The effect of hydatid cyst protoscolex somatic antigens on full-thickness skin wound healing in mouse.

BACKGROUND: Wound healing has evolved in recent years, resulting in diverse therapeutic options. OBJECTIVE: This study evaluated the effects of the somatic antigen of the hydatid cyst protoscolex on wound healing in mice with full-thickness skin wounds. METHODS: Fifty-four adult mice, weighing 25 ± 5 g and approximately 60 days old, were divided into three groups (A, B, and C), each further divided into three subgroups. Subgroups A1, A2, and A3 were assigned negative controls. B1, B2, and B3 received hydatid cyst somatic antigen tests at 10 µg/SC, whereas C1, C2, and C3 received somatic antigen tests at 20 µg/SC. Under general anesthesia, a wound biopsy puncture of 9.8 mm in diameter was performed on the mice's back and spine. In the experimental group, antigen and alum adjuvant were administered subcutaneously around the wound, while the control group received Phosphate-Buffered Saline (PBS). Using digital images, a geometric assessment was conducted on days 0, 1, 3, 6, 9, 12, 15, 18, and 21 post-wounding. The obtained images were analyzed by Image J software and after analyzing the data by SPSS software. RESULTS: A significant difference in terms of epithelization was observed in the antigen treatment group with a dose of 20 µg on days 3 and 6 (P < 0.05). Furthermore, the 20 µg antigen group was significantly higher than the 10 µg antigen group in terms of this factor on day 3 (P < 0.05). Skin samples were taken from all wounds on days 3, 10 and 21 for microscopic evaluation. Regarding epithelization, on day 10, a significant difference was observed in the treatment group with a concentration of 10 µg with the control group and the treatment group with a concentration of 20 µg (P < 0.05). CONCLUSION: Based on the results of the present study, it can be concluded that somatic antigens of protoscolex hydatid cyst are dose-dependent and antigens with a dose of 20 µg by subcutaneous injection accelerate wound healing and epithelialization.

[Preliminary study on the effect of Echinococcus multilocaris on phenotypic transformations of glucose metabolism and polarization types in macrophages].

OBJECTIVE: To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. METHODS: Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. RESULTS: There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1ß (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-ß mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1ß, TNF-α and TGF-ß in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-ß mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). CONCLUSIONS: Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.

Comparative characterization of microRNA-71 of Echinococcus granulosus exosomes.

Cystic echinococcosis (CE) is a global zoonotic disease caused by Echinococcus granulosus, posing a great threat to human and animal health. MiRNAs are small regulatory noncoding RNA involved in the pathogenesis of parasitic diseases, possibly via exosomes. Egr-miR-71 has been identified as one of the miRNAs in the blood of CE patients, but its secretory characteristics and functions remains unclear. Herein, we studied the secretory and biological activity of exosomal egr-miR-71 and its immunoregulatory functions in sheep peripheral blood mononuclear cells (PBMCs). Our results showed that egr-miR-71 was enriched in the exosome secreted by protoscoleces with biological activity. These egr-miR-71-containing exosomes were easily internalized and then induced the dysregulation of cytokines (IL-10 and TNF-α), nitric oxide (NO) and key components (CD14 and IRF5) in the LPS/TLR4 pathway in the coincubated sheep PBMCs. Similarly, egr-miR-71 overexpression also altered the immune functions but exhibited obvious differences in regulation of the cytokines and key components, preferably inhibiting proinflammatory cytokines (IL-1α, IL-1ß and TNF-α). These results demonstrate that exosomal egr-miR-71 is bioactive and capacity of immunomodulation of PBMCs, potentially being involved in immune responses during E. granulosus infection.

Title: Caractérisation comparative du microARN-71 des exosomes d'Echinococcus granulosus. Abstract: L'échinococcose kystique (EK) est une maladie zoonotique mondiale causée par Echinococcus granulosus, représentant une grande menace pour la santé humaine et animale. Les miARN sont des petits ARN régulateurs non codants impliqués dans la pathogenèse des maladies parasitaires, éventuellement via les exosomes. Egr-miR-71 a été identifié comme l'un des miARN présents dans le sang des patients atteints d'EK, mais ses caractéristiques et fonctions sécrétoires restent floues. Ici, nous avons étudié l'activité sécrétoire et biologique du egr-miR-71 exosomal et ses fonctions immunorégulatrices dans les cellules mononucléées du sang périphérique (CMSP) de mouton. Nos résultats ont montré qu'egr-miR-71 était enrichi dans l'exosome sécrété par les protoscolex ayant une activité biologique. Ces exosomes contenant egr-miR-71 ont été facilement internalisés et ont ensuite induit la dérégulation des cytokines (IL-10 et TNF-α), de l'oxyde nitrique (NO) et des composants clés (CD14 et IRF5) de la voie LPS/TLR4 dans les CMSP de mouton co-incubées. De même, la surexpression d'egr-miR-71 a également modifié les fonctions immunitaires mais a montré des différences évidentes dans la régulation des cytokines et des composants clés, inhibant de préférence les cytokines pro-inflammatoires (IL-1α, IL-1ß et TNF-α). Ces résultats démontrent que l'egr-miR-71 exosomal est bioactif et possède une capacité d'immunomodulation des CMSP, potentiellement impliquée dans les réponses immunitaires lors d'une infection à E. granulosus.

