scholarly journals The susceptibility analysis of Echinococcus multilocularis protoscoleces tubulin to mebendazole and RNA interference

2020 ◽  
Author(s):  
Qiqi Shi ◽  
Lele Huo ◽  
Bin Jiang ◽  
Haijun Gao ◽  
Jinxin Zheng ◽  
...  
Keyword(s):  
Rna Interference ◽  
Echinococcus Multilocularis ◽  
Drug Target ◽  
Alveolar Echinococcosis ◽  
Molecular Level ◽  
Benzimidazole Derivatives ◽  
Flame Cell ◽  
Reverse Transcription Pcr ◽  
Transmission Electron

ABSTRACTAlveolar echinococcosis, caused by the larval (metacestode) stage of the tapeworm Echinococcus multilocularis, is a lethal parasitosis of the liver prevalent in the Northern Hemisphere. For chemotherapy the benzimidazole derivatives mebendazole and albendazole were introduced, which were found to disrupt the microtubules by inhibition of the polymerization of tubulin into microtubules, and β-tubulin was determined to be the drug target molecule. In the present study, we evaluated the chemosensitivity of E. multilocularis protoscoleces tubulin to mebendazole and RNA interference in vitro, and to explore whether the molecular level and ultrastructure of E. multilocularis protoscoleces microtubules change post-mebendazole and RNA interference. We identified that mebendazole is parasitostatic to E. multilocularis protoscoleces through suppression the tubulin expression and change the flame cell morphology in molecular level, besides RNA interference indicated that β 2 tubulin is probably one of the vital tubulin gene to form the flame cell and the protonephridial system tubules (collective tubes) of E. multilocularis protoscoleces. Molecular level and ultrastructure detection were performed by reverse transcription-PCR, western blotting and transmission electron microscope. The RNA interference would be probably as a parasitocidal method to disrupt the survival of PSCs, extend that the relevant tubulin maybe as potential target for drug development against AE.

scholarly journals In Vitro Activities of Benzimidazoles against Echinococcus multilocularis Metacestodes

1998 ◽  
Vol 42 (5) ◽  
pp. 1052-1056 ◽  
Author(s):  
Heike Jura ◽  
Augustinus Bader ◽  
Matthias Frosch
Keyword(s):  
Echinococcus Multilocularis ◽  
Alveolar Echinococcosis ◽  
Survival Rates ◽  
Meriones Unguiculatus ◽  
Benzimidazole Derivatives ◽  
Reverse Transcription Pcr ◽  
Clinical Observations ◽  
Vitro Model ◽  
First Time

ABSTRACT Alveolar echinococcosis, caused by the larval (metacestode) stage of the tapeworm Echinococcus multilocularis, is a lethal parasitosis of the liver prevalent in the Northern Hemisphere. For chemotherapy the benzimidazole derivatives mebendazole and albendazole were introduced, and their use has resulted in a significant improvement in the survival rates. However, data from experiments with animals and clinical observations indicate that these drugs elicit only parasitostatic activity and in most cases are not able to completely eliminate the parasitic metacestode tissue. In the present study, we applied a culture system for the in vitro growth and proliferation ofE. multilocularis metacestodes to analyze the parasitostatic and parasitocidal potential of mebendazole. Here, we demonstrate for the first time that at concentrations of >0.1 μM, i.e., at concentrations used for therapy of human alveolar echinococcosis, this antihelminth drug is parasitocidal in vitro. Viability assessment was performed by infection experiments withMeriones unguiculatus and mebendazole-treated metacestode tissue and by reverse transcription-PCR for the detection of E. multilocularis mRNA. The E. multilocularis in vitro model proved to be a valuable tool for the analysis of the potential of antihelminth drugs.

Long-term (35 years) cryopreservation of Echinococcus multilocularis metacestodes

Parasitology ◽  
2020 ◽  
Vol 147 (9) ◽  
pp. 1048-1054
Author(s):  
Teivi Laurimäe ◽  
Philipp A. Kronenberg ◽  
Cristian A. Alvarez Rojas ◽  
Theodor W. Ramp ◽  
Johannes Eckert ◽  
...  
Keyword(s):  
Echinococcus Multilocularis ◽  
Alveolar Echinococcosis ◽  
Geographic Origin ◽  
Experimental Animals ◽  
Long Term Storage ◽  
Term Maintenance ◽  
Term Storage

AbstractThe metacestode of Echinococcus multilocularis is the etiological agent of alveolar echinococcosis. The metacestode stage used for research is maintained in rodents by serial passages. In order to determine whether cryopreservation of E. multilocularis metacestodes would be suitable for long-term maintenance and replace serial passages, isolates of different geographic origin were cryopreserved in 1984–1986. The aim of the current study was to test the viability of cryopreserved isolates following long-term cryopreservation (up to 35 years) and to determine the phylogenetic clades these isolates belonged to. Cryopreserved isolates were tested for viability in vitro and in vivo in gerbils. In vitro results of 5 isolates indicated protoscolex survival in 13 of 17 experiments (76%) and metacestode survival in 5 of 12 (42%) in vivo experiments. In vivo results showed ‘abortive lesions’ in 13 of the 36 animals, 15 were negative and 8 harboured proliferating metacestode tissue containing protoscoleces. Genetic analysis confirmed the isolates belonged to European, Asian and North-American clades. In conclusion, the results of the current study indicate that metacestodes of E. multilocularis are able to survive long-term cryopreservation. Therefore, cryopreservation is a suitable method for long-term storage of E. multilocularis metacestode isolates and reduces the number of experimental animals.

