Dihydroartemisinin induces ER stress-dependent apoptosis of Echinococcus protoscoleces in vitro.

In this study, we investigated the effect of dihydroartemisinin on Echinococcus protoscoleces and explored the role of endoplasmic reticulum stress in this process. Echinococcus protoscoleces were collected and cultured in RPMI 1640 medium. Changes in the expressions of glucose-regulated protein 78 (GRP-78), caspase-12, and C/EBP homologous protein (CHOP) were assessed through confocal immunofluorescence and western blot analysis. Cell viability and morphological changes were observed under a light microscope. The ultrastructure of protoscoleces was observed by scanning electron microscopy and transmission electron microscopy. Caspase-3 activity was detected using an enzyme assay kit. After dihydroartemisinin treatment, the protoscoleces showed loss of viability, and morphological changes including soma contraction, blebs formation, hooks loss, microtrichia destruction, and development of lipid droplets was observed. The levels of caspase-12 and CHOP were increased within 2 days of dihydroartemisinin treatment. However, the levels of GRP-78, caspase-12, and CHOP were decreased in 4 days. Furthermore, caspase-3 activity was increased after treatment with different concentrations of dihydroartemisinin. Dihydroartemisinin can induce apoptosis in protoscoleces via the ER stress-caspase-3 apoptotic pathway in vitro. These results indicate that dihydroartemisinin is a potentially valuable therapeutic agent against echinococcosis.

Ultrastructural changes in hydatid cyst walls obtained from human cases, exposed to different therapeutic approaches.

Recurrence of cystic echinococcosis as a result of treatment failure is frequently reported to cause a major problem in management of such serious parasitic infection. The deeply seated innermost germinal layer of hydatid cysts is a relatively delicate layer, yet responsible for viability maintenance of this parasitic stage. In this study, a trial was done to explore the ultrastructural changes in germinal and laminated layer of the hydatid cyst for the first time in human cases exposed to different therapeutic approaches which were done earlier to the final open surgical intervention. Four groups were included: group 1 did not receive any earlier form of treatment; group 2 was previously treated with only medical therapy; group 3 was treated with a single course of medical treatment, plus a single PAIR technique; group 4 was treated with multiple courses of medical treatment plus multiple PAIR techniques. Complete alteration of ultrastructural features of germinal and laminated layers were observed only with samples from group 4, indicating a kind of failure of the therapeutic approaches used in group, 1, 2, and 3, unless repeated in group 4 to achieve a real change regarding the fitness of the parasitic cystic lesions. Searching for more effective, safe, therapeutic method is highly recommended which may end the suffering of the affected patients.

First study about the development of adult Echinococcus canadensis G6 genotype of goat origin in experimentally infected dogs.

Echinococcus granulosus sensu lato (E. granulosus sl) must be considered as a species complex, comprising Echinococcus granulosus sensu stricto (E. granulosus ss, genotypes G1-G3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and Echinococcus canadensis (G6-G10) although the species status of E. canadensis is still controversial. These genotypes closely match the intermediate hosts associated strains described in earlier times among which E. canadensis G6 corresponds to the camel strain. As there are no studies concerning the development of adult stages of the G6 genotype from non-camel origin, the aims of the present study were: to characterize for the first time the development of E. canadensis G6 in dogs experimentally infected with protoscoleces derived from goats, to describe the resultant adult morphology, to evaluate the growth of their rostellar hooks from larval to adult stages and to determine the prepatent period of the strobilar stage of E. canadensis G6 derived from goats. The development of the strobilar stage of E. canadensis G6 genotype of goat origin was examined by studying the growth (variation of the total worm length) and segmentation in experimentally infected dogs at 14, 25, 35 and 56days post infection. A morphological characterization of 35-day-old worms as well as of larval and adult rostellar hooks was also carried out by conventional optical microscopic observations and/or by scanning electron microscopy. The prepatent period of the strobilar stage was assessed by microscopic examination of faeces from 2 infected dogs. Our results were compared with published data from the camel and other strains. The roles of the host, genotype and species in morphological and developmental features as well as the taxonomic position of E. canadensis G6 were discussed. The prepatent period of E. canadensis G6 genotype of goat origin was determined as at least, 41days. The obtained results contribute to increase the knowledge about the biology and genetics of E. granulosus sl complex and are also of practical usefulness for the design of disease control strategies.