Effect of sunitinib against Echinococcus multilocularis through inhibition of VEGFA-induced angiogenesis.

BACKGROUND: Alveolar echinococcosis (AE) is a lethal zoonosis caused by the fox tapeworm Echinococcus multilocularis. The disease is difficult to treat, and an effective therapeutic drug is urgently needed. Echinococcus multilocularis-associated angiogenesis is required by the parasite for growth and metastasis; however, whether antiangiogenic therapy is effective for treating AE is unclear. METHODS: The in vivo efficacy of sunitinib malate (SU11248) was evaluated in mice by secondary infection with E. multilocularis. Enzyme-linked immunosorbent assays (ELISAs) were used to evaluate treatment effects on serum IL-4 and vascular endothelial growth factor A (VEGFA) levels after SU11248 treatment. Gross morphological observations and immunohistochemical staining were used to evaluate the impact of SU11248 on angiogenesis and the expression of pro-angiogenic factors VEGFA and VEGF receptor 2 (VEGFR2) in the metacestode tissues. Furthermore, the anthelmintic effects of SU11248 were tested on E. multilocularis metacestodes in vitro. The effect of SU11248 on the expression of VEGFA, VEGFR2, and phosphorylated VEGFR2 (p-VEGFR2) in liver cells infected with protoscoleces in vitro was detected by western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). The influence of SU11248 on endothelial progenitor cell (EPC) proliferation and migration was determined using CCK8 and transwell assays. RESULTS: In vivo, SU11248 treatment markedly reduced neovascular lesion formation and substantially inhibited E. multilocularis metacestode growth in mice. Further, it exhibited high anti-hydatid activity as efficiently as albendazole (ABZ), and the treatment resulted in reduced protoscolex development. In addition, VEGFA, VEGFR2, and p-VEGFR2 expression was significantly decreased in the metacestode tissues after SU11248 treatment. However, no effect of SU11248 on serum IL-4 levels was observed. In vitro, SU11248 exhibited some anthelmintic effects and damaged the cellular structure in the germinal layer of metacestodes at concentrations below those generally considered acceptable for treatment (0.12-0.5 µM). Western blotting, RT-qPCR, and ELISA showed that in co-cultured systems, only p-VEGFR2 levels tended to decrease with increasing SU11248 concentrations. Furthermore, SU11248 was less toxic to Reuber rat hepatoma (RH) cells and metacestodes than to EPCs, and 0.1 µM SU11248 completely inhibited EPC migration to the supernatants of liver cell and protoscolex co-cultures. CONCLUSIONS: SU11248 is a potential candidate drug for the treatment of AE, which predominantly inhibits parasite-induced angiogenesis. Host-targeted anti-angiogenesis treatment strategies constitute a new avenue for the treatment of AE.

Establishment of a secondary infection laboratory model of Echinococcus shiquicus metacestode using BALB/c mice and Mongolian jirds (Meriones unguiculatus).

Echinococcus shiquicus is peculiar to the Qinghai­Tibet plateau of China. Research on this parasite has mainly focused on epidemiological surveys and life cycle studies. So far, limited laboratory studies have been reported. Here, experimental infection of E. shiquicus metacestode in BALB/c mice and Mongolian jirds (Meriones unguiculatus) was carried out to establish alternative laboratory animal models. Intraperitoneal inoculation of metacestode material containing protoscoleces (PSCs) obtained from infected plateau pikas were conducted on BALB/c mice. Furthermore, metacestode material without PSCs deriving from infected BALB/c mice was intraperitoneally inoculated to Mongolian jirds. Experimental animals were dissected for macroscopic and histopathological examination. The growth of cysts in BALB/c mice was infiltrative, and they invaded the murine entire body. Most of the metacestode cysts were multicystic, but a few were unilocular. The cysts contained sterile vesicles, which had no PSCs. The metacestode materials were able to successfully infect new mice. In the jirds model, E. shiquicus cysts were typically formed freely in the peritoneal cavity; the majority of these cysts were free while a small portion adhered loosely to nearby organs. The proportion of fertile cysts was high, and contained many PSCs. The PSCs produced in Mongolian jirds also successfully infected new ones, which confirms that jirds can serve as an alternative experimental intermediate host. In conclusion, a laboratory animal infection was successfully established for E. shiquicus using BALB/c mice and Mongolian jirds. These results provide new models for the in-depth study of Echinococcus metacestode survival strategy, host interactions and immune escape mechanism.

