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PubMed1999

[Effect of liposomal albendazole on the ultrastructure of Echinococcus granulosus cysts in mice].

AIM: To observe the histopathological changes of Echinococcus granulosus cysts in mice treated with liposomal albendazole and co-administration with cimetidine by light microscopy and electron microscopy. METHODS: An oral dose of liposomal ABZ with different formulations was given at 200 mg/kg.d. Cimetidine was administered daily at an oral dose of 100 mg/kg.d. Sixty-seven mice were orally given different drugs six days per week for a total of twelve weeks. RESULTS: The histopathological changes indicated that there were significant differences (P < 0.01) between treated groups and control group. The degeneration and necrosis of E. granulosus cysts were marked in liposomal albendazole combined with cimetidin group. CONCLUSION: Liposomal albendazole was more effective against E. granulosus cyst than albendazole. Cimetidine had an apparent synergistic effect when given in combination with liposomal albendazle.

Albendazole↗
PubMed2003

Regionalization of DNA and protein synthesis in developing stages of the parasitic platyhelminth Echinococcus granulosus.

Echinococcus granulosus is a parasitic platyhelminth, which causes cystid hydatid disease, a major zoonosis involving canids as definitive hosts, and both human and herbivorous domestic animals as intermediate hosts. The disease is caused in intermediate hosts by hydatid cysts, formed upon ingestion of E. granulosus eggs excreted by canids. Protoscoleces, the developmental forms of the parasite infective to canids, are formed in the germinal cellular layer of hydatid cysts. We have found that protoscoleces develop from patches of proliferating cells present in the germinal layer of the hydatid cyst, while most of the other cells of the germinal layer are in a resting state. Further, patches of proliferating cells form buds, which elongate and develop a separate population of cycling cells. In these elongated buds, cell differentiation leads to the main structures of the protoscolex. Protein synthesis is very active among cells of early buds and coincides with their proliferating activity. By contrast, protein synthesis presents a much lower activity in the resting cells of the germinal layer surrounding the growing protoscoleces. In elongated buds at different stages of development, protein synthesis is found mainly close to cellular territories in which cell differentiation occurs. In free infective protoscoleces, cells in DNA synthesis are concentrated in the body of the larva while protein synthesis occurs in the entire larva. This is the first description of the regionalization of DNA and protein synthesis in developing stages of E. granulosus.

Animals↗
PubMed2004

Unique precipitation and exocytosis of a calcium salt of myo-inositol hexakisphosphate in larval Echinococcus granulosus.

The ubiquitous intracellular molecule myo-inositol hexakisphosphate (IP6) is present extracellularly in the hydatid cyst wall (HCW) of the parasitic cestode Echinococcus granulosus. This study shows that extracellular IP6 is present as its solid calcium salt, in the form of deposits that are observed, at the ultrastructural level, as naturally electron dense granules some tens of nanometers in diameter. The presence of a calcium salt of IP6 in these structures was determined by two different electron microscopy techniques: (i) the analysis of the spatial distribution of phosphorus and calcium in the outer, acellular layer of the HCW (the laminated layer, LL) through electron energy loss spectroscopy, and (ii) the observation, by transmission electron microscopy, of HCW that were selectively depleted of IP6 by treatment with EGTA or phytase, an enzyme that catalyses the dephosphorylation of IP6. The deposits of the IP6-Ca(II) salt are also observed inside membrane vesicles in cells of the germinal layer (the inner, cellular layer of the HCW), indicating that IP6 precipitates with calcium within a cellular vesicular compartment and is then secreted to the LL. Thus, much as in plants (that produce vesicular IP6 deposits), the existence of transporters for IP6 or its precursors in internal membranes is needed to explain the compound's cellular localisation in E. granulosus.

6-Phytase↗
PubMed2005

Cellular organization and appearance of differentiated structures in developing stages of the parasitic platyhelminth Echinococcus granulosus.

