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.

Echinococcus felidis in hippopotamus, South Africa.

Hydatid cysts of Echinococcus felidis are described from the hippopotamus (Hippopotamus amphibius) from Mpumalanga Province, South Africa. Among six hippopotami investigated, hepatic hydatids were found in three. The identification was based on mitochondrial and nuclear DNA sequences. In addition, the rostellar hook morphology was analysed. This is the first morphological description of the metacestode of E. felidis, and the first molecularly confirmed report of the intermediate host of E. felidis in South Africa. The definitive host of E. felidis in South Africa is the lion (Panthera leo).

On the importance of targeting parasite stem cells in anti-echinococcosis drug development.

The life-threatening diseases alveolar and cystic echinococcoses are caused by larvae of the tapeworms Echinococcus multilocularis and E. granulosus, respectively. In both cases, intermediate hosts, such as humans, are infected by oral uptake of oncosphere larvae, followed by asexual multiplication and almost unrestricted growth of the metacestode within host organs. Besides surgery, echinococcosis treatment relies on benzimidazole-based chemotherapy, directed against parasite beta-tubulin. However, since beta-tubulins are highly similar between cestodes and humans, benzimidazoles can only be applied at parasitostatic doses and are associated with adverse side effects. Mostly aiming at identifying alternative drug targets, the nuclear genome sequences of E. multilocularis and E. granulosus have recently been characterized, revealing a large number of druggable targets that are expressed by the metacestode. Furthermore, recent cell biological investigations have demonstrated that E. multilocularis employs pluripotent stem cells, called germinative cells, which are the only parasite cells capable of proliferation and which give rise to all differentiated cells. Hence, the germinative cells are the crucial cell type mediating proliferation of E. multilocularis, and most likely also E. granulosus, within host organs and should also be responsible for parasite recurrence upon discontinuation of chemotherapy. Interestingly, recent investigations have also indicated that germinative cells might be less sensitive to chemotherapy because they express a beta-tubulin isoform with limited affinity to benzimidazoles. In this article, we briefly review the recent findings concerning Echinococcus genomics and stem cell research and propose that future research into anti-echinococcosis drugs should also focus on the parasite's stem cell population.

Intraventricular hydatid cyst in a child.

Hydatid disease is caused by the infestation of the larvae of tapeworms of the genus Echinococcus. The definitive hosts of Echinococcus are various carnivores, the common being the dog. All mammals (more often sheep and cattle) are intermediate hosts. Humans get infected through the feco-oral route by ingestion of food or milk contaminated by dog feces containing ova of the parasite or by direct contact with dogs. The most common sites of infestation are the liver (75%) and lungs (15%). Various authors state a frequency of hydatidosis of the brain ranging between only 0.2-4% of cases. [1],[2],[3] The prognosis following surgical intervention is good, especially in pediatric age. [2],[3] Hence early diagnosis of this condition is crucial. We report a rare case of a child with a large intraventricular hydatid cyst that had an excellent recovery following surgery.

Morphological study of anomalous "laminated" brood capsules in cystic echinococcosis in humans and sheep.

"Anomalous", as yet unreported forms of brood capsules (BC) in cystic echinococcosis larvae from humans and sheep are described, thought to be the likely effect of degenerative involution of the parasite. Morphological examination showed an inner laminated layer (LL) in the brood capsules in intimate contact with the internal wall surface, suggesting the term "laminated brood capsules" (LBC). Analysis conducted on LBC from cysts of different typology invariably revealed them to contain just dead and highly degenerated protoscoleces (PSC). Structural and ultrastructural aspects of these peculiar forms are described and possible processes of formation discussed. Comparison of germinal layer (GL), BC wall and PSC organization recognize a "tegumental" and a "non-tegumental" pole, coinciding in GL and PSC and inverted in BC. A quite reversible PSC-or-LL switch governed by complex mechanisms is proposed, of which PSC viability/death is assumed to be an essential part, that under certain conditions makes in vivo LL production inwards possible in BC.

Bone hydatid disease.

