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.

Prolonged liver fluke infection combined with alcoholization: An experimental mouse model.

Liver fluke infections disrupt the bile-excreting function of the human liver. Worldwide, excessive alcohol consumption also leads primarily to liver diseases. Our aim was to comprehensively assess the liver state in mice in parallel with the characterization of inflammation when the two adverse factors were combined. C57BL/6 mice were used for the experimental modeling; half of them beforehand were gradually accustomed to consumption of increasing doses of ethanol (from 5% to 20%). Then, half of the animals in each subgroup was infected with Opisthorchis felineus helminths. Finally, the infected (OF), 20% ethanol-consuming (Eth), and subjected to both factors (Eth + OF) mice were compared with no-treatment control. In OF and especially Eth + OF mice, relative liver weight was greater, activities of alanine aminotransferase and aspartate aminotransferase were higher, and bile ducts were considerably enlarged. Eth + OF mice contained noticeably more helminths in the liver than OF mice did. Massive cholangiofibrosis and periductal fibrosis were noted in the liver of the infected mice, especially Eth + OF ones. The liver fluke infection caused inflammatory infiltration and bile duct proliferation. Splenomegaly due to structural changes in the spleen as well as increased levels of interleukin 6 and leukocyte and monocyte counts in the blood reflected substantial inflammation in Eth + OF mice. Thus, in the proposed experimental model, it is shown that a double hit to the liver, i.e., the combination of O. felineus infection with prolonged alcoholization, can be detrimental to both the liver and whole body.

Characteristics of liver fibrosis associated with chronic Opisthorchis felineus infection in Syrian hamsters and humans.

The food-borne liver trematode Opisthorchis felineus causes severe liver damage, including fibrosis. This study shows a comparison of the characteristics between cholangiofibrosis and periductal fibrosis in infected people and in the golden hamster as an experimental model. Comparative evaluation was carried out regarding collagen composition, the number of basic-producing cells, and extracellular-matrix degradation. The results revealed that characteristics of chronic opisthorchiasis due to O. felineus infection in humans and in Syrian hamsters are similar and include well-pronounced development of fibrotic complications in the liver parenchyma. Besides, a difference in fibrogenesis development was demonstrated between chronic O. felineus infection and noninfectious cholecystitis. In this study, we for the first time compared fibrogenesis between humans and model animals against the background of chronic O. felineus infection.

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.

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.

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.

The pathogenic potential of the combined action of chronic Opisthorchis felineus infection and repeated social defeat stress in C57BL/6 mice.

Parasitic food-borne diseases and chronic social stress are frequent attributes of day-to-day human life. Therefore, our aim was to model the combined action of chronic Opisthorchis felineus infection and repeated social defeat stress in C57BL/6 mice. Histological examination of the liver revealed inflammation sites, pronounced periductal fibrosis, and cholangiofibrosis together with proliferation of bile ducts and hepatocyte dystrophy in the infected mice, especially in the stress-exposed ones. Simultaneously with liver pathology, we detected significant structural changes in the cerebral cortex. Immunohistochemical analysis of the hippocampus indicated the highest increase in numerical density of Iba 1-, IL-6-, iNOS-, and Arg1-positive cells in mice simultaneously subjected to the two adverse factors. The number of GFAP-positive cells rose during repeated social defeat stress, most strongly in the mice subjected to both infection and stress. Real-time PCR analysis showed that the expression of genes Aif1 and Il6 differed among the analysed brain regions (hippocampus, hypothalamus, and frontal cortex) and depended on the adverse factors applied. In addition, among the brain regions, there was no consistent increase or decrease in these parameters when the two adverse treatments were combined: (i) in the hippocampus, there was upregulation of Aif1 and no change in Il6 expression; (ii) in the hypothalamus, expression levels of Aif1 and Il6 were not different from controls; and (iii) in the frontal cortex, Aif1 expression did not change while Il6 expression increased. It can be concluded that a combination of two long-lasting adverse factors, O. felineus infection and repeated social defeat stress, worsens not only the hepatic but also brain state, as evidenced behaviorally by disturbances of the startle response in mice.

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.

Repeated and single maternal separation specifically alter microglial morphology in the prefrontal cortex and neurogenesis in the hippocampus of 15-day-old male mice.

