Silver Nanoparticles: Green Synthesis, Characterization, Blood Compatibility and Protoscolicidal Efficacy against Echinococcus granulosus

Spillage of protoscoleces within hydatid fluid during surgery for hydatid cyst is the main reason for its recurrence. Therefore, to inactivate the protoscoleces, various scolicidal substances have been tested. However, novel and more efficient agents are needed owing to several associated complications. This study focused on the effects of green synthetic Silver Nanoparticles (AgNPs) from Zizyphus spina- christi leaves on Echinococcus granulosus protoscoleces. Also, to evaluate the blood compatibility of Ag NPs. The Ag NPs were identified by ultraviolet-visible (UV-Visible) spectrophotometer, X-ray diffraction (XRD), Scanning electron microscopy imaging, and Energy-dispersive X-ray spectroscopy (EDX). Hydatid fluid was aspirated aseptically from cysts of infected sheep liver. The protoscoleces were exposed to Ag NPs at several concentrations. Also, scanning electron microscopy for ultrastructural changes and in vitro erythrocytes lysis was performed. The Ag NPs were spherical; the particles' size reached 50 nm, and presented a surface plasmon peak around 460 nm. The current study's findings indicated the powerful in vitro scolicidal efficacy of the green biosynthesized AgNPs. Several morphological alterations were observed on the protoscoleces by optical and scanning electron microscopy. Lysis of RBCs at different doses of Ag NPs was significantly (P≤0.05) less than the positive control value, thus proposing its biocompatibility. This work suggests that chemicals like polyphenols present in the extract of Z. spina- christi act as reducing and stabilizers agents to create Ag NPs Nevertheless, further investigations are needed to investigate the Ag NPs scolicidial effects in animal models.

Identification of Different Extracellular Vesicles in the Hydatid Fluid of Echinococcus granulosus and Immunomodulatory Effects of 110 K EVs on Sheep PBMCs

Echinococcosis, mainly caused by Echinococcus granulosus, is one of the 17 neglected tropical diseases. Extracellular vesicles (EVs) play an essential role in the host–parasite interplay. However, the EVs in the hydatid fluid (HF) of E. granulosus are not fully characterized. Herein, three different types of HF EVs, designated as 2 K, 10 K, and 110 K EVs based on the centrifugal force used, were morphologically identified. A total of 97, 80, and 581 proteins were identified in 2 K, 10 K, and 110 K EVs, respectively, 39 of which were commonly shared. Moreover, 11, 8, and 25 miRNAs were detected, respectively, and all of the 7 selected miRNAs were validated by qPCR to be significantly lower abundant than that in protoscoleces. It was further deemed that 110 K EVs were internalized by sheep peripheral blood mononuclear cells (PBMCs) in a time-dependent manner and thus induced interleukin (IL)-10, tumor necrosis factor-α (TNF-α), and IRF5 were significantly upregulated and IL-1β, IL-17, and CD14 were significantly downregulated (p < 0.05). These data demonstrate the physical discrepancy of three HF EVs and an immunomodulatory effect of 110 K EVs on sheep PMBCs, suggesting a role in immune responses during E. granulosus infection.

The secreted protein signature of hydatid fluid from pulmonary cystic echinococcosis

The vast majority of cystic echinococcosis cases in Southern Brazil are caused by Echinococcus granulosus and Echinococcus ortleppi. Comparative proteomic studies of helminths have increased the knowledge about the molecular survival strategies adopted by parasites. Here, we surveyed the protein contents of the hydatid fluid compartment of E. granulosus and E. ortleppi pulmonary bovine cysts, in an attempt to compare their molecular arsenal in this host-parasite interface. Hydatid fluid samples from three isolates of each species were analyzed by trypsin digestion and mass spectrometry. We identified 280 proteins in E. granulosus and 251 proteins in E. ortleppi, highlighting a core of 52 proteins common to all samples of hydatid fluid. The in silico functional analysis revealed important molecular functions and processes active in pulmonary cystic echinococcosis. Some were more evident in one species, such as apoptosis in E. ortleppi, and cysteine protease activity in E. granulosus, while many molecular activities have been found in fluids of both species, such as proteolysis, development signaling and extracellular structures organization. The similar molecular tools employed by E. granulosus and E. ortleppi for their survival within the host are potential targets for new therapeutic approaches to deal with cystic echinococcosis and other larval cestodiases.

In Vitro and in Vivo Scolicidal Activities of Holothuria leuco-spilota Extract and CeO2 Nanoparticles against Hydatid Cyst

Background: We aimed to investigate the scolicidal effects of Holothuria leucospilota extract and CeO2 nanoparticles against protoscoleces of hydatid cysts in-vitro and in-vivo. Methods: Hydatid cysts were collected from, Urmia slaughterhouses between years 2016-2017 and the hydatid fluid aspirated from the fertile cysts. Various concentration of H. leucospilota extract, CeO2 NPs and combination of CeO2-NPs/H. leucospilota were used for 10-60 min to evaluate the viability of protoscoleces by 0.1% eosin method. CASPASE -3 activity measured for assessment of cell apoptosis in treated protoscoleces. BALB/c mice were infected intraperitoneally with 2000 viable protoscoleces and treated daily for 4 wk by intragastrical inoculation with H. leucospilota, CeO2 NPs, combination of CeO2 NPs/H. leucospilota and Albendazole. Cyst development was macroscopically analyzed. Results: H. leucospilota extract and combination of CeO2 NPs/H. leucospilota have potent scolicidal activity at concentration of 20 mg/ml and 15 mg/ml after 60 min treatment. Maximum caspase-3 activity was observed when protoscoleces expose with H. leucospilota and combination H. leucospilota & CeO2 NPs. After treatment of cyst infected mice with extract and CeO2 NPs, combination of CeO2 NPs/H leucospilota and albendazole, a significant decrease in number of cysts, size and volume of cyst (P<0.05) was observed. Conclusion: This result shows an antihydatic and scolicidal effects of H. leucospilota extract and CeO2 NPs.