Report of a two-headed protoscolex of Echinococcus granulosus revealed by phase contrast microscopy in Shiraz, Iran.

Echinococcus granulosus sensu lato can produce cystic echinococcosis (CE)/hydatidosis in different hosts. Each CE cyst can produce numerous protoscoleces by its germinal layer in two forms: evaginated and invaginated. Usually, each protoscolex has one head consisting of a rostellum with two rows of hooks and four suckers. During a study of protoscolicidal agents on sheep Echinococcus granulosus protoscoleces, and investigations on their microscopic changes using Phase contrast microscopy, we observed a case of two- headed evaginated protoscolex. Two heads were attached to a unique bigger size of protoscolex body. Morphological observations showed its dimension around two times of usual protoscolex. There was no space between the bodies, hence one fused body was observed which was clearly shown by Phase contrast microscopy. Each head possessed two rows of hooks. Using micrometry all parts of the two-headed protoscolex, especially hooks were measured and photographed showing all aspects of its morphology including tegument, hooks, and two heads. Specific parts of two-headed protoscolex including suckers, hooks, also calcareous corpuscles were measured. The measurements on the two-headed protoscolex showed that the small hook blade length (SBL) and large hook blade length (LBL) were almost langer than one head protoscolex. A total of 120 calcareous corpuscles in two headed-protoscolex is much higher than one head protoscoleces.

Pulmonary cystic echinococcosis in a child.

This case report describes the presentation, diagnosis, and treatment of a 13-year-old boy with pulmonary cystic echinococcosis. The patient presented with low-volume hemoptysis, and lung imaging revealed a large cystic mass, as well as smaller pseudo-nodular lesions, suggesting a large intrathoracic hydatid cyst and ruptured cysts. The diagnosis was confirmed by a positive echinococcosis Western Blot assay, despite equivocal serology. The treatment consisted of surgical removal of the large cyst using thoracoscopy, along with a two-week course of albendazole and praziquantel, followed by albendazole alone for two years. Analysis of the cyst membrane revealed an Echinococcus granulosus protoscolex. The patient had a successful recovery.

Transforming growth factor-ß signalling regulates protoscolex formation in the Echinococcus multilocularis metacestode.

The lethal zoonosis alveolar echinococcosis (AE) is caused by tumor-like, infiltrative growth of the metacestode larval stage of the tapeworm Echinococcus multilocularis. We previously showed that the metacestode is composed of posteriorized tissue and that the production of the subsequent larval stage, the protoscolex, depends on re-establishment of anterior identities within the metacestode germinative layer. It is, however, unclear so far how protoscolex differentiation in Echinococcus is regulated. We herein characterized the full complement of E. multilocularis TGFß/BMP receptors, which is composed of one type II and three type I receptor serine/threonine kinases. Functional analyzes showed that all Echinococcus TGFß/BMP receptors are enzymatically active and respond to host derived TGFß/BMP ligands for activating downstream Smad transcription factors. In situ hybridization experiments demonstrated that the Echinococcus TGFß/BMP receptors are mainly expressed by nerve and muscle cells within the germinative layer and in developing brood capsules. Interestingly, the production of brood capsules, which later give rise to protoscoleces, was strongly suppressed in the presence of inhibitors directed against TGFß/BMP receptors, whereas protoscolex differentiation was accelerated in response to host BMP2 and TGFß. Apart from being responsive to host TGFß/BMP ligands, protoscolex production also correlated with the expression of a parasite-derived TGFß-like ligand, EmACT, which is expressed in early brood capsules and which is strongly expressed in anterior domains during protoscolex development. Taken together, these data indicate an important role of TGFß/BMP signalling in Echinococcus anterior pole formation and protoscolex development. Since TGFß is accumulating around metacestode lesions at later stages of the infection, the host immune response could thus serve as a signal by which the parasite senses the time point at which protoscoleces must be produced. Overall, our data shed new light on molecular mechanisms of host-parasite interaction during AE and are relevant for the development of novel treatment strategies.

