Effect of the exposure period and concentration of aqueous and alcoholic zingiber (Zingiber officinale) extract on the mean of mortality for hydatid cyst's protocolises.

OBJECTIVE: To determine the anti-protocolises effect of aqueous and alcoholic zingiber extract. METHODS: The present experimental study was conducted from 01, July 2021 to 15. December, 2021 at the College of Education for Pure Sciences, Iraq, and comprised 25mg/mL, 50mg/mL, 100mg/mL and 200mg/mL concentrations of both aqueous and alcoholic extracts of zingiber officinale that were prepared and used in vitro on hydatid cyst protocolises with 10min, 20min, 30min, 40min, 50min and 60min exposure times. The data was analysed using statistical package for the social sciences (SPSS) version 10 by using ANOVA and t- test to determine the differences between groups. RESULTS: The concentration of 200mg/ml was more efficient against the protocolises for aqueous extract, while the alcoholic extract was effective at both 100mg/ml and 200mg/ml concentrations. There was a positive relationship of the degree of killing with both exposure time and concentration. CONCLUSIONS: Zingiber officinale could be a potential bioactive natural product and its active constituents could be isolated and used as anti-protoscolex without any side effects.

Repurposing of a library for high-content screening of inhibitors against Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis (CE) is a zoonotic disease caused by the larval stage of the dog tapeworm Echinococcus granulosus sensu lato (E. granulosus), with a worldwide distribution. The current treatment strategy for CE is insufficient. Limited drug screening models severely hamper the discovery of effective anti-echinococcosis drugs. METHODS: In the present study, using high-content screening technology, we developed a novel high-throughput screening (HTS) assay by counting the ratio of propidium iodide-stained dead protoscoleces (PSCs) to the total number of PSCs. In vitro and ex vivo cyst viability assays were utilized to determine the effect of drugs on cyst viability. RESULTS: Using the newly established HTS assay, we screened approximately 12,000 clinical-stage or The Food and Drug Administration (FDA)-approved small molecules from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library, as well as the LOPAC1280 and SelleckChem libraries, as a strategic approach to facilitate the drug discovery process. Initial screening yielded 173 compounds with anti-echinococcal properties, 52 of which demonstrated dose-response efficacy against E. granulosus PSCs in vitro. Notably, two agents, omaveloxolone and niclosamide, showed complete inhibition upon further validation in cyst and microcyst viability assays in vitro after incubation for 3 days, and in an ex vivo cyst viability assay using cysts isolated from the livers of mice infected with E. granulosus, as determined by morphological assessment. CONCLUSIONS: Through the development of a novel HTS assay and by repurposing libraries, we identified omaveloxolone and niclosamide as potent inhibitors against E. granulosus. These compounds show promise as potential anti-echinococcal drugs, and our strategic approach has the potential to promote drug discovery for parasitic infections.

Comparative study of the anthelmintic activity of ethanolic and aqueous extracts of Atriplex halimus, the anti-echinococcal plant used in Algeria.

Cystic echinococcosis (CE) remains a major human public health problem in Algeria. Surgical treatment is the gold standard. However, one of the most critical complications of surgery is a recurrence of the disease, which is attributed to the dissemination of protoscoleces during the operation. This study aimed to compare the scolicidal effect of two ethanolic and aqueous extracts of Atriplex halimus, a plant used by the Algerian population against CE. The comparison involved the assessment of the phytochemical content, making screening using GC-MS/MS, and the evaluation of the antioxidant activity using DPPH and the in vitro scolicidal effect of A. halimus extracts. The aqueous and ethanolic extracts of leaves contained 19.601 ± 0.016 and 15.406 ± 0.003 mg of gallic acid equivalent per g of extract (GAE/g extract) of total phenolic. However, the flavonoid content in ethanolic and aqueous extracts was 4.350 ± 0.023 and 1.995 ± 0.026 mg of quercetin equivalent per g (QE/g extract), respectively. Four compounds were identified by GC-MS/MS for the ethanolic extract, while twenty-three compounds were determined for the aqueous extract. The ethanolic and aqueous extracts of A. halimus demonstrated an antioxidant activity with IC50 = 0.850 ± 0.026 mg/ml and IC50 = 0.897 ± 0.060 mg/ml, respectively. The higher in vitro scolicidal effect was 100% after 90 min and 10 min at 100 and 150 mg/ml, respectively for the ethanolic extract and after 120 and 60 min at 100 and 150 mg/ml, respectively for the aqueous extract. Atriplex halimus could be used as a natural source for the production of a potential scolicidal agent for use during CE surgery and/or percutaneous interventions.