Echinococcus granulosus is the causative agent of hydatidosis, a major zoonoses that affects humans and herbivorous domestic animals. The disease is caused by the pressure exerted on viscera by hydatid cysts that are formed upon ingestion of E. granulosus eggs excreted by canine. Protoscoleces, larval forms infective to canine, develop asynchronously and clonally from the germinal layer (GL) of hydatid cysts. In this report, we describe the cellular organization and the appearance of differentiated structures both in nascent buds and developed protoscoleces attached to the GL. Early protoscolex morphogenesis is a highly complex and dynamic process starting from the constitution of a foramen in the early bud, around which nuclei are distributed mainly at the lateral and apical regions. Similarly, distribution of nuclei in mature protoscoleces is not homogenous but underlies three cellular territories: the suckers, the rostellar pad, and the body, that surrounds the foramen. Several nuclei are associated to calcareous corpuscles (Cc), differentiated structures that are absent in the earlier bud stages. The number of nuclei is similar from the grown, elongated bud stage to the mature protoscolex attached to the GL, strongly suggesting that there is no significant cellular proliferation during final protoscolex development. The amount of DNA per nucleus is in the same range to the one described for most other platyhelminthes. Our results point to a sequential series of events involving cell proliferation, spatial cell organization, and differentiation, starting in early buds at the GL of fertile hydatid cysts leading to mature protoscoleces infective to canine.

Animals↗
PubMed2007

Apoptosis as a possible mechanism of infertility in Echinococcus granulosus hydatid cysts.

Echinococcus granulosus is a parasitic cestode causing hydatidosis in intermediate hosts (human and herbivorous). Most symptoms of the disease occur by the pressure exerted on viscera by cysts that are formed upon ingestion of the parasite eggs excreted by definitive hosts (canines). Protoscoleces, the developmental form of the parasite infective to definitive hosts, are formed in the germinal nucleated layer of fertile hydatid cysts. For unknown reasons, some cysts are unable to produce protoscoleces (infertile hydatid cysts). In this study, analysis of DNA fragmentation using TUNEL and agarose gel electrophoresis showed higher levels of apoptosis in infertile cysts as compared to fertile cysts. Additionally, caspase 3 was detected both in fertile and infertile cysts; the activity of this enzyme was found to be higher in infertile cysts. We conclude that apoptosis may be involved in hydatid cyst infertility. This is the first report on the presence of programmed cell death in E. granulosus.

Animals↗
PubMed1993

Interleukin-1-dependent mitogenic responses induced by protoscoleces of Echinococcus multilocularis in murine lymphocytes.

Mitogenic effects of protoscoleces (PSCs) of Echinococcus multilocularis on murine lymphocytes were studied. Spleen cells from normal BALB/c mice showed significant proliferative responses when cocultured with PSCs. Proliferative responses were observed in both the T and B cell populations. The PSCs also stimulated cells of the macrophage/monocyte lineage to secrete interleukin-1 (IL-1). Depletion of plastic- and Sephadex G-10-adherent cells from the spleen cell population significantly reduced the proliferative responses to PSCs and the low responsiveness was restored by addition of plastic-adherent cells to these cultures. Furthermore, addition of anti-IL-1 serum to the spleen cell cultures stimulated with PSCs completely suppressed the proliferative responses. These findings demonstrate that the mitogenic effect of PSC on lymphocytes depends on IL-1 secreted by cells of macrophage/monocyte lineage.

Animals↗
PubMed1990

In vivo morphological damage induced by a new benzimidazole prodrug in Echinococcus multilocularis metacestodes.

Benzimidazoles are the most widely used compounds in chemotherapy of alveolar hydatid disease (AHD), but long-term administration is required to reach detectable plasma levels. N-Methoxycarbonyl N'-2-nitro 4-trifluoromethyl phenyl thiourea (2) was prepared as a potential prodrug of (5-trifluoromethyl-1H-benzimidazole-2-yl)-carbamic acid methyl ester. Biological effects of 2 were evaluated in gerbils (Meriones unguiculatus) against the causative agent of AHD, Echinococcus multilocularis metacestodes, through a transmission electron microscopy study. Significant morphological damage of tegument and protoscolices occurred after an 18-day treatment. The treatment of AHD by using the prodrug form of the benzimidazole deserves further study.

Animals↗
PubMed1993

Synthesis of propargylic alcohols and biological effects on Echinococcus multilocularis metacestodes.