In-vitro evaluation of Nigella sativa and Punica granatum effect on protoscolices of hydatid cysts

Cystic echinococcosis (CE) are commonly found in the liver and lungs of affected hosts. The treatment approach is usually surgical, or giving drugs in conjunction before surgery to kill protoscolices, to avoid anaphylactic shock from leakage of hydatid fluid into the peritoneum and to decrease opportunities for recurrences. The present study was to evaluate the in vitro scolicidal efficacy of hydroalcoholic extract of Punica granatum peel and Nigella sativa, on the protoscolices of CE that collected from the lungs of infected camels. Different concentrations of extracts with different exposure times were used and a viability assay was applied to measure the scolicidal effect. N. sativa showed its highest scolicidal efficacy at 100 mg/mL and 10 mg/mL concentrations after 30 and 60 min. P. granatum peel extract showed its maximum scolicidal efficacy at 100 mg/mL concentration after 120 min. All experiments of the current study revealed that the extracts of both N. sativa and P. granatum had a scolicidal effects on the protoscolices of camel hydatid cysts. It could be concluded that N. sativa extract is more potent than P. granatum peel extract regarding scolicidal effect, but the efficacies of both extracts were of moderate significant correlation to exposure time and concentrations.

Antigen B from Echinococcus granulosus enters mammalian cells by endocytic pathways

Cystic hydatid disease is a zoonosis caused by the larval stage (hydatid cyst) of Echinococcus granulosus (Cestoda, Taeniidae). The hydatid cyst develops in the viscera of intermediate host as a unilocular structure filled by the hydatid fluid, which contains parasitic excretory/secretory products. Antigen B (AgB) is the major component of E. granulosus metacestode hydatid fluid. Functionally, AgB has been implicated in immunomodulation and lipid transport. However, the mechanisms underlying AgB functions are not completely known. In this study, we investigated AgB interactions with different mammalian cell types and the pathways involved in its internalization. AgB uptake was observed in four different cell lines, NIH-3T3, A549, J774 and RH. Inhibition of raft-mediated endocytosis causes about 50 and 69% decrease in AgB internalization by RH and A549 cells, respectively. Interestingly, AgB colocalized with the raft endocytic marker, but also showed a partial colocalization with the clathrin endocytic marker. The results indicate that raft-mediated endocytosis is the main route to AgB internalization, and that a clathrin-mediated entry may also occur at a lower frequency. Cellular internalization could be a requirement for AgB functions as a lipid carrier and/or immunomodulatory molecule, contributing to create a more permissive microenvironment to metacestode development and survival.Author summaryAntigen B (AgB) is an oligomeric lipoprotein highly abundant in Echinococcus granulosus hydatid fluid. AgB has already been characterized as an immunomodulatory protein, capable of inducing a permissive immune response to parasite development. Also, an important role in lipid acquisition is attributed to AgB, because it has been found associated to different classes of host lipids. However, the mechanisms of interaction employed by AgB to perform its functions remain undetermined. In this study, we demonstrate that mammalian cells are able to internalize E. granulosus AgB in culture and found that specific mechanisms of endocytosis are involved. Our results extend the understanding of AgB biological role indicating cellular internalization as a mechanism of interaction, which in turn, may represent a target to intervention.

Management of cysto-biliary communication in hydatid cyst of liver

Introduction: Biliary communication of hepatic hydatosis is one of the important complications. It may be minor or major communication. Minor communication is usually diagnosed during operation by the presence of bile stained hydatid fluid on aspiration or bile leak during surgery. However, in major communication, patients present with obstructive jaundice, abdominal pain and cholangitis. Surgical management of major communication is difficult and is associated with high morbidity and mortality.Methods: This is a retrospective study at Tribhuvan University Teaching Hospital, Kathmandu, Nepal over a period of two year (April 15th 2012 to April 15th 2014) and analyzed the medical records of 30 patients admitted and treated for hydatid cyst of liver in surgical ward during that period. Demography, clinical features, management and outcome were analyzed.Results: Of 30 patients with hepatic hydatid cyst operated over a period of two years, thirteen patients had cystobiliary communication. Amongst them, five minor communications were diagnosed during operation and managed with suture plication and omentopexy. Out of the eight major communications, seven were diagnosed preoperatively and all had cholangitis, and one had hydatid cyst of gallbladder and was diagnosed intraoperatively. Among six cases of major communication, five cases required CBD exploration and bile duct reconstruction while partial cholecystectomy was done for hydatid cyst of gall bladder and one case was managed by ERCP sphincterotomy, evacuation of cyst content and stenting. one case was managed with pigtail drain as the patient had infected hydatid cyst and not fit for surgery.Conclusion: Cystobiliary communication is a common complication of hydatid cyst of liver. Therapeutic options are related to size and location of the cyst and size of communication.Journal of Society of Surgeons of Nepal Vol.17(1) 2014: 31-34