Anti-echinococcal effect of metformin in advanced experimental cystic echinococcosis: reprogrammed intermediary carbon metabolism in the parasite.

Metformin, a safe biguanide derivative with antiproliferative properties, has shown antiparasitic efficacy against the Echinococcus larval stage. Hence, we assessed the efficacy of a dose of 250 mg kg-1 day-1 in experimental models of advanced CE, at 6 and 12 months post-infection with oral and intraperitoneal administration, respectively. At this high dose, metformin reached intracystic concentrations between 0.7 and 1.7 mM and triggered Eg-TOR inhibition through AMPK activation by AMP-independent and -dependent mechanisms, which are dependent on drug dose. Cystic metformin uptake was controlled by increased expression of organic cation transporters in the presence of the drug. In both experimental models, metformin reduced the weight of parasite cysts, altered the ultrastructural integrity of their germinal layers, and reduced the intracystic availability of glucose, limiting the cellular carbon and energy charge and the proliferative capacity of metacestodes. This glucose depletion in the parasite was associated with a slight increase in cystic uptake of 2-deoxiglucose and the transcriptional induction of GLUT genes in metacestodes. In this context, drastic glycogen consumption led to increased lactate production and altered intermediary metabolism in treated metacestodes. Specifically, the fraction of reducing soluble sugars decreased twofold, and the levels of non-reducing soluble sugars, such as sucrose and trehalose, were modified in both cystic fluid and germinal cells. Taken together, our findings highlight the relevance of metformin as a promising candidate for CE treatment and warrant further research to improve the therapeutic conditions of this chronic zoonosis in humans.

Efficacy and mechanism of energy metabolism dual-regulated nanoparticles (atovaquone-albendazole nanoparticles) against cystic echinococcosis.

BACKGROUND: Albendazole (ABZ) and atovaquone (ATO) achieve killing efficacy on Echinococcus granulosus (Egs) by inhibiting energy metabolism, but their utilization rate is low. This study aims to analyze the killing efficacy of ABZ-ATO loading nanoparticles (ABZ-ATO NPs) on Egs. METHODS: Physicochemical properties of NPs were evaluated by ultraviolet spectroscopy and nanoparticle size potentiometer. In vitro experiments exmianed the efficacy of ATO, ABZ, or ATO-ABZ NPs on protoscolex activity, drug toxicity on liver cell LO2, ROS production, and energy metabolism indexes (lactic dehydrogenase, lactic acid, pyruvic acid, and ATP). In vivo of Egs-infected mouse model exmianed the efficacy of ATO, ABZ, or ATO-ABZ NPs on vesicle growth and organ toxicity. RESULTS: Drug NPs are characterized by uniform particle size, stability, high drug loading, and - 21.6mV of zeta potential. ABZ or ATO NPs are more potent than free drugs in inhibiting protoscolex activity. The protoscolex-killing effect of ATO-ABZ NPs was stronger than that of free drugs. In vivo Egs-infected mice experiment showed that ATO-ABZ NPs reduced vesicle size and could protect various organs. The results of energy metabolism showed that ATO-ABZ NPs significantly increased the ROS level and pyruvic acid content, and decreased lactate dehydrogenase, lactic acid content, and ATP production in the larvae. In addition, ATO-ABZ NPs promoted a decrease in DHODH protein expression in protoscolexes. CONCLUSION: ATO-ABZ NPs exhibits anti-CE in vitro and in vivo, possibly by inhibiting energy production and promoting pyruvic acid aggregation.

Activating the iNOS regulatory pathway by arginine deprivation targets energy metabolism to induce autophagy-dependent apoptosis against spinal echinococcosis.