Ultrastructure of oncosphere and early stages of metacestode development of Echinococcus granulosus.

The ultrastructure of the oncospheres and developing metacestodes in vitro of Echinococcus granulosus was studied with emphasis on the origin of the laminated layer. The activated oncospheres measured 28 microns (S.D. = 1.83), and by day 5 of in vitro culture the metacestodes attained diameters up to 52 microns (S.D. = 2.66). The oncospheral plasma membrane of the tegument appeared to be formed by a microtubular cytoskeleton arranged in a predetermined pattern. By day 2 there were three main types of vesicles in the perikaryon: Vd, Vg1 and Vg2. Vesicles appeared to be synthesized in the perikaryon and continuously transported to the periphery, where the Vd vesicles could contribute to the ground substance of the syncytial tegument. We suggest that the Vg1 and Vg2 vesicles contribute to the laminated layer where, by day 5, they were seen in increased density and forming aggregations on the outer border.

Echinococcus granulosus: the mechanism of oncosphere lysis by sheep complement and antibody.

A heat-labile component of normal sheep serum (56 degrees C for 30 min but not 50 degrees C for 30 min) was able to lyse oncospheres in vitro. The degree of effect, and the proportion of oncospheres lysed, was related to the concentration of normal unheated sheep serum complement, or other sources of complement (rabbit, mouse) in the culture. Lower concentrations were required for lysis if the culture serum was obtained from sheep immune to E. granulosus infection. Heat inactivation of normal or immune sheep serum removed any lytic ability. No lysis occurred in any concentration of unheated foetal lamb serum. However, unheated foetal lamb serum was able to restore the lytic effect to heated normal or immune serum. This suggests that lysis in both immune and normal serum is antibody-dependent and complement-mediated. The lysis in normal serum would appear to be due to natural cross-reacting antibodies that can fix complement at the oncosphere surface. The complement lesion resulted in damage to the plasma membrane. This then peeled back, predisposing the oncosphere to osmotic destruction. The use of bleach to dissolve the embryophore caused damage to the plasma membrane similar to that caused by complement. Developing metacestodes at 3 days of age in vitro in immune serum were susceptible to the addition of complement at that time.

Echinococcus granulosus: ultrastructure of epithelial changes during the first 8 days of metacestode development in vitro.

The epithelium of artificially hatched and activated oncospheres of E. granulosus was studied ultrastructurally over the first 8 days of metacestode development in vitro. Within 4 h of activation, the epithelium was transformed from a thin cytoplasmic layer into a much wider layer packed with penetration gland granules and containing mitochondria and Golgi apparatus. Microvilli were extended from the outer plasma membrane and the basal lamina on the inner epithelial surface virtually disappeared. Microvilli increased in number and length over the first 24 h of development while granules in both the epithelium and penetration gland decreased in number. The granules appear to be involved in microvilli formation. After 3 days of development, the first lamination resolved ultrastructurally as shortened microvilli and some microtriches extending from the epithelium surrounded by an electron-dense microfibrillate material containing sloughed microvilli. By 6 days post-activation, no microvilli remained and only double-walled truncated microtriches extended from the epithelium. The microfibrillate material had become more electron-dense and was closer to the epithelium than at day 1. Within 8 days of metacestode development, a second lamination had developed. Both microfibrillate and particulate material of a greater electron density than the first lamination was added to the microthrix side of the first lamination.

Ultrastructural study of in vitro larval development of Echinococcus granulosus protoscoleces.

Through ultrastructural study of the morphological forms developed in vitro during protoscolex culture, we describe larval E. granulosus histogenesis. The transformation of the spined microtriches in the protoscolex into truncated microtriches that develop within the hydatid cyst is discussed. The paper also describes the mitochondria location change that occurs during the evolution; the mitochondria pass from the most internal area of the distal cytoplasm along the cytoplasmic extensions into the cytoplasm of tegumental cells. The ultrastructures of both the vesiculated protoscolex and the posterior bladder demonstrate that each state corresponds to the initial step on one of the two paths of in vitro vesicular development.

Viability of Echinococcus granulosus cysts in mice following cultivation in vitro.