This study describes the synthesis of propargylic alcohols derived from isatin and their biochemical and morphological effects on Echinococcus multilocularis metacestodes in Meriones unguiculatus. Propargylic alcohols decreased the alkaline phosphatase and the lactate dehydrogenase activities of the metacestode selectively. The most effective compound, 1b, decreased the lactate dehydrogenase enzymatic activity, and the glucose concentration in the parasite increased, whereas the glycogen content was partially decreased. Furthermore, the ultrastructure study revealed several damages. The host-parasite relationships are very important in the intrahepatic cestodes as shown by the biochemical side effects observed in the host's liver during the treatment. An in vitro enzymatic study was performed with alcohols 1b and 1c.

Alkaline Phosphatase↗
PubMed2003

Proteomic analysis of the larval stage of the parasite Echinococcus granulosus: causative agent of cystic hydatid disease.

We describe the preparation of Echinococcus granulosus metacestode protein extracts for two-dimensional electrophoresis (2-DE). Protoscoleces and hydatid fluid were prepared by precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants. Compared to the untreated control, TCA precipitation improved the 2-DE gel profile of the protoscoleces proteins. Comparison of 2-DE gels from insoluble and soluble fractions of the protoscoleces protein extract showed that most proteins are insoluble after lysis by sonication. Host serum proteins, especially albumin and globulins, caused horizontal streaking problems on the hydatid fluid 2-DE gels due to their high content in this sample. Even after the preparation of a hydatid fluid parasite enriched fraction, the high amount of bovine serum albumin and globulins made parasite-specific proteins difficult to detect by 2-DE. Despite the absence of an E. granulosus genome sequencing or expressed sequence tag (EST) projects, it was possible to identify 15 prominent protein spots from a whole protein protoscoleces 2-DE gel by peptide mass fingerprinting. These include actins, tropomyosin, paramyosin, thioredoxin reductase, antigen P-29, cyclophilin, and the heat shock proteins hsp70 and hsp20. This work demonstrates that 2-DE and PMF are important tools to identify proteins from the hydatid fluid and protoscoleces and for the comparative analysis of cysts from different hosts or between active and resting cysts.

Animals↗
PubMed1997

A protein with a novel calcium-binding domain associated with calcareous corpuscles in Echinococcus granulosus.

A novel intracellular calcium-binding protein from Echinococcus granulosus is described in this work. A cDNA was isolated from a lambdagt11 protoscolex expression library and the deduced amino acid sequence has at least fifteen sequentially repeated twelve-residue repeats that resemble the calcium-binding loop of EF-hands; however, the dodecamer motif has no flanking helices. The cDNA was expressed in Escherichia coli using the pGEX vector, and a recombinant fusion protein (EgCaBP1-GST) was obtained. The recombinant fusion protein binds calcium when assayed with 45Ca. It is possible that the calcium-binding motifs present a secondary structure similar to the parallel beta roll structure described for an alkaline protease from Pseudomonas aeruginosa. A native protein of more than 300 kDa was recognized by an anti-EgCaBP1 monoclonal antibody by Western-blot analysis. Immunohistochemistry using a pool of anti-EgCaBP1-GST mouse sera demonstrated a strong association of the protein with calcareous corpuscles. The possible role of this protein and that of the calcareous corpuscles in the protoscolex are discussed.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterization of a thioredoxin gene from Echinococcus granulosus.

The insert of a clone from a lambdagt11 Echinococcus granulosus (Platyhelminth, Cestoda) protoscolex cDNA library, showed an open reading frame whose deduced protein sequence presents a high homology with all described thioredoxins (TRX). The TRX active site (Trp-Cys-Gly-Pro-Cys) is completely conserved. With a monospecific antibody, selected from a total anti-protoscolex sera by the isolated clone, a 12 kDa polypeptide was immunoprecipitated from a protoscolex total protein extract. Furthermore, an antiserum raised against a recombinant EgTRX also recognizes a 12 kDa band in these extracts. The recombinant protein presents TRX activity, using the insulin reduction assay. Finally, a TRX activity was characterized in protoscolex extracts. In all organisms where TRXs were studied, they participate in a cascade of redox exchanges, contributing to the maintaining of cell homeostasis. Considering that the parasitic flatworm E. granulosus is probably submitted to an important oxidative stress due to host defences, EgTRX protein could be involved in the survival strategies of this parasite.

