver fluke infection, opisthorchiasis felinea.

Fish-borne trematode infections affect the health of more than 18 million people in Russia and Asian countries. Infection of humans and other mammals with the liver fluke Opisthorchis felineus (Rivolta, 1884) is accompanied by gradual development of liver disorders. Although there is indirect evidence that opisthorchiasis may be associated with damage to other organs, direct evidence of the connection between opisthorchiasis felinea and a kidney pathology has not yet been reported. To gain first insights into the possible relation, we investigated time course profiles of blood markers of renal failure as well as renal histological changes during opisthorchiasis from 1 month to 1.5 years postinfection in golden hamsters Mesocricetus auratus. For the first time, we showed that opisthorchiasis felinea leads to the development of glomerulopathy. In particular, O. felineus infection provoked gradual increases in serum creatinine, serum glucose, and urine protein concentrations. Moreover, there was gradual accumulation of renal tubular casts and of the mesangial matrix. Although the mechanisms underlying these renal pathologies remain unclear and require further research, we can conclude that O. felineus infection causes gradual progression of glomerulopathy accompanied by tubulopathy. Thus, overall, these aberrations correlate with the time course of hepatic pathological changes in opisthorchiasis felinea.

Antioxidants resveratrol and SkQ1 attenuate praziquantel adverse effects on the liver in Opisthorchis felineus infected hamsters.

Anthelmintic praziquantel (PZQ) is the drug of the choice for opisthorchiasis, schistosomiasis and other trematodiases therapy for several decades. Despite its good therapeutic performance and effective control of trematode infections, PZQ has some shortcomings; its inability to counteract disease sequelae necessitates novel therapeutic strategies. Testing of antioxidants that have proven themselves in clinical practice, in combination with this anthelmintic drug, offers new opportunities for developing alternatives to PZQ monotherapy. The effects of two antioxidants combined with PZQ on histological parameters of liver tissue were evaluated in a hamster model of opisthorchiasis felinea. Liver pathology including the parenchyma state, accumulation of neutral lipids and 4-Hydroxy-2-nonenal as a lipid peroxidation product, biochemical characteristics of hamster blood serum, and mRNA expression of inflammation- and fibrogenesis-associated genes were determined. PZQ and opisthorchiasis caused liver accumulation of lipids and glycogen. The combination of PZQ with resveratrol (RSV) or 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) significantly reduced hepatocyte changes (P = 0.009 and P = 0.009, respectively, Mann-Whitney U test) as compared with infected hamsters treated only with PZQ. RSV and SkQ1 significantly reduced cholangiocyte hyperplasia, bile duct proliferation, fibrosis, and lipid droplet and glycogen granule accumulation. The downregulation of 4-hydroxynonenal was also observed. The combinations of the anthelmintic drug with antioxidants RSV and SkQ1 ameliorate host oxidative stress and mitigate adverse effects of PZQ on hepatic parenchyma. The use of drug combinations may improve the action of standard anthelmintic agents, such as PZQ, which still remains the most effective agent against adult trematodes.

A tumorigenic cell line derived from a hamster cholangiocarcinoma associated with Opisthorchis felineus liver fluke infection.

AIMS: The food-born trematode Opisthorchis felineus colonizes bile ducts of the liver of fish-eating mammals including humans. There is growing evidence that this liver fluke is a risk factor for cholangiocarcinoma (CCA). Cancer cell lines are necessary for drug screening and for identifying protein markers of CCA. The aim was to establish a cell line derived from cholangiocarcinoma associated with opisthorchiasis felinea. MAIN METHODS: Allotransplantation, immunohistochemistry, karyotype analysis, cell culture techniques, immunocytochemistry and real-time PCR. KEY FINDINGS: Here we repot the establishment of first CCA cell line, CCA-OF, from a primary tumor of an experimental CCA in Syrian hamsters treated with low doses of dimethyl nitrosamine and associated with O. felineus infection. The cell line was found to be allotransplantable. Expression of epithelial and mesenchymal markers (cytokeratin 7, glycosyltransferase exostosin 1, Ca2+-dependent phospholipid-binding protein annexin A1 and vimentin) was demonstrated by immunostaining of the primary tumors, CCA-OF cells, and allotransplants. CCA-OF cells were found to express presumed CCA biomarkers previously detected in both human and experimental tumors associated with the liver fluke infection. The cells were diploid-like (2n = 42-46) with complex chromosomal rearrangements and have morphological features of epithelial-like cells. The usefulness of the CCA-OF cell model for antitumor activity testing was demonstrated by an analysis of effects of resveratrol treatment. It was shown that resveratrol treatment inhibited the proliferation and the migration ability of CCA-OF cells. SIGNIFICANCE: Thus, the allotransplantable CCA-OF cell line can be used in studies on helminth-associated cholangiocarcinogenesis and for the testing of antitumor drugs.

Anthelmintic Activity of Antioxidants: In Vitro Effects on the Liver Fluke Opisthorchis felineus.