S100 proteins and their implication in the thalamus: an integrative review / Las proteínas S100 y su implicación en el tálamo: una revisión integradora

SUMMARY: S100 proteins belong group of calcium-binding proteins and are present in physiological intracellular and extracellular regulatory activities, such as cell differentiation, and act in inflammatory and neoplastic pathological processes. Recently, its expressions in the nervous system have been extensively studied, seeking to elucidate its action at the level of the thalamus: A structure of the central nervous system that is part of important circuits, such as somatosensory, behavioral, memory and cognitive, as well as being responsible for the transmission and regulation of information to the cerebral cortex. This article is an integrative review of scientific literature, which analyzed 12 studies present in Pubmed. The analysis showed that the relationship of S100 proteins and the thalamus has been described in neoplastic processes, mental disorders, hypoxia, trauma, stress, infection, Parkinson's disease and epilepsy. In summary, it is possible to conclude that this protein family is relevant as a marker in processes of thalamic injury, requiring further studies to better understand its clinical, preclinical meanings and its prognostic value.

Las proteínas S100 pertenecen al grupo de proteínas fijadoras de calcio y están presentes en actividades reguladoras fisiológicas intracelulares y extracelulares, como la diferenciación celular, y actúan en procesos patológicos inflamatorios y neoplásicos. Recientemente, sus expresiones en el sistema nervioso han sido ampliamente estudiadas, buscando dilucidar su acción a nivel del tálamo: una estructura del sistema nervioso central que forma parte de importantes circuitos, como el somatosensorial, conductual, de memoria y cognitivo, así como además de ser responsable de la transmisión y regulación de la información a la corteza cerebral. Este artículo es una revisión integradora de la literatura científica, que analizó 12 estudios presentes en Pubmed. El análisis mostró que la relación de las proteínas S100 y el tálamo ha sido descrita en procesos neoplásicos, trastornos mentales, hipoxia, trauma, estrés, infección, enfermedad de Parkinson y epilepsia. En resumen, es posible concluir que esta familia de proteínas es relevante como marcador en procesos de lesión talámica, requiriendo más estudios para comprender mejor su significado clínico, preclínico y su valor pronóstico.

Morphology of Echinococcus granulosus protoscolex / Morfología de los Protoescolex de echinococcus granulosus

SUMMARY: Echinococcus granulosus (E. granulosus), is a tapeworm that spreads between intermediate and definitive hosts through the ingestion of fecal matter contaminated with the parasite's eggs. The life cycle consists of differentiation from eggs to oncospheres to embryos, which eventually form cysts in organs like the liver, lungs and others. Within these cysts are protoscolices, an intermediate stage of the parasite which develop into adult tapeworms once they infect their definitive hosts. When these hydatid cysts form in humans, it is known as Cystic Echinococcosis (CE). This disease is treated through surgical excision of the cysts and or chemotherapy with benzimidazole compounds. Understanding the morphology of the intermediate developmental stage of E. granulosus, protoscolex stage, can allow researchers to identify defining structural changes and protein functions that could be used to develop treatment modalities for CE. Unique characteristics in the tegumental surface during the protoescolex stage and proteins associated with cyst fertility have all been identified in previous research studies and bring researchers closer to understanding the underlying mechanisms of E. granulosus development, and consequently, means to disrupt it to achieve better control of the disease.

El Echinococcus granulosus (E. granulosus), es un cestodo que se propaga entre huéspedes intermedios y definitivos a través de la ingestión de materia fecal contaminada con los huevos del parásito. El ciclo de vida consiste en la diferenciación de huevos a oncosferas y embriones, que finalmente forman quistes en órganos como el hígado, los pulmones y otros. Dentro de estos quistes hay protoescólices, una etapa intermedia del parásito que se convierte en su forma adulta (tenia), una vez que infectan a sus huéspedes definitivos. Cuando estos quistes hidatídicos se desarrollan en seres humanos, se les conoce como equinococosis quística (EC). Esta enfermedad se trata mediante la extirpación quirúrgica de los quistes o la quimioterapia con compuestos benzimidazólicos. La comprensión de la morfología de la etapa de desarrollo intermedia del E. granulosus y la etapa de protosclex, puede permitir a los investigadores identificar cambios estructurales definidos y funciones de proteínas que podrían usarse para desarrollar modalidades de tratamiento para la CE. Las características únicas en la superficie tegumentaria durante la etapa de protoescolex y las proteínas asociadas con la fertilidad del quiste se han identificado en estudios de investigación anteriores y acercan a los investigadores a la comprensión de los mecanismos subyacentes del desarrollo del E. granulosus y, en consecuencia, los medios para interrumpirlo para lograr un mejor control de la enfermedad.