Amino Acid Sequence↗
PubMed1994

Echinococcus granulosus: characterization of the electrical potential of the syncytial tegument of protoscoleces incubated in vitro--effect of inhibitors.

Upon microelectrode impalements of the tegument of Echinococcus granulosus protoscoleces incubated in Ringer Krebs solution, electrical potential differences of -49 +/- 1 mV, using procedure I, and -53 +/- 1 mV, using procedure II, were recorded. The changes in the electrical potential difference as well as the structural alterations produced by sodium deoxicholate indicate that the observed potentials are established across the apical membrane of the syncytial tegument. The electrical potential difference is primarily dependent on the K+ gradient across the tegumental membrane: a 10-fold increase in the external K+ causes a 30-mV change in the electrical potential. External Na+ concentration changes also affected the electrical potential difference, but altering the external Cl- has no detectable effect. Amiloride, a very well-known blocker of Na+ epithelia channels, produced a reversible hyperpolarization that reached the maximum response at 10(-3) M. Ouabain, 10(-4) M, caused a depolarization in both fresh and Na-rich protoscoleces, although this depolarization was greater and showed a faster onset under the latter condition. It is concluded that the electrical potential difference of E. granulosus protoscolex is generated at the apical tegumental membrane and that it can be experimentally divided into two main components: One of them depends on ionic gradients and membrane permeabilities in accordance with the electrodiffusion predicted by the Goldman, Hodgkin, and Katz equation, while the other depends on the electrogenicity of an active Na+/K+ transport system.

Amiloride↗
PubMed1995

Echinococcus granulosus: partial characterization of the conductive properties of two cation channels from protoscoleces of the ovine strain, reconstituted on planar lipid bilayers.

Two cationic channels present in the microsomal fraction from Echinococcus granulosus protoscoleces of the sheep strain were studied in planar bilayer reconstitution experiments. A whole-worm homogenate was subjected to differential centrifugation and the postmitochondrial supernatant was laid on the top of a discontinuous sucrose gradient. The 15-30% (w/v) membrane fraction, enriched in NADPH-cytochrome c reductase, exhibited the highest fusion rate, two cationic channels being most frequently reconstituted. Both of them were highly cation-selective and had high conductances (244 and 107 pS in symmetrical 150 mM KCl). In most experiments, none of them displayed voltage dependence. The 244-pS channel was activated by Ca2+ and blocked by Ba2+, both in the micromolar range, thus partially resembling the Ca(2+)-activated K+ channel from more highly evolved animals. The 107-pS channel exhibited a Cs+ approximately equal to K+ > Na+ > Li+ > Ca2+ selectivity sequence (as measured by permeability ratios) and, most frequently, a high open probability (> 0.9) irrespective of the experimental conditions used, therefore sharing many properties of Schistosoma mansoni outer tegumental membrane cation channels.

Animals↗
PubMed1997

Echinococcus multilocularis metacestodes: immunological and immunocytochemical analysis of the relationships between alkaline phosphatase and the Em2 antigen.

Echinococcus multilocularis metacestodes possess an alkaline phosphatase (EmAP) which has been extensively characterized at the biochemical level in previous studies. The apparent molecular weight of the enzyme monomer and its isoelectric point matched those originally described for the Em2 antigen, a reference antigen currently used for the immunodiagnosis of E. multilocularis infection. These observations raised questions about the molecular relationship between the two molecules. In order to investigate the relations between EmAP and the Em2 antigen, immunoblotting and ELISA were carried out using polyclonal and monoclonal antibodies directed against EmAP and the Em2 antigen, respectively. In addition, the localization of EmAP and the Em2 antigen was compared by immunofluorescence and immunogold electron microscopy in in vitro-generated E. multilocularis metacestodes. The results show that common epitopes between EmAP and Em2 exist, which are predominantly of a peptidic nature. Both antigens are localized in an acellular parasite structure, the laminated layer, with additional locations for the EmAP on the glycocalyx and in the central region of invaginated protoscoleces. These results suggest a putative functional relationship between the two antigens and that Em2 could originate from EmAP.

Alkaline Phosphatase↗
PubMed1998

Echinococcus granulosus: an intraperitoneal diffusion chamber model of secondary infection in mice.