Spinal echinococcosis is one of the most overlooked zoonotic parasitic diseases worldwide. There is currently no safe and effective treatment to eradicate it, and research based on the physiological-metabolic signature of the disease is lacking. Herein, we repurposed agrimol B as a potent anti-hydatid compound and validated its pharmacological mechanism based on arginine uptake as a target through multi-omics sequencing. This herbal component suppressed energy metabolism and activated ROS aggregation by inducing mitochondrial membrane potential depolarization, which subsequently triggered autophagy-dependent apoptosis leading to parasite death. Moreover, we discovered that arginine deprivation induced metabolic changes led to a shift from ornithine to nitrogen oxide synthesis, thus boosting the iNOS enzyme-regulated dominant metabolic pathway. The excess NO targeted the mitochondrial respiratory chain complex IV to disrupt energy metabolic homeostasis and induced a downstream pathological waterfall effect to kill the hydatid. A novel metabolic regulatory mechanism targeting mitochondrial damage for arginine starvation therapy was discovered. Finally, arginine depletion was found to be superior to the anti-spinal echinococcosis effect of albendazole and accompanied by the potential for disc protection. This study unveils the role of arginine in the physiological metabolism of Echinococcus granulosus and reveals the value of targeting arginine metabolism as a potential therapy. In addition, agrimol B is proposed as a promising therapeutic strategy for spinal echinococcosis to block arginine uptake and break this parasite's metabolic balance.

Usefulness of a murine model of hepatic cystic echinococcosis for preclinical drug trials: Efficacy of albendazole vs albendazole nanocrystals.

Cystic echinococcosis is a zoonotic infection caused by the larval stage of Echinococcus granulosus sensu lato. The disease is characterized by the long-term growth of cysts, most commonly in the liver and lungs. Although an ideal model of cystic echinococcosis should induce the development of cysts in the liver and imitate the natural infection route, the murine model of intraperitoneal is still widely used in the field of experimental theraphy. The aim of the present work was to evaluate the usefulness of the murine model of hepatic CE for preclinical drug trials. The effectiveness of albendazole could also be assessed by measuring the diameter of the hepatic cyst. The albendazole significantly reduced the size of the cysts. The ultrastructural alterations of the germinal layer of hepatic cysts provoked by albendazole coincided with those observed in the intraperitoneal model. Similar results were obtained with both albendazole doses. Therefore, the efficacy of albendazole nanocrystals in the murine model of hepatic cystic echinococcosis was carried out at albendazole doses of 25 mg/kg. The abdominal ultrasound allows us to assess the response of cysts to drugs only in a qualitative manner. Although the size of cysts in the albendazole nanocrystal group was not significantly lower than that observed with albendazole, at the ultrastructural level, a greater extent of damage was observed. The murine model of hepatic cystic echinococcosis can be effectively used for assessing the effect of novel formulations or compounds. The main advantage of this model is that cysts are located in the orthotopic organ, which resembles the location most commonly found in human cases. In future studies, the usefulness of the model for pharmacokinetics studies in hepatic cysts will be evaluated.

In vitro and ex vivo protoscolicidal effect of poly(amidoamine) nanoemulsion against Echinococcus granulosus.

Hydatidosis causes a serious health hazard to humans and animals leading to significant economic and veterinary and public health concern worldwide. The present study aimed to evaluate the in vitro and ex vivo protoscolicidal effects of synthesized poly(amidoamine), PAMAM, nanoemulsion. In this study, PAMAM was characterized through dynamic light scattering technique to investigate the particle size and zeta potential of nanoemulsified polymer. For the in vitro and ex vivo assays, we used eosin dye exclusion test and scanning electron microscope (SEM) to evaluate the effects of the prepared and characterized PAMAM nanoemulsion against protoscoleces from Echinococcus granulosus sensu lato G6 (GenBank: OQ443068.1) isolated from livers of naturally infected camels. Various concentrations (0.5, 1, 1.5 and 2 mg/mL) of PAMAM nanoemulsion at different exposure times (5, 10, 20 and 30 min) were tested against protoscolices. Our findings showed that PAMAM nanoemulsion had considerable concentration- and time-dependent protoscolicidal effect at both in vitro and ex vivo experiments. Regarding in vitro assay, PAMAM nanoemulsion had a potent protoscolicidal effect when compared with the control group with a highest protoscolicidal activity observed at the concentration of 2 mg/mL at all exposure times, such that 100% of protoscolices were killed after 20 min of exposure. Also, the mortality of protoscolices was 100% after 30 min of exposure to 1 and 1.5 mg/mL of PAMAM nanoemulsion, in vitro. Concerning ex vivo assay PAMAM nanoemulsion recorded the highest mortality rates at the concentration of 2 mg/mL (55, 99.4 and 100% at 10, 20, 30 min, respectively). Ultrastructure examination of examined protoscolices after 20 min of exposure to PAMAM nanoemulsion showed a complete loss of rostellar hooks, disruption of suckers with disorganization of hooks with partial or complete loss of them, and damage of protoscolices tegument with loss of their integrity in the form of holes and contraction of the soma region were observed in 1.5 and 2 mg/mL of PAMAM, in vitro and ex vivo, showing more damage in the in vitro conditions. It can be concluded that PAMAM nanoemulsion is a promising protoscolicidal agent offering a high protoscolicidal effect at a short exposure time. Further in vivo studies and preclinical animal trials are required to evaluate its efficacy and clinical applications against hydatid cysts.