Early-life adversity impairs neuronal plasticity of the developing brain. In rodents, brain maturation processes, including neuro- and synaptogenesis, myelination, microglial maturation, and hypothalamic-pituitary-adrenal (HPA) axis development continue in the postnatal period. In our study, two models of early-life stress were used: repeated maternal separation (MS) from postnatal day (PND) 2 to PND14 for 3 h daily and single maternal deprivation (MD) on PND9 for 24 h. Effects of each type of early-life stress on neuron density, neurogenesis, microglial morphology, and HPA axis programming were studied in 15-day-old male mice. Neither early-life stress paradigm affected the expression of stress-related genes (Crh, Avp, Crhr1, Crhr2, Nr3c1, and Nr3c2) and the serum level of corticosterone on PND15. Immunohistochemical analysis was performed on slices of the hippocampus and prefrontal cortex (PFC) with antibodies against a marker of mature neurons (NeuN), of microglia (Iba1), proliferating cells (Ki67), and immature neurons (DCX). We found higher density of ameboid microglia and intermediate microglia in the PFC in groups MS and MD, respectively, than in a control group. In both stressed groups, a higher number of Ki67-positive cells was noted in the dentate gyrus. Thus, in mice, the process of transformation of ameboid microglia into ramified ones as well as a neurogenesis reduction take place during the second postnatal week, whereas early-life stress can disturb these processes in a stress- and region-dependent manner.

Stress early in life leads to cognitive impairments, reduced numbers of CA3 neurons and altered maternal behavior in adult female mice.

The hippocampus is a crucial part of the limbic system involved both in cognitive processing and in the regulation of responses to stress. Adverse experiences early in life can disrupt hippocampal development and lead to impairment of the hypothalamic-pituitary-adrenal axis response to subsequent stressors. In our study, two types of early-life stress were used: prolonged separation of pups from their mothers (for 3 hours/day, maternal separation, MS) and brief separation (for 15 minutes/day, handling, HD). In the first part of our study, we found that adult female mice (F0) who had experienced MS showed reduced locomotor activity and impairment of long-term spatial and recognition memory. Analysis of various hippocampal regions showed that MS reduced the number of mature neurons in CA3 of females, which is perhaps a crucial hippocampal region for learning and memory; however, neurogenesis remained unchanged. In the second part, we measured maternal care in female mice with a history of early-life stress (F0) as well as the behavior of their adult offspring (F1). Our results indicated that MS reduced the level of maternal care in adult females (F0) toward their own progeny and caused sex-specific changes in the social behavior of adult offspring (F1). In contrast to MS, HD had no influence on female behavior or hippocampal plasticity. Overall, our results suggest that prolonged MS early in life affects the adult behavior of F0 female mice and hippocampal neuronal plasticity, whereas the mothers' previous experience has effects on the behavior of their F1 offspring through disturbances of mother-infant interactions.

Inhibition of Opisthorchis felineus glutathione-dependent prostaglandin synthase by resveratrol correlates with attenuation of cholangiocyte neoplasia in a hamster model of opisthorchiasis.

Food-borne trematodiases represent major neglected parasitic diseases. Trematodes of the family Opisthorchiidae including Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis are ranked eight on the global list of the 24 most prevalent food-borne parasites. Chronic O. felineus infection symptoms include precancerous lesions with the potential for malignancy. In recent decades, liver flukes of the family Opisthorchiidae have been extensively scientifically explored, however despite this the molecular mechanisms of O. felineus pathogenicity and its carcinogenic potential have not been studied. Opisthorchis felineus glutathione-dependent prostaglandin synthase (GST σ) is the major component of the excretory-secretory product of this liver fluke. We hypothesised that the activity of this enzyme is involved in the infection pathogenesis, including the formation of precancerous lesions. To test this hypothesis and to gain insights into the mechanisms of precancerous lesion formation, we (i) investigated whether excretory parasitic GST σ retains its enzymatic activity, (ii) tested resveratrol (RSV) as a possible inhibitor of this enzyme, and (iii) assessed biliary neoplasia and oxidative DNA damage as well as the expression of neoplasia and fibrogenesis marker genes after prolonged administration of RSV in a hamster model. RSV was found to inhibit GST σ enzymatic activity in a dose-dependent manner (R = 0.85, P < 0.001; half-maximal effective dose (ED50) = 48.6 µM). Prolonged administration of RSV significantly suppressed high-grade biliary neoplasia (P = 0.008), attenuated upregulation of hyperplasia and fibrogenesis-related genes (Tgfb, α-SMA and CK7), and decreased the elevated oxidative DNA damage. Taking into account that RSV can influence a wide range of pathways, further research is needed to confirm the role of GST σ in O. felineus pathogenicity. Nevertheless, the chemopreventive effect of RSV targeting biliary neoplasia formation might be useful for improving the outcomes in infected populations and represents a compelling rationale for RSV testing in combination chemotherapy of opisthorchiasis.

Effects of supramolecular complexation of praziquantel with disodium glycyrrhizinate on the liver fluke Opisthorchis felineus: An in vitro and in vivo study.