Estudio morfocuantitativo de protoescólices y otras estructuras fundamentales de Echinococcus granulosus en hospederos humanos / Morphoquantitative study of protoscolices and other fundamental structures of Echinococcus granulosus in human hosts

La equinococosis quística (EQ) a pesar de ser una enfermedad endémica en diversos lugares del planeta, presenta pocos estudios morfológicos y cuantitativos de las estructuras fundamentales del Echinococcus granulosus en humanos, en especial de los protoescólices. El objetivo de este estudio fue analizar morfocuantitativamente protoescólices y otras estructuras fundamentales de E. granulosus obtenidos de hospederos humanos. Estudio de corte transversal. Se estudiaron 8 especímenes de EQ hepática humana, aplicando un muestreo no probabilístico por conveniencia. Se evaluó las capas quísticas, el líquido y la arenilla hidatídica. Las capas fueron fijadas en formaldehido tamponado a 10 % y procesadas para su inclusión en paraplast. Se realizaron cortes de 5 µm de grosor y fueron teñidas con H-E para su análisis con microscopía óptica. El líquido y arenilla fueron centrifugados y al sedimento obtenido se le realizó análisis directo para determinar las medidas morfométricas de los protoescólices y de los ganchos grandes y pequeños. Se utilizó estadística descriptiva. De los 8 quistes estudiados, 6 eran quistes univesicular, uno multivesicular y un quiste abscedado, cuyas capas laminada y germinativa se encontraban bien definidas. Las vesículas prolígeras presentaban forma redondeada con protoescólices en su interior. Los protoescólices invaginados presentaron un largo y ancho promedio de 140,8 ± 34,3 µm y 106,2 ± 29,5 µm, respectivamente; y los desarrollados un largo de 237,2 ± 53,0 µm y ancho de 128,7 ± 32,0 µm. Los ganchos rostelares presentaron contornos suaves distribuidos en dos filas regulares. El promedio del largo total de los ganchos grandes y pequeños fue 20,1 ± 2,7 µm; el promedio del ancho total fue 7,4 ± 1,2 µm. En conclusión, las características morfocuantitativas de los ganchos de E. granulosus en humanos, son distintos a otras especies hospederas intermediarias y de otros Echinococcus spp. Es posible que el abandono del estado de resistencia de los protoescólices invaginado hasta su desarrollo genere implicancias epidemiológicas de interés.

SUMMARY: Although cystic echinococcosis (CE) is an endemic disease in several parts of the world, few morphological and quantitative studies of the fundamental structures of Echinococcus granulosus in humans, especially protoscolices. The aim of this study was to perform a morphoquantitative analysis of protoescolex and other fundamental structures of E. granulosus from human hosts. Cross- sectional study. Eight human hepatic EQ specimens were studied, applying non-probabilistic convenience sampling. Cystic layers, fluid and hydatid grit were evaluated. The layers were fixed in 10% buffered formaldehyde and processed for embedding in paraplast. Slices of 5 µm thickness were made and stained with H-E for light microscopic analysis. The liquid and grit were centrifuged and the sediment obtained was analyzed directly to determine the morphometric measurements of the protoscolices and the large and small hooks. Descriptive statistics were used. Of the 8 cysts studied, 6 were univesicular cysts, one multivesicular and one abscessed cyst, whose lamellar and germinative layers were well defined. The proligerous vesicles had a rounded shape with protoscolices inside. The invaginated protoscolices had an average length and width of 140.8 ± 34.3 µm and 106.2 ± 29.5 µm, respectively; and the developed ones had a length of 237.2 ± 53.0 µm and width of 128.7 ± 32.0 µm. The rostellar hooks presented smooth contours distributed in two regular rows. The average total length of the large and small hooks was 20.1 ± 2.7 µm; the average total width was 7.4 ± 1.2 µm. In conclusion, the morphoquantitative characteristics of E. granulosus hooks in humans are distinct from other intermediate host species and from other Echinococcus spp. It is possible that the abandonment of the resistance state of the invaginated protoscolices until their development generates epidemiological implications of interest.

Bioinformatics analysis and experimental verification of Notch signalling pathway-related miRNA-mRNA subnetwork in extracellular vesicles during Echinococcus granulosus encystation.