Bone hydatid disease lacks a typical clinical appearance and image characteristics on x ray or CT are similar to those of tuberculosis, metastases and giant cell tumour or bone cysts. However, MRI does show distinctive diagnostic features of bone hydatid disease, especially in the spine. Until recently, treatment of osseous hydatid disease has been entirely surgical. Effective chemotherapy using benzimidazoles, particularly mebendazole, albendazole and combination treatments, has now been achieved in experimental studies and clinical practice. However, most of these drugs are still in the experimental stage or are in the early stages of clinical use.

Clinical considerations of primary hydatid disease of the pancreas.

BACKGROUND: The pancreas is a rare primary location of hydatid disease. The purpose of our study is to gain more insight into this entity and to focus on the management and the diagnostic approach to the disease. METHODS: The medical records of 5 patients with hydatid cysts of the pancreas were reviewed. RESULTS: Four of the cysts were primary, while in 1 case a coexisting cyst was found in the liver. The body and tail of the pancreas were the most common locations. Clinical presentation varied according to the anatomic location of the cyst. Abdominal pain, discomfort and vomiting were the main clinical symptoms. One patient presented with obstructive jaundice, while another patient manifested a mild episode of anaphylactic shock. The indirect hemagglutination test was positive in 3 of 4 cases. A computed tomography scan successfully imaged all cysts and calcification of the cystic wall was found in 3 of 4 cases. All patients underwent surgical therapy. Hydatid cysts in the tail of the pancreas were successfully treated with distal pancreatectomy, while cysts in the body and head of pancreas were treated with proper evacuation, pericystectomy and omentoplasty. The postoperative course was uneventful in all patients except 1 who presented a pancreatic fistula and was re-operated. The mean length of hospitalization after surgery was 11-12 (range 10-13) days, except for the patient who needed to be re-operated. No evidence of cyst recurrence was observed during the follow-up period. CONCLUSIONS: Hydatid cystic masses of the upper abdomen might also originate from the pancreas especially in endemic countries. Surgical excision of the entire cystic lesion remains the optimal treatment, offering hope for a complete cure.

Polymerase chain reaction for detection of patent infections of Echinococcus granulosus ("sheep strain") in naturally infected dogs.

Polymerase chain reaction (PCR) for the identification of eggs of the tapeworm Echinococcus granulosus ("sheep strain") was evaluated with primers derived from mitochondrial sequences. Specificity of these primers was confirmed by investigating DNA of other strains of E. granulosus and of 14 helminth species which inhabit the intestines of dogs. This PCR assay was used to investigate 131 purged dogs from Kazakhstan. Eighteen dogs harboured Echinococcus worms, ten of them in mixed infections with Taenia spp. Coproantigen detection was positive in 15 and taeniid eggs could be recovered from 13 of these specimens. Eight of the egg-containing samples were positive in the PCR for E. granulosus and four in a Echinococcus multilocularis -specific PCR revealing one mixed infection. Egg-containing faeces from two dogs harbouring both Taenia spp. and Echinococcus spp. were negative in both PCRs. The combination of egg isolation and PCR will also be of value in epidemiological studies when investigating environmental samples.

[Parasitological examinations of the hydatoid cysts of liver]. / Diagnostyka parazytologiczna bablowcowych torbieli watroby.

Liver cysts taken from 9 patients, operated on in the Medical Academy Clinic in 2000, have been examined. Cyst layers, their thickness and content have been analysed. We have searched for the cestoid scolexes and rostellar hooks. In 6 patients Echinococcus granulosus larvae have been found. In 4 cases there have been protoscolexes discovered, however with some signs of degeneration of the larva. In one case dead larva has been diagnosed on the basis of the presence of single hooks and in one patient one larva alive with unchanged structures has been revealed. Profound parasitological examination is a good supplement to the examination of the histopathological slides, especially in the case of degenerating larvae, when the tissues of the parasite lose their contact with the connective tissue layer of the host. Separated layer fragments, as well as protoscolexes and rostellar hooks need not be exposed to histological cutting.

[Establishment of germinal cell line of Echinococcus granulosus].