The viability of hydatid cysts developed in vitro for 90 days was assessed by implantation into mice. Cysts removed from mice at 270 days post-infection (p.i.) increased their size 13.5-fold and contained several brood capsules containing protoscoleces. Thus, cysts remain viable after prolonged in vitro culture. The implantation in mice of 15 cysts developed in vitro yielded an average of 10 cysts per mouse, which is indicative of a high survival rate in these experimental infections. The ultrastructural study of cysts recovered from mice 270 days p.i. showed that the germinal membrane was more compact than before implantation and several layers of tegumental cells had developed. Observations of cysts removed from mice indicated that the plasma membrane surrounding microtriches had prolongations opening into the laminated layer.

High molecular mass glycans are major structural elements associated with the laminated layer of in vitro cultivated Echinococcus multilocularis metacestodes.

The laminated layer of the larval stage (metacestode) of the cestode parasite Echinococcus multilocularis is composed largely of carbohydrates, which form a tight microfibrillar meshwork around the entire metacestode. Since this laminated layer is the only parasite structure which is in constant contact with host immune and non-immune cells, and appears largely resistant to physiological and immunological reactions of the host, it most likely carries out important functions with regard to host-parasite interactions. In infected hosts, the metacestode is usually concentrically covered by host connective tissue cells and large amounts of collagen, causing a dense scar-like fibrosis, and it is likely that host-derived components are incorporated into the laminated layer at the host-parasite interface. Therefore, in order to obtain information on the molecular composition of this structure, we used parasite larvae which were generated through in vitro cultivation and thus were largely devoid of interfering host components. Lectin fluorescence on section-labelling of metacestodes embedded in LR-White suggested that the laminated layer is largely composed of N-acetyl-beta-D-galactosaminyl, and alpha- and beta-D-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine-beta-1.3-galactose, while N-linked glycopeptides and alpha-D-mannosyl residues and/or glucosyl residues were found mainly within the germinal layer, and within the cellular mass and the surface of developing protoscoleces. Lectin-gold EM confirmed these findings. The laminated layer was isolated from in vitro cultivated metacestodes by urea extraction, and the ultrastructure of the purified laminated layer was assessed comparatively with respect to the laminated layer of intact parasites. The glycan composition was determined using SDS-PAGE and lectin blotting. This work has laid the basis for a more detailed dissection of the molecular composition of the laminated layer.

In vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces.

The in vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces were studied by means of light and electron microscopy. Both drugs had a protoscolicidal activity that increased proportionally with increasing concentrations of the drugs. Ivermectin showed the more rapid effects and caused contraction and paralysis of protoscoleces. A paralyzing effect was also observed with levamisole, followed by irreversible tissue vacuolation leading to death.

The effects of albendazole and albendazole sulphoxide combination-therapy on Echinococcus granulosus in vitro.

Protoscoleces of Echinococcus granulosus were incubated in vitro with decreasing concentrations of either albendazole (ABZ) or albendazole sulphoxide (ABZ.SO) (50, 10, 1 and 0.1 micrograms ml-1), and in combination. Viability was assessed by the methylene blue exclusion test and establishment of infection in mice. Protoscolex ultrastructure was determined by means of transmission and scanning electron microscopy. ABZ and ABZ.SO, when used separately had protoscolicidal activity after a longer incubation period (30 days) than when used as combined compounds. When incubated in the presence of ABZ + ABZ.SO, protoscolex viability dropped rapidly. That is, protoscoleces were all non-viable at 12 days of exposure, with no cyst developing following their inoculation into mice. The ultrastructural changes induced by ABZ or ABZ.SO alone, were: (a) rostellar disorganization, (b) formation of numerous blebs on the tegument, (c) loss of the microtriches, (d) increased vesiculation within the tegumentary cytons together, (e) an increase in lipid deposits and (f) depletion of glycogen reserves. After incubation with combined ABZ and ABZ.SO the tegument contained numerous blebs which became detached, leaving debris only, some intact nuclei being discernible in the protoscolex parenchyma.

Anthelmintic effects of cyclosporin A on protoscoleces and secondary hydatid cysts of Echinococcus granulosus in the mouse.