The present work describes a new experimental model of secondary infection which allows, through the recovery of the parasite together with its local in vivo environment, examination of the local nonadaptive immune response of the infected host and the differentiation of the parasite from protoscoleces to cysts. In this model we administered protoscoleces within silicone diffusion chambers, previously implanted into the peritoneal cavities of mice. The process of designing the model involved, first, determination of the optimal time postimplantation to infect the mice and, second, evaluation of the parasite's ability to establish infection within the chambers. The optimal time for infection was considered to be after the inflammation caused by implantation of the chambers had subsided. Our results showed that by day 20 postsurgery, three parameters used as indications of inflammation (complement C3, serum amyloid P protein, and polymorphonuclear cells in the peritoneum and in the chamber contents) had reverted to their normal levels. In our study of parasite differentiation, we found that 2-3% of the total number of parasites inoculated into the chambers were recovered as viable cysts after 100 days. Throughout the infection period, the population of parasites recovered was heterogeneous; certain parasite morphologies that have not been described previously were observed. In conclusion, the use of intraperitoneal diffusion chambers offers a potential tool for investigating the in vivo differentiation process of secondary cysts of Echinococcus granulosus in mice and the early local interactions between host and parasite during this process.

Animals↗
PubMed2000

Echinococcus granulosus: DNA extraction from germinal layers allows strain determination in fertile and nonfertile hydatid cysts.

A method for the isolation of Echinococcus granulosus DNA from germinal layers of hydatid cysts is described. The method includes a hexadecyltrimethylammonium bromide/chloroform extraction and an adsorption to diatomaceous earth suspension. DNA suitable for polymerase chain reaction was obtained and used for parasite strain determination by mitochondrial cytochrome c oxidase I gene sequencing. Fertile and nonfertile cyst isolates from sheep, cattle, pigs, and humans were characterized. Hitherto, no direct parasite strain characterization has been made on nonfertile hydatid cysts, whereas here we report that nonfertile hydatid cysts were produced by sheep strain (G1 genotype) in sheep, cattle, and humans and by pig strain (G7 genotype) in pigs.

Animals↗
PubMed2001

Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein.

We report the isolation and characterization of an Echinococcus granulosus gene that codes for a protein with actin filament fragmenting and nucleating activities (EgAFFP). The genomic region corresponding to the EgAFFP gene presents a coding sequence of 1110 bp that is interrupted by eight introns. The EgAFFP deduced amino acid sequence is about 40% homologous to those of several members of the gelsolin family, such as Physarum polycephalum fragmin, Dictyostelium discoideum severin, and Lumbricus terrestris actin modulator. As do other proteins of the same family, EgAFFP presents three repeated domains, each one characterized by internal conserved amino acid motifs. Assays with fluorescence-labeled actin showed that the full-length recombinant EgAFFP effectively binds actin monomers in both a calcium-dependent and calcium-independent manner and also presents actin nucleating and severing activities.

Actins↗
PubMed2001

O-glycosylation in Echinococcus granulosus: identification and characterization of the carcinoma-associated Tn antigen.

In the present work we demonstrate that the cancer-associated O-glycosylated Tn antigen (GalNAc-O-Ser/Thr) is expressed by the cestode Echinococcus granulosus. This antigen was detected in both larval and adult worm extracts, with the highest specific activity observed in the adult excretion/secretion preparation. Histochemical analysis showed that Tn is preferentially expressed in the parenchyma in both parasite stages and the external part of tegument in adult worms. A similar pattern was observed for sialyl-Tn, a related O-linked antigen. Tn glycoproteins from protoscoleces were resolved by SDS-PAGE in two main components of 43 and 49 kDa. After purification, this material was reactive with lectins which bind GlcNAc/sialic acid, GalNAc, and T antigen. In a preliminary evaluation, high levels of Tn antigen were detected in serum samples from patients with hydatid cyst, suggesting that the measure of Tn in serum could be a biomarker of this disease, although extensive work is necessary in order to determine the clinical usefulness of this assay. The results reported here, the first evidence of O-glycosylation pathways in E. granulosus and the presence of Tn antigen in cestodes, suggest that the evaluation of O-glycosylated antigens might give new insights in the host-parasite relationship.

Adult↗