Baiting not-owned dogs against Echinococcus granulosus: innovative tools for integrated control.

Cystic echinococcosis (CE), caused by the larval stage of the cestode Echinococcus granulosus, is one of the most widespread zoonoses in Mediterranean countries. Baiting not-owned dogs with praziquantel (PZQ), due to their key role in the maintaining the transmission of CE, currently appears to be the most effective way to limit the transmission of CE, as well as an important aspect to introduce for the control of this parasitic disease. Therefore, this study aims to test 3 types of PZQ-based baits by evaluating different parameters (integrity over time, attractiveness and palatability for dogs, and mechanical resistance after release to different altitudes) and the bait acceptance in field by target animals, i.e. not-owned dogs, by using camera traps. The double PZQ-laced baits (with a double layer of highly palatable chews) showed the greatest resistance in the environment while also preserving the attractiveness and palatability up to 10 days, also withstood heights of 25 m, thus resulting as the most suitable also for drone delivery. The results on the field showed that most of the baits were consumed by not-owned dogs (82.2%), while the remaining were consumed by wild boars (8.9%), foxes (6.7%), badgers (1.1%) and hedgehogs (1.1%), confirming the specific and high attractiveness of the double PZQ-laced baits for the target population and highlights how an anthelmintic baiting programme may be a viable tool for the management of E. granulosus among free-ranging dog populations in endemic rural areas.

Comparison of the Scolicidal Activity of Two Leaves Extracts of Myrtus communis from Algeria Against Echinococcus granulosus Sensu Lato Protoscoleces.

PURPOSE: During cystic echinococcosis surgery, the use of scolicidal agents such as hypertonic saline (20%) aims to reduce the risk of infection recurrence, but most of the used agents are associated with undesirable side effects. Therefore, the use of natural scolicidal agents such as medicinal plant extracts could reduce these medical issues. The present study aimed to compare in vitro the scolicidal activity between two extracts of the medicinal plant Myrtus communis from Algeria against Echinococcus granulosus sensu lato protoscoleces. METHODS: The ethanolic and aqueous extraction of plant leaves was performed. Phytochemical analysis by gas chromatography-tandem mass spectrometry (GC-MS/MS), determination of total phenolic and flavonoid contents, and in vitro antioxidant activity by DPPH were evaluated for both extracts. Finally, the in vitro scolicidal activity was tested by different concentrations. The viability was evaluated by the eosin exclusion test. RESULTS: The phytochemical analysis revealed 28 components for the ethanolic extract and 44 components for the aqueous extract. The major components were 2'-hydroxy-5'-methoxyacetophenone and 4-amino-2-methylphenol, respectively. The total phenolic and flavonoid contents were 45.9 ± 0.085 mg of gallic acid equivalent per g of extract (GAE/g E) and 16.5 ± 0.004 mg of quercetin equivalent per g (QE/g E) for the ethanolic extract, and 36.5 ± 0.016 mg GAE/g E and 18.2 ± 0.023 mg QE/g E for the aqueous extract, respectively. Furthermore, ethanolic and aqueous extracts of M. communis gave a value of IC50 = 0.009 ± 0.0004 mg/ml and IC50 = 0.012 ± 0.0003 mg/ml for the antioxidant activity, respectively. The in vitro scolicidal activity with concentrations of 50, 75, 100, and 150 mg/ml was tested for 5, 10, 15, and 30 min, and 5, 10, 15, 30, 60, 90, and 120 min for ethanolic and aqueous extracts, respectively. The mortality rate of protoscoleces at concentrations of 100 and 150 mg/ml was 98.8 and 100%, respectively, after 5 min of exposure to the ethanolic extract, while this rate was 100% at the same concentrations only after 60 min of exposure to the aqueous extract. CONCLUSIONS: The ethanolic extract showed a stronger scolicidal activity against E. granulosus s.l protoscoleces than the aqueous extract. In the future, other investigations are necessary to elucidate the mechanism of action and the possible toxicity on human cells. Moreover, experimental animal studies are required to investigate the efficacy of different extracts of this plant and its components as natural anti-parasitic alternatives for the treatment of human cystic echinococcosis.