Cyclosporin A (CsA), employed primarily as an immunosuppressant during the management of organ and graft transplants, exhibits anthelmintic properties. However, its efficacy against tapeworm infections in laboratory models is variable. A preliminary investigation has been undertaken to assess the action of CsA on the establishment and growth of protoscoleces and secondary hydatid cysts of ovine Echinococcus granulosus in mice. Administration of CsA in five consecutive daily doses, beginning 2 days prior to infection, resulted in significant reduction in cyst establishment (measured in terms of cyst masses, cyst numbers and cyst wet weights), when mice were autopsied 20 weeks post-infection. None of these parameters were significantly reduced when the drug was administered 18 weeks post-infection, although wet weight decreased by 42%. Ultrastructural examination of the germinal membrane and laminated layer of late-treated E. granulosus revealed abnormalities in all cysts studied whereas control and early-treated hydatids were normal. A case is made for the consideration of a clinical use for CsA for post-operative control of secondary hydatidosis and its efficacy against hydatid cysts is discussed.

Development of chemotherapeutic model for the in vitro screening of drugs against Echinoccus granulosus cysts: the effects of an albendazole-albendazole sulphoxide combination.

This paper describes a novel experimental model for the screening of putative drugs against the metacestode stage of E. granulosus using hydatid cysts derived from in vitro culture of protoscoleces. The effects of an ABZ+ABZ.SO combination against cultured and murine cysts were studied with this in vitro model system. This treatment produced loss of turgidity of the cultured cysts in less time than in the murine cysts but the ultrastructural tissue damage observed in both cultured and murine cysts was similar. The ultrastructural changes induced by ABZ+ABZ.SO were: (i) vacuolation of the distal cytoplasm that extended to the tegumentary cells of the germinal membrane; (ii) increased number of mitochondria; (iii) partial loss of microtriches; (iv) increased number of autophagosomes; and (v) an increase in lipid deposits.

Enhanced larval cyst growth of Echinococcus multilocularis in praziquantel-treated jirds (Meriones unguiculatus).

Jirds (Meriones unguiculatus) inoculated intraperitoneally with cystic material of Echinococcus multilocularis were given daily oral treatments of praziquantel at 300 mg/kg of body weight (bw) or dimethyl sulfoxide vehicle for five-day treatment regimens starting at 29 days postinoculation (PI) up to 69 days PI. At 39 or 49 days PI, the growth of the larval cystic mass (LCM) in jirds following a single or two five-day treatment regimens was significantly enhanced (P < 0.05) by 129.0% (2.3-fold) or 102.9% (2.0-fold), respectively. At 59 or 69 days PI following three or four five-day treatments with praziquantel, LCM growth was enhanced by 47.8% (1.5-fold) and 44.1% (1.4-fold), respectively, but was no longer significantly different than that in control jirds. A single five-day treatment on 29-33 days PI (with necropsy at 69 days PI) significantly enhanced the growth of the LCM by 87.6% (1.9-fold). Parasites from praziquantel treatment regimens examined ultrastructurally showed consistent damage to the germinal membrane evidenced by vacuolization and rupture of syncytial cytoplasm, rupture and coalescence of the electron-lucent vesicles just below the microvilli of the tegumental surface, and swelling and rounding of mitochondria. At 39 days PI, increased blebbing of the germinal membrane into the lumen of the LCM in praziquantel-treated animals was observed by scanning electron microscopy. The treatment-induced blebs were identified as nucleated germinal cells by transmission electron microscopy and appeared to be responsible for metastasis and enhanced growth of the LCM. Although praziquantel damaged the ultrastructural integrity of the LCM, treatment failed to inhibit larval cyst growth or protoscolex development.(ABSTRACT TRUNCATED AT 250 WORDS)

Echinococcus granulosus:isoprinosine treatment of the metacestode stage.

The efficacy of short- and long-term treatments with Isoprinosine, an immunomodulatory compound, was studied in Echinococcus granulosus cysts developed in NMRI mice intraperitoneally infected with sheep pulmonary cysts. After treatment, a reduction in the size and number of cysts with macroscopic modifications was observed. The structural alterations included damage or destruction of the protoscoleces and partial destruction of the cyst wall, which predominated at the inner germinal layer level. The efficacy of this drug was evaluated after long-term and short-term treatment. Short-term treatment with a dose of 1 g/kg/day gave better results, with a loss of infectivity of the larval tissue. The well-tolerated long-term treatment with a dose of 2 g/kg/day showed the absence of toxicity of this compound. The survival time of treated animals was greater than that of untreated controls.

Increases in the effects of albendazole on Echinococcus multilocularis metacestodes by the dipeptide methyl ester (Phe-Phe-OMe).