AIM: To establish a cell line of Echinococcus granulosus. METHODS: The proliferating membranes striped from liver cysts in a naturally infected sheep from Urumq were released to monodispersed germinal cells by grinding. The germinal cells were cultivated in the RPMI 1640 medium supplemented with 10%-20% calf serum on collagen-coated 24-well culture plate alternately from passage 1 to passage 14, and then continued. The morphological feature and growth situation were observed by light microscopy. The cultured cells were inoculated into BALB/c mice to identify the infectivity. ELISA was used to determine the immunogenicity of the cells. RESULTS: The germinal cells have been cultivated continuously up to passage 75. The subcultured cells were circular in shape with smooth surface and had the tendency to form syncytia and tissue-like masses. The cells from this cell line could be stored for at least 15 days in refrigerator at 4 degrees C and 10 months in liquid nitrogen. No cyst materials were detected in the mice inoculated with cells. The antigens from cell line could react with positive sera from mice infected with protoscoleces and sera against secreted antigens of cyst membrane, soluble antigens from cyst membrane, soluble antigens from protoscoleces and SHF. CONCLUSION: A germinal cell line of Echinococcus granulosus was successfully established.

Establishment of a primary culture of Echinococcus multilocularis germinal cells.

This study was designed to establish an in vitro primary culture of germinal cells of Echinococcus multilocularis, a parasite that causes alveolar echinococcosis of the liver (AEL). We also investigated the temperature-dependency of the cultured cells. The germinal cells, which originated from a human lesion, were cultured by an original fluid-suspension method at 25 degrees C or 37 degrees C for 4 weeks. Anchorage-dependent and -independent cells were observed by light microscopy, transmission electron microscopy, and immunocytochemistry to confirm their origin. Cell number and viability were examined by immunocytochemistry and mitochondrial exclusion test. The cultured cells were also inoculated into jirds (Meriones unguiculatus) to evaluate metacestode formation. Morphology and immunocytochemistry showed that the cultured cells were typically germinal cells. The cell number declined gradually over the 4-week culture period, but viability remained at 50% at 3 weeks. These findings were not associated with either of the two culture temperatures; moreover, host-associated cells were not noted in the cultured cells at 25 degrees C. The implanted cells formed metacestodes in the jird peritoneal cavity, and their histology demonstrated mature and typical alveolar-type echinococcal cysts. We successfully established an in vitro primary culture of germinal cells. This should contribute to future studies, and, hence, a better outcome for patients with AEL.

Differentiation of Spanish strains of Echinococcus granulosus using larval rostellar hook morphometry.

Two multivariate statistical procedures (Principal Component Analysis and Discriminant Function Analysis) were used to analyse morphometric data from larval hooks of Spanish samples of Echinococcus granulosus previously characterized in physiological, biochemical and genetic studies. The 5 physical variables considered could be reduced to 2 functional variables ("length of hooks" and "number of hooks"). The number of hooks was found not to be a reliable character to differentiate between strains of this parasite. Three Spanish strains (sheep-cattle, pig, and horse strains) were identified; these results are compatible with those obtained using other techniques. We consider larval hook morphology as a valid criterion for identifying E. granulosus strains in Spain, with potential use for epidemiological studies.

Cytochemical study of the germinal membrane of the Echinococcus granulosus cyst.

The present cytochemical study was undertaken to provide more information on the localization of enzymatic and glycoconjugates in the germinal membrane of the Echinococcus granulosus cyst. The distinctive distribution of binding sites for two lectins (peanut agglutinin and Dilochos biflorus agglutinin) in the germinal membrane are described. An investigation is made of the distribution and specific activity of adenosine triphosphatase, alkaline phosphatase and acid phosphatase. The results suggest that cells located in the deeper layer of the germinal membrane are intrinsic in the cellular differentiation process. The dissimilarities detected in both the enzymatic activity and the lectin-binding receptors could be associated with metacestode development or degeneration.

Possible formation of new brood capsule by the previously formed brood capsule in Echinococcus multilocularis metacestodes.

In Echinococcus multilocularis metacestodes obtained from the peritoneal cavity of an experimentally infected jird, cellular accumulations were found not only on the inner surfaces of germinal layers but also on the outer surfaces of brood capsules. These cellular accumulations are believed to represent at incipient form of the brood capsule. It has been thought that brood capsules are produced by the germinal layer, but we speculate that the brood capsule itself, as well as the germinal layer, may have the potential to produce new brood capsules.