A comparative in vivo assessment of the artemisinin derivatives artesunate and artemether (150 and 300 mg/kg) and praziquantel (400 mg/kg) was conducted in a hamster model of opisthorchiasis caused by Opisthorchis felineus. Physiological, biochemical, and hematological parameters, including complete blood counts and peripheral blood cell composition, were evaluated in all experimental groups. The animals were infected with 100 metacercariae of O. felineus and 1.5 months post-infection received a single oral dose of the tested compounds. Food consumption was monitored before the experiment ended, after which worm burdens were quantified. The mean infestation intensity in hamsters was 41.9 ± 1.75 worms per animal. Artesunate and artemether produced mean worm burden reductions of 60% and 58% at 150 mg/kg and 84.5% and 82% at 300 mg/kg, respectively (p < 0.001). Artesunate showed statistically significant efficacy, and artemether demonstrated a trend-level effect, both exceeding that of praziquantel (65%). Unlike praziquantel, these compounds normalized alanine aminotransferase activity, which may suggest an absence of hepatotoxicity. Administration of artesunate and artemether at both doses normalized platelet, eosinophil, and basophil counts in infected animals. The results indicate that artemisinin derivatives may serve as promising alternatives for the treatment of O. felineus opisthorchiasis and warrant further preclinical and clinical evaluation.

BackgroundThe 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.ResultsDose- 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.ConclusionsArtemisinin 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.

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.

The Ku autoantigen is a human nuclear, DNA-binding heterodimer of 70kDa and 86kDa proteins. It is the target of autoantibodies in several autoimmune diseases. We now report the expression of a cDNA encoding the 70kDa Ku protein. Large amounts of protein were obtained using a recombinant baculovirus vector, in contrast with earlier unsuccessful attempts using other expression systems. We demonstrate that the 70kDa Ku protein is targeted to the nucleus and is associated with the nuclear matrix when expressed in the absence of the 86kDa Ku component. No post-translational modifications were observed. The 70kDa protein binds double and single-stranded DNA with very high affinity. Our results suggest that the baculovirus expression system may be of widespread use in the production and characterization of human autoantigens.

The combination of 4-week repeated social defeat stress (RSDS) and Opisthorchis felineus infection was modeled in C57BL/6 mice. Various parameters were compared between three experimental groups of male mice (SS: mice subjected to RSDS, OF: mice infected with O. felineus, and OF + SS: mice subjected to both adverse factors) and behavior-tested and intact (INT) controls. The combination caused liver hypertrophy and increased the blood level of proinflammatory cytokine interleukin 6 and proteolytic activity of cathepsin B in the hippocampus. Meanwhile, hypertrophy of the spleen and of adrenal glands was noticeable. Anxious behavior in the elevated plus-maze test was predominantly due to the infection, with synergistic effects of an interaction of the two adverse factors on multiple parameters in OF + SS mice. Depression-like behavior in the forced swimming test was caused only by RSDS and was equally pronounced in SS mice and OF + SS mice. Helminths attenuated the activities of cathepsin B in the liver and hypothalamus (which were high in SS mice) and increased cathepsin L activity in the liver. The highest blood level of corticosterone was seen in SS mice but was decreased to control levels by the trematode infection. OF mice had the lowest level of corticosterone, comparable to that in INT mice. Thus, the first data were obtained on the ability of O. felineus helminths-even at the immature stage-to modulate the effects of RSDS, thereby affecting functional connections of the host, namely "helminths -> liver <-> brain axis."

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.

We report here studies of the behavior of male mice of the inbred strain C57BL/6 after prolonged exposure to two factors – social stress and infections with Opisthorchis, a combination which is often seen in humans. Mice of four groups were compared: 1) mice with prolonged experience of defeat in intermale confrontations (30 days) (SS); 2) those infected with O. felineus helminths (six months) (OF); 3) animals subjected to both factors (SS + OF); and 4) mice experiencing neither factor (CON). The behavior of all animals was evaluated in an open field test including a box, which was empty for the first 3 min of the test and contained an unfamiliar male of the inbred strain BALB/c during the second 3 min. Social stress had a stronger influence on the behavioral parameters evaluated in this test than infection. SS mice were more active than all others in exploring the box containing an unfamiliar male: they climbed onto it much more frequently and had longer mean durations of time spent close to the box. In addition, during the first 3 min of the test, these animals displayed elevated exploratory activity (number of rearings by the wall), and greater numbers and durations of grooming episodes. Infected mice of the OF group showed no difference in behavior from the CON group in either the first 3 min or the second 3 min of the test. In mice with the combination of factors (SS + OF), nonsocial forms of behavior were also no different from those in controls and reactions to the unfamiliar male were weaker than in SS mice. These data lead to the conclusions that prolonged experience of defeats in intermale confrontations had a stronger influence on social and nonsocial behavior in mice than chronic infection of the animals with O. felineus helminths and that the combination of these factors reduces social interest in mice.

The purpose of the research is to evaluate the effect of albendazole as part of the supramolecular complex with disodium salt of glycyrrhizic acid obtained by solid-phase mechanical treatment.Materials and methods. The anthelmintic activity of the complex and its effect on the host organism was assessed on hamsters infected with Opisthorchis felineus by single and 7-fold administration at 45 days after infection. After 21 days, we counted the number of helminthes in the liver, and conducted a morphometric analysis of the liver and spleen, and detected biochemically the activity of alanine aminotransferase and aspartate aminotransferase in the animals’ blood serum.Results and discussion. The number of O. felineus significantly decreased after 7-fold, but not a single, administration of albendazole (ABZ) and ABZ-Na2GA complex (1 : 10). The administrated substances had no effect on the weight gain of the animals and the daily consumption of the pellets. At the same time, ABZ only as part of the complex normalized the weight of the liver and spleen in hamsters infected with O. felineus and reduced the alanine aminotransferase activity. Consequently, a longer administration of ABZ as part of the complex with disodium glycyrrhizinate has not only a pronounced anthelmintic effect, but also improves some of the physiological parameters of hamsters to a greater extent than a pure substance.