BACKGROUND: Encystation of the protoscoleces (PSCs) of Echinococcus granulosus is the main cause of secondary hydatid dissemination in the intermediate host. Extracellular vesicles (EVs) can transfer miRNAs into parasite cells to regulate mRNA expression. However, loading of developmental pathway-related miRNAs, such as those related to the Notch signalling pathway in EVs is unclear. Thus, we screened the miRNA-mRNA subnetwork involved in the Notch pathway during E. granulosus encystation in vitro and assessed changes in expression in the parasite and EVs. METHODS: mRNAs and miRNAs differentially expressed (DE) between PSCs and microcysts (MCs) were screened using high-throughput sequencing. DE mRNAs obtained from transcriptome analysis were intersected with mRNAs predicted to be targets of the conserved DE miRNAs of a small RNA library. DE miRNA functions were analysed using public databases, and a miRNA-mRNA subnetwork related to the Notch pathway was established. Notch pathway-related mRNA and miRNA expression of worms and EVs at different times was verified. RESULTS: In total, 1445 DE mRNAs between MCs and PSCs were screened after the intersection between 1586 DE mRNAs from the transcriptome and 9439 target mRNAs predicted using 39 DE miRNAs from the small RNA library. The DE mRNAs were clustered into 94 metabolic pathways, including the Notch pathway. Five DE miRNAs, including the most significantly expressed new DE miRNA, egr-new-mir0694-3p, corresponding to four target mRNAs (EgrG_000892700, EgrG_001029400, EgrG_001081400 and EgrG_000465800) were all enriched in the Notch pathway. The expression of the above mRNAs and miRNAs was consistent with the results of high-throughput sequencing, and the expression of each miRNA in EVs was verified. Annotated as ADAM17/TACE in the Notch pathway, EgrG_000892700 was down-regulated during PSC encystation. egr-miR-4989-3p and egr-miR-277a-3p expression in EVs after encystation was nearly five times that in EVs before encystation, which might regulate the expression of EgrG_000892700. CONCLUSIONS: Five miRNAs corresponding to four target mRNAs may be involved in regulating the Notch pathway during the PSC encystation. EVs may regulate the expression of EgrG_000892700 in PSCs because of continuous targeting of egr-miR-4989-3p and egr-miR-277a-3p and participate in the regulation the Notch pathway. The study might expand new ideas for blocking the secondary infection of E. granulosus PSCs via EVs miRNAs.

Determination of extracellular traps structures from sheep polymorphonuclear leukocytes to Echinococcus granulosus protoscoleces.

It was aimed to detect extracellular traps structures from sheep polymorphonuclear leukocytes (PMN) after being confronted with Echinococcus granulosus protoscoleces in vitro. Also, the effect of cyst fluid was examined on the development of extracellular traps. At the end of the incubation for 1 h, the extracellular traps augmented with neutrophil elastase, histone (H3) and myeloperoxidase were visualized in the protoscoleces-PMN co-culture microscopically. Some protoscoleces lysed and the chitinous hooks released were surrounded by the extracellular traps. The other protoscoleces were still intact and the extracellular trap structures were observed around them. The relationship between the extracellular DNA contents and the protoscoleces concentration was not found statistically significant (P > 0.05). The extracellular DNA amount in the co-cultures diluted in RPMI-1640 increased with the incubation time (P < 0.05). However, the time-dependent relationship was not found in the co-cultures diluted in the cyst fluid (P > 0.05). The difference in the extracellular DNA amount was detected as statistically significant (P < 0.05) between the two co-culture groups (diluted in RPMI-1640 or the cyst fluid), except for 30 min incubation. To the Author's knowledge, NETosis reaction was firstly observed in sheep PMN after being confronted with protoscoleces in vitro. The cyst fluid had some negative effects on the development of extracellular traps from sheep PMNs at the 1-h incubation time. It should be investigated which molecules are responsible for NETosis inhibition in hydatid cyst fluid. Future studies may clarify whether neutrophils fight with protoscoleces by using their different mechanisms.

Echinococcus granulosus Protoscolex DM9 Protein Shows High Potential for Serodiagnosis of Alveolar Echinococcosis.

Alveolar echinococcosis (AE) caused by infection with E. multilocularis metacestode, represents one of the most fatal helminthic diseases. AE is principally manifested with infiltrative, proliferating hepatic mass, resembling primary hepatocellular carcinoma. Sometimes metastatic lesions are found in nearby or remote tissue. AE diagnosis largely depends on imaging studies, but atypical findings of imaging features frequently require differential diagnosis from other hepatic lesions. Serological tests may provide further evidence, while obtaining reliable AE materials is not easy. In this study, alternative antigens, specific to AE were identified by analyzing E. granulosus protoscolex proteins. An immunoblot analysis of E. granulosus protoscolex showed that a group of low-molecular-weight proteins in the range from 14 kDa to 16 kDa exhibited a sensitive and specific immune response to AE patient sera. Partial purification and proteomic analysis indicated that this protein group contained myosin, tubulin polymerization promoting protein, fatty-acid binding protein, uncharacterized DM9, heat shock protein 90 cochaperone tebp P-23, and antigen S. When the serological applicability of recombinant forms of these proteins was assessed using enzyme-linked immunosorbent assay, DM9 protein (rEgDM9) showed 90.1% sensitivity (73/81 sera tested) and 94.5% specificity (172/181 sera tested), respectively. rEgDM9 showed weak cross-reactions with patient sera from the transitional and chronic stages of cystic echinococcosis (3 to 5 stages). rEgDM9 would serve as a useful alternative antigen for serodiagnosis of both early- and advanced-stage AE cases.

Transcriptome analysis of Echinococcus granulosus sensu stricto protoscoleces reveals differences in immune modulation gene expression between cysts found in cattle and sheep.