Assessing the Potential Apoptotic Effects of Different Hydatid Cyst Fluids on Human Healthy Hepatocytes and Hepatocellular Carcinoma Cells.

Cystic Echinococcosis (CE) is a zoonotic infection caused by the larval form of Echinococcus granulosus in humans. Emerging evidence suggests an intriguing inverse association between E. granulosus infection and the occurrence of cancer. This study aimed to investigate the influence of diverse host-derived hydatid cyst fluids (HCF) with distinct genotypes on human liver hepatocytes (HC) and hepatocellular carcinoma cells (HepG2). Specifically, we examined their effects on cell proliferation, apoptosis sensitivity (BAX/BCL-2), apoptosis-related p53 expression, and the expression of cancer-related microRNA (hsa-miR-181b-3p). Cell proliferation assays, real-time PCR, and ELISA studies were conducted to evaluate potential anti-cancer properties. The findings revealed that animal-origin HCF (G1(A)) induced direct cell death by augmenting the susceptibility of HepG2 cells to apoptosis. Treatment with both G1(A) and G1(H) HCF sensitized HepG2 and HC cell lines to apoptosis by modulating the BAX/BCL-2 ratio, accompanied by upregulation of the p53 gene. Additionally, G1(A) HCF and human-derived HCFs (G1(H), G7(H)) reduced the expression of miR-181b-3p in HepG2 cells. Consequently, this study demonstrates the potential anti-cancer effect of HCF in HepG2 cells and provides the first comparative assessment of HCFs from human and animal sources with diverse genotypes, offering novel insights into this field.

Synergism therapeutic and immunoregulatory effects of Albendazole + rAd-mIL-28B against Echinococcosis in experiment-infected mice with protoscoleces.

The metacestode stage of Echinococcus granulosus can cause cystic echinococcosis (CE), which still widely occurs around the world. Since the early 1970s, benzimidazoles have been shown to inhibit the growth of cysts and used to treat CE. However, benzimidazoles are still ineffective in 20%-40% of cases. In order to explore the new agents against CE, we have investigated the therapeutic effect of the recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) on protoscoleces-infected mice. In our study, we successfully established the model mice which infected with protoscoleces intraperitoneally. At 18 weeks post-infection, the mice received rAd-mIL-28B (1×107 PFU) weekly by intramuscular injection for 6 weeks. Compared with the untreated control (13.1 ± 2.2 g), there was a significant reduction in cysts wet weight in rAd-mIL-28B group (8.3 ± 3.5 g) (P < 0.05), especially in Albendazole (ABZ) + rAd-mIL-28B group (5.8 ± 1.4 g) (P < 0.01). We also observed the severe damage of the germinal layer and the laminated layer of cysts after treatment. rAd-mIL-28B group showed a prominent increase in the level of Th1 type cytokines (such as IFN-γ, IL-2 and TNF-α). Meanwhile, the frequency of Foxp3+ T cells was decreased in the rAd-mIL-28B group (4.83 ± 0.81%) and ABZ + rAd-mIL-28B group (4.60 ± 0.51%), comparing with the untreated group (8.13 ± 2.60%) (P < 0.05). In addition, compared with the untreated control (122.14 ± 81.09 pg/ml), the level of IFN-γ significantly increased in peritoneal fluid in the rAd-mIL-28B group (628.87 ± 467.16 pg/ml) (P < 0.05) and ABZ + rAd-mIL-28B group (999.76 ± 587.60 pg/ml) (P < 0.001). Taken together, it suggested that ABZ + IL-28B may be a potential therapeutic agent against CE.

Protoscolicidal activity of Atriplex halimus leaves extract against Echinococcus granulosus protoscoleces.