Three months after infection with Echinococcus multilocularis, Mongolian gerbils were given either the dipeptide methyl ester (Phe-Phe-OMe) or a combination of Phe-Phe-OMe plus albendazole to treat alveolar echinococcosis. Each drug was given orally at the daily dose of 50 mg/kg of body weight following various administration regimens. Histologic and ultrastructural studies of parasites recovered from infected gerbil tissues showed that the dipeptide methyl ester increases the effect of albendazole.

Determination of minimum effective concentration of praziquantel in in vitro cultures of protoscoleces of Echinococcus granulosus.

The minimum effective concentration of praziquantel against protoscoleces of Echinococcus granulosus (ovine strain) was determined in vitro; 20 micrograms/litre gave statistically significant results. No difference was seen between the sensitivity of ovine and equine protoscoleces at 50 micrograms/litre. Morphological observations on treated protocsoleces and passage into gerbils both suggested that the eosin-exclusion technique overestimates viability. Electron microscopy of treated protoscoleces showed severe, disruptive tegumentary damage. Praziquantel is thus an extremely active protoscolicidal agent that may have an important peri-operative role in preventing recurrence.

Echinococcus granulosus: in vitro maintenance of whole cysts and the assessment of the effects of albendazole sulphoxide and praziquantel on the germinal layer.

Small entire cysts of Echinococcus granulosus of human and animal origin were cultured in vitro in the presence or absence of albendazole sulphoxide (1000 micrograms/litre) or praziquantel (500 micrograms/litre) for 10 or 11 d, and subsequently passaged into the peritoneal cavity of gerbils to assess viability by continued cyst growth. Viability was reduced in the presence of albendazole sulphoxide, and disintegration of the germinal layer immediately after culture was demonstrated at the ultrastructural level. Praziquantel had no apparent effect on cyst growth.

In vitro culture of Echinococcus multilocularis and Echinococcus vogeli metacestodes: studies on the host-parasite interface.

The larval stage of Echinococcus multilocularis causes alveolar echinococcosis (AE) in various mammalians including humans, while Echinococcus vogeli larvae cause a related disease which is also occasionally found in man. Traditionally, Echinococcus metacestodes have been maintained in the laboratory by serial transplantation passages into susceptible animals such as mice or gerbils, enabling the parasite to proliferate asexually. These experimental animal models have been used extensively to investigate host-parasite interactions and to study immunological events occurring at the host-parasite interface. However, with the use of laboratory animals it has always been difficult to investigate in more detail those factors modulating metacestode differentiation, and investigations on gene expression and respective regulation have been hampered by the complexity of the host-parasite interplay. There has been a need for an in vitro culture model which would enable researchers to dissect specific parasite compartments involved in the host-parasite relationship in more detail. This review summarises the studies leading to the development and application of a suitable in vitro culture model for the maintenance and proliferation of E. multilocularis and E. vogeli metacestodes, including the formation of protoscoleces, in a chemically defined medium devoid of host influence. These culture models have been used to study the basic parameters of metacestode in vitro proliferation and differentiation, and for the dissection of the ultrastructure and composition of the acellular laminated layer, the structure of which is predominantly involved in the physical interaction between the parasite and host immune and non-immune cells and tissues. For E. multilocularis, in vitro cultured parasites have been more extensively employed to study the localisation of several antigens, and to generate defined antigens for immunological studies. Although in vitro culture will not completely eliminate the need of animal experimentation, a wider application of this technique could significantly reduce the use of animals, and thus the costs and time required for respective experimental investigations.

The scanning electron microscopy of scolices in a case of hydatid cyst from human liver.

The scanning electron microscopic (SEM) features of hydatid scolices from a case of hydatid disease of the liver are described. The scolex when fully everted has a double circle of hooklets which occupy a large area of the parasite. The hooklets are inserted into the rostellum with one row of hooklets overlapping the other. Each hooklet is about 20 to 40 um long, has a basic round structure sharp at the distal end and broadens as it curves towards the point of insertion at the rostellum, where 2 blunt projections correspond to the 2 annular bundles of muscle fibres at the orifice of the scolex. The function of the hooklets is not fully understood but they are possibly used for anchorage and/or for the purpose of propulsion in a fluid medium by the act of invagination and evagination of the hooklets.