Cystic Echinococcosis (CE), a zoonotic parasitic disease, is caused by the cestode Echinococcus granulosus sensu lato. CE inflicts severe damage in cattle, sheep, and human hosts worldwide. Fertile CE cysts are characterized by the presence of viable protoscoleces. These parasite forms are studied with minimal contamination with host molecules. Hosts, cattle and sheep, show differences in their CE cyst fertility. The effect of the host in protoscolex transcriptome is not known. We genotyped and performed transcriptomic analysis on sheep protoscoleces obtained from liver and lung CE cysts. The transcriptomic data of Echinococcus granulosus sensu stricto protoscoleces from 6 lung CE cysts and 6 liver CE cysts were Collected. For host comparison analysis, 4 raw data files belonging to Echinococcus granulosus sensu stricto protoscoleces from cattle liver CE cysts were obtained from the NCBI SRA database. Principal component and differential expression analysis did not reveal any statistical differences between protoscoleces obtained from liver or lung cysts, either within the same sheep or different sheep hosts. Conversely, there are significant differences between cattle and sheep protoscolex samples. We found differential expression of immune-related genes. In cattle, 7 genes were upregulated in protoscoleces from liver cysts. In sheep, 3 genes were upregulated in protoscoleces from liver and lung CE cysts. Noteworthy, are the differential expression of antigen B, tegument antigen, and arginase-2 in samples obtained from sheep CE cysts, and basigin in samples from cattle CE cysts. These findings suggest that the host species is an important factor involved in the differential expression of immune related genes, which in turn is possibly related to the fertility of Echinococcus granulosus sensu stricto cysts.

In vitro evaluation of albendazole-loaded nanostructured lipid carriers on Echinococcus granulosus microcysts and their prophylactic efficacy on experimental secondary hydatidosis.

To enhance the therapeutic effects of albendazole (ABZ) on Echinococcus granulosus protoscoleces and metacestodes, ABZ-loaded nanostructured lipid carriers (ABZ-NLCs) are prepared by the hot high-speed homogenization method. Protoscoleces and microcysts were treated in vitro with free ABZ and ABZ-NLCs (concentrations of 1, 5, and 10 µg/ml), and the corresponding effects were monitored by methylene blue exclusion test and scanning and transmission electron microscopy. Chemoprophylactic treatment was performed on Balb/C mice 1 day before intraperitoneal injection of viable protoscoleces. The drugs were administered daily by intragastric inoculation for a period of 30 days. The prophylactic efficacy was assessed based on the number and weight of cysts developed in treated mice. The ultrastructural alterations in cysts were examined by transmission electron microscopy. After 18 days, all the protoscoleces incubated with 10 µg/ml ABZ-NLCs were killed, while 51.25 ± 4.03% of the protoscoleces incubated with 10 µg/ml free ABZ were still viable. Microcysts treated with ABZ-NLCs underwent degenerative alterations in a shorter time than when free ABZ was applied. The mean weight of the cysts recovered from mice of ABZ-NLCs group was significantly lower than that of the free ABZ group (P < 0.05), yielding prophylactic efficacy of 92.45% and 38.53%, respectively. The cysts treated with ABZ-NLCs showed marked ultrastructural changes in the germinal layer. This study demonstrated that both in vitro and in vivo treatments with ABZ-NLCs are significantly more efficient than treatment with free ABZ against E. granulosus protoscoleces, metacestodes, and prevention of cyst development in mice.

Ultrastructural characterization of the tegument in protoscoleces of Echinococcus ortleppi.

Cystic echinococcosis is a globally distributed zoonosis caused by cestodes of the Echinococcus granulosus sensu lato (s.l.) complex, with Echinococcus ortleppi mainly involved in cattle infection. Protoscoleces show high developmental plasticity, being able to differentiate into either adult worms or metacestodes within definitive or intermediate hosts, respectively. Their outermost cellular layer is called the tegument, which is important in determining the infection outcome through its immunomodulating activities. Herein, we report an in-depth characterization of the tegument of E. ortleppi protoscoleces performed through a combination of scanning and transmission electron microscopy techniques. Using electron tomography, a three-dimensional reconstruction of the tegumental cellular territories was obtained, revealing a novel structure termed the 'tegumental vesicular body' (TVB). Vesicle-like structures, possibly involved in endocytic/exocytic routes, were found within the TVB as well as in the parasite glycocalyx, distal cytoplasm and close inner structures. Furthermore, parasite antigens (GST-1 and AgB) were unevenly localised within tegumental structures, with both being detected in vesicles found within the TBV. Finally, the presence of host (bovine) IgG was also assessed, suggesting a possible endocytic route in protoscoleces. Our data forms the basis for a better understanding of E. ortleppi and E. granulosus s.l. structural biology.