Millions of people worldwide have a chronic infection with the liver fluke Opisthorchis felineus, which causes opisthorchiasis in humans and animals. The only known effective drug for this disease is praziquantel (PrzQ); however, its efficacy is below 100%, and it has some adverse effects. In this study, a water-soluble complex of PrzQ with a disodium salt of glycyrrhizic acid (disodium glycyrrhizinate; Na2GA) in a 1:10 ratio (PrzQ:GA) allowed the PrzQ dose to be decreased 11-fold for effective killing of O. felineus. An in vitro experiment showed a sufficient anthelmintic efficiency of PrzQ:GA against both adult and juvenile O. felineus individuals. A Syrian golden hamster model of opisthorchiasis revealed a considerable anthelmintic effect at all tested PrzQ:GA doses (50, 100, 200, 400, and 1100 mg/kg) with the best performance at 400 and 1100 mg/kg. Further comparison of the effects of PrzQ (400 mg/kg) and PrzQ:GA (400 mg/kg) regarding the state of the host indicated significant advantages of the latter. Histological examination showed that PrzQ:GA was better at decreasing the O. felineus-induced inflammatory infiltration (as compared with PrzQ alone) as well as interfered with the development of epithelial dysplasia and metaplasia in large bile ducts and cholangiofibrosis. Both PrzQ and PrzQ:GA decreased the number of myeloid (CFU-GM) and erythroid (BFU-E + CFU-E) colonies induced by O. felineus infection. The drugs had no negative effect on the animal behavior in an open field test 2-4 h after administration. Thus, PrzQ:GA proved to be a good anthelmintic agent having no evident adverse effects on the host, thereby suggesting that further preclinical and clinical trials would be warranted.

Oncolytic effect of wild-type Newcastle disease virus isolates in cancer cell lines in vitro and in vivo on xenograft model.

Oncolyic virotherapy is one of the modern experimental techniques to treat human cancers. Here we studied the antitumor activity of wild-type Newcastle disease virus (NDV) isolates from Russian migratory birds. We showed that NDV could selectively kill malignant cells without affecting healthy cells. We evaluated the oncolytic effect of 44 NDV isolates in 4 histogenetically different human cell lines (HCT116, HeLa, A549, MCF7). The safety of the isolates was also tested in normal peripheral blood mononuclear (PBMC) cells. The viability of tumor cell lines after incubation with NDV isolates was evaluated by MTT. All cell lines, except for normal PBMC primary cells, had different degrees of susceptibility to NDV infection. Seven NDV strains had the highest oncolytic activity, and some NDV strains demonstrated oncolytic selectivity for different cell lines. In vivo, we described the intratumoral activity of NDV/Altai/pigeon/770/2011 against subcutaneous non-small cell lung carcinoma using xenograft SCID mice model. All animals were responsive to therapy. Histology confirmed therapy-induced destructive changes and growing necrotic bulk density in tumor tissue. Our findings indicate that wild-type NDV strains selectively kill tumor cells with no effect on healthy PBMC cells, and intratumoral virotherapy with NDV suppresses the subcutaneous tumor growth in SCID mice.

Transcriptional Activity of Heparan Sulfate Biosynthetic Machinery is Specifically Impaired in Benign Prostate Hyperplasia and Prostate Cancer.

Heparan sulfates (HSs) are key components of mammalian cells surface and extracellular matrix. Structure and composition of HS, generated by HS-biosynthetic system through non-template-driven process, are significantly altered in cancer tissues. The aim of this study was to investigate the involvement of HS-metabolic machinery in prostate carcinogenesis. Transcriptional patterns of HS-metabolic enzymes (EXT1, EXT2, NDST1, NDST2, GLCE, 3OST1/HS3ST1, SULF1, SULF2, HPSE) were determined in normal, benign, and cancer human prostate tissues and cell lines (PNT2, LNCaP, PC3, DU145). Stability of the HS-metabolic system patterns under the pressure of external or internal stimuli was studied. Overall impairment of transcriptional activity of HS-metabolic machinery was detected in benign prostate hyperplasia, while both significant decrease in the transcriptional activity and changes in the expression patterns of HS metabolism-involved genes were observed in prostate tumors. Prostate cancer cell lines possessed specific transcriptional patterns of HS metabolism-involved genes; however, expression activity of the system was similar to that of normal prostate PNT2 cells. HS-metabolic system was able to dynamically react to different external or internal stimuli in a cell type-dependent manner. LNCaP cells were sensitive to the external stimuli (5-aza-deoxycytidin or Trichostatin A treatments; co-cultivation with human fibroblasts), whereas PC3 cells almost did not respond to the treatments. Ectopic GLCE over-expression resulted in transcriptional activation of HS-biosynthetic machinery in both cell lines, suggesting an existence of a self-regulating mechanism for the coordinated transcription of HS metabolism-involved genes. Taken together, these findings demonstrate impairment of HS-metabolic system in prostate tumors in vivo but not in prostate cancer cells in vitro, and suggest that as a potential microenvironmental biomarker for prostate cancer diagnostics and treatment.