The behavior of male mice of the inbred C57B/6 strain was investigated after prolonged exposure to two factors ‒ social stress and helminth infection, the combination of which is often observed in humans. Four experimental groups of mice were compared: 1) with a long-lasting experience of social defeats in intermale confrontations (30 days) (SS); 2) infected with O. felineus helminths (6 months) (OF); 3) exposed to both factors (SS + OF); 4) intact mice without influence of any factors (CON). The behavior of all animals was assessed in the open field test with a box placed in it, which was empty for the first 3 min of the test and kept an unfamiliar male of the inbred BALB/c strain for the second 3 min. Social stress had a more pronounced effect on the assessed behavioral parameters of mice in this test than infection. Mice SS were the most active in exploring of the box with an unfamiliar male inside than other animals: they climbed on it much more often and had a longer average time spent near the box. In addition, they had increased exploratory (number of wall rearings) and grooming (the number and duration) activities. Behavior of infected OF mice did not differ from CON one in both the first and the second 3 min of the test. In mice with a combined effect of factors (SS + OF), non-social forms of behavior also did not differ from the control indices, and the reaction to an unfamiliar male in the box was weaker than in mice SS. It was concluded that long-lasting experience of social defeats in intermale confrontations has a more pronounced effect on the social and non-social behavior in mice, compared with chronic infection of animals with O. felineus helminths. Moreover, the combination of two influencing factors promotes to the decreasing of social interest in mice.

Interleukin-1 beta (IL-1β) is an indicator of liver infl ammatory processes in opisthorchiasis. Administration of curcumin and new complex of praziquantel with disodium glycyrrhizin ate reduced the parasitic load in O. felineus-infected animals. Moreover, the complex prevented the development of pathomorphological changes and reduced the IL-1β expression in the liver.

Background Nowadays, it is still important to develop effective anti-opisthorchiasis agents. In this work, we tested a complex of praziquantel (PZQ) with a plant origin compound-disodium glycyrrhizinate-in the ratio 1:10 PZQ:Na(2)GA, containing 11-fold less of the active ingredient. Our aim was to study various ways to treat trematode Opisthorchis felineus with this complex in vitro. Additionally, an in vitro comparison of the anthelmintic action was made among racemic-PZQ, (R)-PZQ, and (S)-PZQ on juvenile and adult maritae of O. felineus. Methods Worms extracted from the hamsters were subjected to various regimens of administration of the complex: once a day for 3 days or three times within 1 day. Moreover, mature maritae and juvenile worms of O. felineus were subjected to the comparison the anthelmintic effectiveness of racemic-PZQ, (R)-PZQ, and (S)-PZQ. Results The O. felineus maritae that received PZQ:Na(2)GA (1:10) thrice within 1 day were most strongly affected by the drug. Their motility substantially decreased already on the second day after the last dose, and the percentage of live worms by the end of the experimental period was the lowest. These results indicate a cumulative anthelmintic effect of this substance under the regimen "three times within 1 day." For the first time, we report that among the three substances (racemic-PZQ and two enantiomers), (R)-PZQ has the highest anthelmintic activity, toward both juvenile and sexually mature maritae of O. felineus. Conclusion These findings suggest that the development of a supramolecular complex of (R)-PZQ with disodium glycyrrhizinate and administration of this complex three times within 1 day are promising approaches.

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; Na(2)GA) 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.

Chronic stress is a risk factor for major depression. Social defeat stress is a well-validated murine model of depression. However, little is known about the gene activity dynamics during the development of a depression-like state. We analyzed the effects of social defeat stress of varying duration (10 and 30 days) on the behavioral patterns and prefrontal-cortex transcriptome of C57BL/6 mice. The 10-day exposure to social defeat stress resulted in a high level of social avoidance with no signs of depression-associated behavior. Most animals exposed to 30 days of social defeat stress demonstrated clear hallmarks of depression, including a higher level of social avoidance, increased immobility in the forced swimming test, and anhedonic behavior. The monitoring of transcriptome changes revealed widespread alterations in gene expression on the 10th day. Surprisingly, the expression of only a few genes were affected by the 30th day of stress, apparently due to a reversal of the majority of the early stress-induced changes to the original basal state. Moreover, we have found that glucocorticoid-sensitive genes are clearly stimulated targets on the 10th day of stress, but these genes stop responding to the elevated corticosterone level by the 30th day of stress. The majority of genes altered by the 30-day stress were downregulated, with the most relevant ones participating in chromatin modifications and neuroplasticity (e.g., guanine nucleotide exchange factors of the Rho-family of GTPases). Very different molecular responses occur during short-term and long-term social stress in mice. The early-stress response is associated with social avoidance and with upregulation and downregulation of many genes, including those related to signal transduction and cell adhesion pathways. Downregulation of a few genes, in particular, genes for histone-modifying methyltransferases, is a signature response to prolonged stress that induces symptoms of depression. Altogether, our data show that the development of depression under social stress conditions is correlated with suppression of the overactive molecular response to induced stress, involving gene regulatory resistance to glucocorticoid molecules, potentially via a chromatin remodeling mechanism.