Scolicidal effects of Cassia fistula and Urtica dioica extracts on protoscoleces of hydatid cysts.

Echinococcosis is among the most underestimated parasitic diseases that have universal distribution. The primary treatment is surgery. Hence, the development of new and more effective scolicidal agents with lower side effects is crucial. This study evaluated the therapeutic effects of Urtica dioica and Cassia fistula extracts as a scolicidal herbal drug in vitro. Suspension of protoscoleces was obtained from the infected livers of sheep in Khorramabad, Iran. Hydro-alcoholic solution was extracted from the leaves and stems of Urtica dioica and the fruit of Cassia fistula. Echinococcus granulosus protoscoleces were treated with the essential oils at concentrations of 10, 25, 50, and 100 mg/mL for 10, 20, 30, and 60 min and their viability was evaluated by the eosin staining test. The extract of Urtica dioica at a concentration of 100 mg/mL killed 90.51% of protoscoleces after 60 min. Cassia fistula also killed 67.74% of protoscoleces after 60 min. This study obtained satisfactory results. Urtica dioica and Cassia fistula extracts are promising protoscolicides and can be used in the treatment of hydatid cysts and pre-surgically to prevent secondary infections.

Promising proteins detected by Western blot from Echinococcus granulosus protoscoleces for predicting early post-surgical outcomes in CE-affected Tunisian children.

BACKGROUND: Cystic echinococcosis (CE) affects predominantly young patients in highly endemic areas. Improved serological methods are needed for the follow-up of CE cases, especially given the high rates of post-surgical relapse that require detection as soon as possible. METHODS: We designed a study to investigate the value of antigenic proteins extracted from Echinococcus granulosus (E. granulosus) protoscoleces, and of recombinant B2t and 2B2t proteins, for assessing the efficacy of surgical treatment carried out on CE-affected children. This study was performed on 278 plasma samples collected from 59 Tunisian children surgically treated for CE and monitored for 3 years post-surgery. The patients were classified according to post-surgical outcomes into a "non-relapsed" (NRCE) and a "relapsed" (RCE) group. We performed in-house ELISAs to measure anti-B2t and anti-2B2t IgG and immunoblotting for the detection of IgG against SDS-PAGE-resolved E. granulosus protoscoleces-specific antigens. The Wilcoxon test was applied to assess anti-B2t and anti-2B2t IgG levels. We applied the Cochran Q test to compare the distribution of immunoblotting antigenic bands between 1-month and 1-year post-surgery. RESULTS: The probability of being "relapse-free" when a decrease in antibody titers occurred between 1 month and 1 year post-surgery was 81% and 75%, respectively, for anti-B2t and anti-2B2t IgG. We identified five protoscolex protein bands of 20, 26/27, 30, 40 and 46 kDa as highly immunoreactive by immunoblot for both RCE and NRCE patients at 1 month post-surgery, and significantly lower immunoreactivity after 1 year (p < 10-4) for NRCE compared to RCE patients. The proteins at 26/27 and 40 kDa displayed the best performance in predicting the outcome, with an 84% probability of being relapse-free when the reactivity against the 40 kDa antigen, the doublet at 26/27 kDa, or both was absent or disappeared between 1 month and 1 year post-surgery, and a 93% probability of being relapsed when both bands remained reactive or increased in intensity between the two time points. CONCLUSIONS: The B2t protein could be useful for the prediction of CE early post-surgical outcomes. The proteins of E. granulosus protoscoleces, especially the doublet P26/27 and P40, could be promising predictive biomarkers for the post-surgical follow-up of CE cases as well.

Mobile Phone Radiation: Its Efficacy as Protoscolicidals.

OBJECTIVE: There are different types of radiations, such as microwaves and mobile waves. Certain types of radiofrequency were evaluated in hydatid cyst ablation or as protoscolicidals. This study aimed to assess the influence of mobile waves on hydatid cyst protoscolices. METHODS: Hydatid cysts were collected from the slaughterhouse and transferred to the laboratory. The contents of the cysts were drained in sterile conditions and the protoscolices were rinsed three times with phosphate buffered saline. Equal volumes of protoscolex suspensions were aliquoted into similar tubes. Based on the distance of the samples from the mobile generation waves, the tubes containing the parasitic suspensions were classified into three groups, each of which was further categorised into nine subgroups according to the time of the radiation exposure. The subgroup with zero exposure time was considered the control. RESULTS: It was found that the mortality rate of the protoscolices increases as the distance of the sample from the wave-generation source decreases (p<0.0001). Increasing the time of exposure also improves the mortality rate of protoscolices. CONCLUSION: The mortality rate of protoscolices was directly proportional to the time of exposure and inversely proportional to the distance from the mobile generation waves.