Currently, molecular parasitologists are searching for new agents against trematodiases. Redox metabolism is important for parasites as far as long-lived adult parasites inside a mammalian host are exposed to redox challenges. Antioxidants have been poorly studied as anthelmintic agents, in particular against the foodborne trematodes. Study of in vitro anthelmintic activity of nonenzymatic natural and synthetic antioxidants of various chemical structures was performed using standard motility and mortality assays against juvenile and adult Opisthorchis felineus worms. Promising agents have been found among both natural and synthetic compounds. The mitochondria-targeted antioxidant SkQ1 [10-(6'-plastoquinonyl)decyltriphenylphosphonium] in motility assays was as effective (half-maximal inhibitory concentration [IC50] 0.6-1.4 µM) as praziquantel (IC50 0.47-1.4 µM), and SkQ1 was significantly more effective than praziquantel in mortality assays. Moreover, extensive tegument damage of the adult fluke was revealed after SkQ1 treatment. Flavonoids manifested potency too, with IC50 values in a micromolar range (5.1-17.4 µM). Other natural and synthetic compounds tested against helminths were significantly less effective than praziquantel. Results of our study indicate that SkQ1 and flavonoids have high anthelmintic activities against the liver flukes. We propose that structure-activity relationship research might be worthwhile based on the structures of the most effective substances.

A comparative study of Helicobacter pylori infection in hamsters experimentally infected with liver flukes Opisthorchis felineus, Opisthorchis viverrini, or Clonorchis sinensis.

Helicobacter pylori causes a wide range of human diseases including cancer. Carcinogenic foodborne trematodes Opisthorchis viverrini, Clonorchis sinensis, and O. felineus might promote transmission and spread of H. pylori infection in the definitive mammalian host, which in turn might contribute to the liver fluke-associated malignancy. Our objectives were to find out whether liver flukes O. felineus, O. viverrini, and C. sinensis are carriers of Helicobacter pylori and to determine whether H. pylori is present in feces, bile, and stomach samples from the experimentally infected hamsters. We found that liver flukes are not reservoirs of H. pylori. Nevertheless, the prevalence of H. pylori and the H. pylori ureA gene copy number were significantly elevated after the infection. Overall, although the liver flukes O. felineus, C. sinensis, and O. viverrini are not reservoirs of H. pylori, the infection with the liver flukes significantly modifies the biliary and gut microbiota by increasing H. pylori abundance. This may be a feature of any liver fluke pathogenesis that have not previously been taken into account. Our findings appear to be novel in terms of comparative assessment of the host microbiota and Helicobacter abundance during epidemiologically important liver fluke infections.

Effects of Three-time Administration of a Supramolecular Complex of Praziquantel with Disodium Glycyrrhizinate on Trematode Opisthorchis felineus in Hamsters.

BACKGROUND: Praziquantel (PZQ) is the most commonly used anthelmintic drug for treating trematodiases. It was shown here that PZQ in complex with disodium glycyrrhizinate (PZQ-Na2GA, in the 1:10 ratio) has higher bioavailability than PZQ alone. Our aim was to determine the effects of three-time administration of PZQ-Na2GA in an experimental opisthorchiasis felinea model. METHODS: The PZQ-Na2GA complex (1:10) at a 400 mg/kg dose (meaning 36.4 mg/kg PZQ) was administered to Opisthorchis felineus-infected hamsters three times under a "9:00 am-6:00 pm-9:00 am" regimen (PZQ-Na2GA × 3). Effects of treatment were assessed as a reduction of helminth load in the hamsters and as changes in physiological, hematological, and blood biochemical parameters. The helminths extracted from the liver of the hamsters that received PZQ-Na2GA thrice were assayed for sensitivity to PZQ in vitro. RESULTS: PZQ-Na2GA × 3 reduced the number of O. felineus helminths in the liver by 87%, which is 30% better than a previously reported effect of one-time administration of the complex. Meanwhile, relative weights of the liver and thymus diminished, and some hematological parameters improved. The helminths extracted from the hamsters 1 month after the PZQ-Na2GA × 3 treatment showed elevated sensitivity to PZQ, as determined in vitro. CONCLUSION: Compared with previously published data on the effectiveness of various drugs in experimental opisthorchiasis felinea, PZQ-Na2GA × 3 exerts the most potent anthelmintic effect. In addition, PZQ-Na2GA × 3 improves physiological status of O. felineus-infected hamsters and sensitizes the surviving parasites to subsequent PZQ treatment.

EV-transported microRNAs of Schistosoma mansoni and Fasciola hepatica: Potential targets in definitive hosts.

Trematodes are widespread parasitic flatworms that significantly affect mankind either directly as human parasites, or indirectly via the infection of livestock and the related economic damage. The two most important trematode taxa are the blood flukes Schistosoma and the liver flukes Fasciola, but detection and differentiation of these parasites remains a challenge. Recently, microRNAs (miRNAs) were described from extracellular vesicles (EV) for both parasites secreted into respective hosts. These molecules have been proposed as mediators of parasite-host communication, and potential biomarkers for the detection of parasitic infections from host blood. Our aim here was to study similarities and differences in the miRNA complements of Schistosoma mansoni and Fasciola hepatica, EV-load in particular, to predict their targets and potential functions in the parasite-host interaction. We reanalyzed the known miRNA complements of S. mansoni and F. hepatica and found 16 and 4 previously overlooked, but deeply conserved miRNAs, respectively, further moving their complements closer together. We found distinct miRNA enrichment patterns in EVs both showing high levels of flatworm miRNAs with potential for the detection of an infection from blood. Two miRNAs of the protostome specific MIR-71 and MIR-277 families were highly expressed in EVs and could, therefore, have potential as biomarkers for trematode infection. Curiously, we identified nucleotide differences in the sequence of Mir-277-P2 between S. mansoni and F. hepatica that hold great promise for the distinction of both parasites. To test whether the EV-miRNAs of S. mansoni and F. hepatica could be modulating the expression of host genes, we predicted miRNA targets in 321 human and cattle messenger RNAs that overlapped between both hosts. Of several predicted targets, wnt signaling pathway genes stood out and their suppression likely leads to changes in the glucose concentration in host blood and the reduction of inflammatory and immune responses.