Cystic echinococcosis, an endemic zoonosis in Algeria, is caused by the development of the helminth Echinococcus granulosus. Surgery remains the main treatment despite inducing relapse and several adverse reactions. In this context, natural scolicidal agents seem to be promising tools to overcome these reactions. In our study, we evaluated the phytochemical contents, antioxidant activity and scolicidal effect of Atriplex halimus. In this context, the aqueous extract from AH leaves (AHE) was subjected to preliminary phytochemical screening by HPLC. The in vitro antioxidant activity was determined by DPPH test. The cytotoxicity of AHE was evaluated in murine peritoneal macrophages and cell viability was examined by MTT assay. Moreover, different concentrations of AHE (20, 40, 50, 60 and 100 mg/ml) were tested on E. granulosus protoscoleces (PSC) cultures, during different times of incubation (15, 30, 60, 90, 120 and 180 min). The viability was evaluated by eosin exclusion test. The morphological and ultrastructural damages were evaluated by SEM. Our results indicate that total phenolic and flavonoids contents were 37.93 µg of Gallic acid equivalent per mg of extract (GAE/mg E) and 18.86 µg of Quercetin equivalent per mg (QE/mg E) respectively. Furthermore, AHE has an antioxidant activity with an IC50 of 0.95 mg/ml. Interestingly, the extracts did not exhibit any cytotoxic effect against murine peritoneal macrophages. Moreover, our study indicated a significant scolicidal activity time- and dose-dependent. At 60 and 100 mg/ml; and after 120 min of incubation; the mortality rate was 99.36 and 100%, respectively. The parasite's tegument is one of the plant's targets as demonstrated by SEM. Our findings show the benefits of Atriplex halimus extract as a new promising scolicidal tool in hydatid cyst treatment.

In vitro effects of harmine against Echinococcus granulosus protoscoleces by stimulating DNA damage.

Cystic echinococcosis (CE), a parasitic larval cystic stage of a small taeniid-type tapeworm (Echinococcus granulosus), causes illness in intermediate hosts and has become a threat to global public health. Currently, chemical compounds recommended by the WHO targeting CE are albendazole and mebendazole, however, none of them shows enhanced efficacy. Novel molecular compounds are urgently required to treat this disease. Our group uncover a drug, termed harmine (HM), that may be capable of treating CE. In this study, we aim to evaluate the anti-parasitic efficacy and the mechanism of DNA damage of HM against E. granulosus. In vitro, the results indicated that, within two and three days of treatment, ABZ killed 30.4% and 35.3% of protoscoleces, whereas HM killed 52.7% and 100% of protoscoleces, respectively. Furthermore, the presence of abnormalities in the internal structure of protoscoleces was examined by ultrastructural images of TEM, and the result showed that there were scattered nucleoli and heterochromatin margination phenomenon by HM treatment. DNA damage of protoscoleces was examined by using the comet assay, and results showed the DNA of protoscoleces was damaged. Moreover, EgATM, EgP53, EgTopo2a and EgRad54 genes were used to support the DNA damage by HM treatment, and results showed that all four genes were upregulated expression. In further, the result of HM treatment was tested by using designed siRNA to inhibit the expression of EgTopo2a and EgRad54. The results demonstrated that the viability was 88.75 ± 2.11% after suppressing the expression of EgTopo2a, which was significantly higher than that for HM alone group (P < 0.01). The viability was 10.11 ± 2.60% after transfected with EgRad54 siRNA, which was significantly lower compared with the HM alone group (P < 0.01). Based on our preliminary data, HM demonstrated significant parasiticidal activity against E. granulosus in vitro without obvious toxicity towards its host cells, suggesting that HM can be a potential anti-echinococcosis drug. HM was found to induce DNA damages of CE by activating the EgATM-EgP53-EgTopo2a signaling pathway. We therefore surmise that DNA damage response may be one of the mechanisms of HM against the parasite.

Effects of Zataria multiflora essential oil on the germinative cells of Echinococcus granulosus.

BACKGROUND: Novel and more efficient compounds are urgently required for medical treatment of cystic echinococcosis (CE). Germinative cell culture of Echinococcus granulosus could be used for anti-echinococcosis agent tests and other biological studies on CE. This study was performed to establish an in vitro cell culture model for E. granulosus germinative cells and to evaluate the lethal effect of Zataria multiflora essential oil (ZMEO) on the cultured cells. METHODS: The inner surface of germinal layers of CE cysts was scraped, and the obtained materials were trypsinized to obtain a suspension of single germinative cells. Medium 199 was used as the basic culture medium and was supplemented with fetal bovine serum, 2-mercaptoethanol, L-cysteine, L-glutamine, glucose, sodium pyruvate, hydatid fluid, amphotericin B and antibiotics. The cells were cultured at a concentration of 104 cells/ml of culture medium and incubated at 37 °C. The culture medium was replaced every 7 days. Chemical composition of ZMEO was identified by GC-MS analysis. ZMEO was tested at concentrations of 0.5-8 mg/ml. Viability of the cells was assessed by trypan blue exclusion assay. RESULTS: A significant increase in the cell number was evident at 20, 30 and 45 days after cultivation. At 45 days of cultivation, the number of cells was approximately five-fold higher than on the first day. In GC-MC analysis, carvacrol, p-cymene, g-terpinene and thymol were found to be the main compounds of ZMEO. The lethal effect of ZMEO on the germinative cells at concentrations of 6, 7 and 8 mg/ml was 100% after 60, 25 and 7 min of exposure, respectively. CONCLUSIONS: At 45 days of cultivation, the cell concentration was suitable for the desired in vitro experiments. A high lethal effect of ZMEO on the germinative cells of E. granulosus may be considered an opportunity for the introduction of a novel, more effective and safer therapeutic agent for treatment of CE using an herbal product.

In vitro effects of tropisetron and granisetron against Echinococcus granulosus (s.s.) protoscoleces by involvement of calcineurin and calmodulin.

BACKGROUND: Cystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato  (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs. METHODS: Protoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively. RESULTS: At 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 µM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 µM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations. CONCLUSIONS: Tropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE.

The Effects of Nitric Oxide and Inhibitor, and Combination of Albendazole and Praziquantel On Liver in Mice Injected with Echinococcus granulosus Larvae.

In this study, the role of nitric oxide (NO) in the pathogenesis of hydatidosis and the interaction with effects of anthelmintic drugs, albendazole and praziquantel, were examined in larval infection caused by protoscolices obtained from hydatid cysts of sheep liver in Albino Balb/c mice. Animals were divided into ten groups including controls and infected groups. Larval infection was established with intraperitoneal injection of protoscolices. Eight months after infection with protoscolices, the infected animals were divided into 6 groups. The infected animals were given a selective inhibitor of inducible nitric oxide synthase (iNOS) L-N6-(1-Iminoethyl) lysine-hydrochloride (L-NIL), NO donor sodium nitroprusside (SNP), albendazole and praziquantel as anthelmintic drugs for 7 days. In addition, control groups were composed of intact group, control, anthelmintic drugs + L-NIL, and anthelmintic drugs + SNP. The liver and blood samples were taken for cytological, histological, immunohistochemical and biochemical analyses 7 days after treatments at the end of experiment. The animals injected with protoscolices showed histopathological changes including inflammation areas, infiltration and accumulation of leukocytes, dilation of sinusoids, and damage in endothelial cells and hepatocytes at light microscopy. Electron microscopy were revealed severe damage in sinusoidal endothelial cells, leukocytes especially eosinophils in sinusoid lumens and disorganization in endoplasmic reticulum and nuclear membrane. Endothelial nitric oxide synthase (eNOS) and iNOS reactions were increased in the tissue. Anthelmintic drugs decreased inflammation areas and damages; however, it did not change NOS reactions in the animals given protoscolices. L-NIL and SNP diminished both iNOS and eNOS reactions. Unlike the group administered the inhibitor, SNP treated group exhibited less inflammation areas. Combination of these substances and drugs resulted in decreased inflammation areas. eNOS and iNOS reactions decreased in the drugs and SNP administered group, while only iNOS reaction was decreased in L-NIL given infection group. In addition, the infected groups which received SNP displayed expanded sinusoids and hepatocytes with vacuoles, intriguingly. While levels of serum nitrite/nitrate elevated only in the infection group given drugs and SNP, it decreased in the L-NIL administered group. Tissue level of malondialdehyde increased in infection groups with drugs and SNP. In conclusion, the results indicated that NO plays an important role in the pathogenesis of hydatidosis.

Chemical Composition, Apoptotic Activity, and Antiparasitic Effects of Ferula macrecolea Essential Oil against Echinococcus granulosus Protoscoleces.

BACKGROUND: Today, the present protoscolicidals used to minimize the serious risks during hydatid cyst surgery are not completely safe and have various adverse side effects. The present study aimed to evaluate the chemical composition and apoptotic activity of Ferula macrecolea essential oil (FMEO) as well as its in vitro and ex vivo protoscolicidal effects against hydatid cyst protoscoleces. METHODS: Gas chromatography/mass spectrometry (GC/MS) analysis was performed to determine the chemical composition of FMEO. Protoscoleces of hydatid cysts were collected from liver fertile hydatid cysts of infected sheep and were then treated with various concentrations of the essential oil (75, 150, and 300 µL/mL) for 5-60 min in vitro and ex vivo. Then, by using the eosin exclusion test, the viability of the protoscoleces was studied. The caspase-3-like activity of the FMEO-treated protoscoleces was also evaluated through the colorimetric protease assay Sigma Kit based on the manufacturer's instructions. RESULTS: According to GC/MS, the main constituents of the essential oil were terpinolene (77.72%), n-nonanal (4.47%), and linalool (4.35%), respectively. In vitro, the maximum protoscolicidal activity of FMEO was observed at the concentrations of 150 and 300 µL/mL, such that 100% of the protoscoleces were killed after 30 and 20 min of exposure, respectively. Based on the obtained findings, the results demonstrate that FMEO required a longer time to kill protoscoleces ex vivo; after 12 min of exposure to FMEO, only 13.4% of the protoscoleces remained alive. After 48 h of the treatment of protoscoleces, FMEO, in a dose-dependent manner and at doses of 75, 150, and 300 µL/mL, induced the activation of the caspase enzyme by 24.3, 35.3, and 48.3%, respectively. CONCLUSIONS: Our findings demonstrate the potent protoscolicidal effects of FMEO in vitro and ex vivo; however, further studies are required to assess the safety and the efficiency of FMEO as a promising scolicidal agent in a preclinical model and clinical setting.

Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model.

BACKGROUND: Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. METHODS: The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. RESULTS: In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 µg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. CONCLUSIONS: Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

Copper nanoparticles: Biosynthesis, characterization, and protoscolicidal effects alone and combined with albendazole against hydatid cyst protoscoleces.

BACKGROUND: Surgery remains the preferred treatment option for hydatid cyst (cystic echinococcosis); however, recent studies have demonstrated that the current protoscolicidal agents used during surgery are associated with some adverse side effects such as biliary fibrosis, hepatic necrosis, and cirrhosis. The present study aims to evaluate the in vitro and ex vivo anti-parasitic effects of copper nanoparticles (CuNPs) alone and combined with albendazole on hydatid cyst protoscoleces. METHODS: CuNPs was green synthesized using C. spinosa extract. Various concentrations of CuNPs (250, 500, and 750 mg/mL) alone and combined with albendazole (ALZ, 200 mg/mL) were exposed to protoscoleces collected from the liver fertile hydatid cysts of infected sheep for 5-60 min in vitro and ex vivo. Next, the eosin exclusion test was applied to determine the viability of protoscoleces. Caspase-3 like activity of CuNPs-treated protoscoleces was then evaluated using the colorimetric protease assay Sigma Kit based on the manufacturer's instructions. RESULTS: Scanning electron microscopy (SEM) results showed that the particle size of CuNPs was 17 and 41 nm with the maximum peak at the wavelength of 414 nm. The maximum protoscolicidal activity of CuNPs was observed at the concentration of 750 mg/mL in vitro, so that 73.3 % of protoscoleces were killed after 60 min of exposure. Meanwhile, the mortality of protoscoleces was 100 % after 10 min of exposure to 750 mg/mL of CuNPs along with ALZ (200 mg/mL). Nevertheless, the findings proved that CuNPs even in combination with ALZ required a longer time to kill protoscoleces ex vivo. After 48 h of treating protoscoleces, CuNPs in a dose-dependent manner and at doses of 250, 500, and 750 mg/mL induced the caspase enzyme activation by 20.5 %, 32.3 %, and 36.1 %, respectively. CONCLUSION: The findings of the present investigation showed potent protoscolicidal effects of CuNPs, especially combined with albendazole, which entirely eliminated the parasite after 10-20 min of exposure. The results also showed that although the possible protoscolicidal mechanisms of CuNPs are not clearly understood, the inducing apoptosis through caspases is one of the main protoscolicidal mechanisms of CuNPs. However, supplementary studies, especially in animal models and clinical settings, are needed to approve these results.