scholarly journals In vivo and in vitro efficacy of crocin against Echinococcus multilocularis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Chuanchuan Liu ◽  
Haining Fan ◽  
Lu Guan ◽  
Ri-li Ge ◽  
Lan Ma
Keyword(s):  
Echinococcus Multilocularis ◽  
Structural Damage ◽  
Secondary Infection ◽  
Treatment Options ◽  
Collagen Deposition ◽  
Mammalian Cell Lines ◽  
Human Foreskin Fibroblasts ◽  
Transmission Electron

Abstract Background Alveolar echinococcosis (AE) is a fatal zoonosis caused by the larvae of Echinococcus multilocularis. However, current chemotherapy treatment options are based on benzimidazoles [albendazole (ABZ) and mebendazole], which have limited efficacy. Therefore, novel drugs are necessary for the treatment of this disease. Methods The anthelmintic effects of crocin were tested on E. multilocularis metacestodes, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFFs) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. The in vivo efficacy of crocin was investigated in mice following secondary infection with E. multilocularis. Furthermore, collagen deposition and degradation in host tissues around the metacestodes were evaluated. Results In vitro, crocin had a median effective concentration of 11.36 μM against cultured E. multilocularis metacestodes, while it reduced germinal cell viability at a median inhibitory concentration of 10.05 μM. Crocin was less toxic to HFFs and RH mammalian cell lines than to metacestodes. Transmission electron microscopy revealed that crocin treatment resulted in structural damage in the germinal layer. In addition, 60.33 ± 3.06% of protoscoleces were killed by treatment with 10 μM crocin for 7 days, indicating that crocin has a parasiticidal effect. In vivo, the metacestode weight was significantly reduced after the administration of crocin at 50 mg/kg and 100 mg/kg (55.1 and 68.1%, respectively). Metacestode pathology showed structural disruption of the germinal and laminated layers after crocin treatment. The crocin- and ABZ-treated groups presented significant increases in the levels of interleukin (IL)-2 and IL-4. Furthermore, crocin inhibited the expression of matrix metalloproteinases (MMPs) (MMP2 and MMP9) and promoted collagen deposition in the metacestode. Conclusions Crocin was demonstrated to exert parasiticidal activity against E. multilocularis in vitro and in vivo, and can be developed as a novel drug for the treatment of AE. Graphical abstract

scholarly journals A secreted Echinococcus multilocularis activin A homologue promotes regulatory T cell expansion

2019 ◽  
Author(s):  
Justin Komguep Nono ◽  
Manfred B. Lutz ◽  
Klaus Brehm
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Larval Stage ◽  
Echinococcus Multilocularis ◽  
Alveolar Echinococcosis ◽  
Activin A ◽  
Dependent Manner ◽  
Cell Type

ABSTRACTBackgroundAlveolar echinococcosis (AE), caused by the metacestode larval stage of the fox-tapeworm Echinococcus multilocularis, is a chronic zoonosis associated with significant modulation of the host immune response. A role of regulatory T-cells (Treg) in generating an immunosuppressive environment around the metacestode during chronic disease has been reported, but the molecular mechanisms of Treg induction by E. multilocularis remain elusive so far.Methodology/Principal findingsWe herein demonstrate that excretory/secretory (E/S) products of the E. multilocularis metacestode promote the formation of Foxp3+ Treg from CD4+ T-cells in vitro in a TGF-β-dependent manner. We also show that host T-cells secrete elevated levels of the immunosuppressive cytokine IL-10 in response to metacestode E/S products. Within the E/S fraction of the metacestode we identified an E. multilocularis activin A homolog (EmACT) that displays significant similarities to mammalian Transforming Growth Factor-β (TGF-β)/activin subfamily members. EmACT obtained from heterologous expression promoted host TGF-β-driven CD4+ Foxp3+ Treg conversion in vitro. Furthermore, like in the case of metacestode E/S products, EmACT-treated CD4+ T-cells secreted higher levels of IL-10. These observations suggest a contribution of EmACT in the in vitro expansion of Foxp3+ Treg by the E. multilocularis metacestode. Using infection experiments we show that intraperitoneally injected metacestode tissue expands host Foxp3+ Treg, confirming the expansion of this cell type in vivo during parasite establishment.Conclusions/SignificanceIn conclusion, we herein show that E. multilocularis larvae secrete a factor with clear structural and functional homologies to mammalian activin A. Like its mammalian homolog, this protein induces the secretion of IL-10 by T-cells and contributes to the expansion of TGF-β-driven Foxp3+ Treg, a cell type that has been reported crucial for generating a tolerogenic environment to support parasite establishment and proliferation.AUTHOR SUMMARYThe metacestode larval stage of the tapeworm E. multilocularis grows infiltratively, like a malignant tumor, within the organs of its human host, thus causing the lethal disease alveolar echinococcosis (AE). Immunosuppression plays an important role in both survival and proliferation of the metacestode, which mainly depends on factors that are released by the parasite. These parasite-derived molecules are potential targets for developing new anti-echinococcosis drugs and/or improving the effectiveness of current therapies. Additionally, an optimized use of such factors could help minimize pathologies resulting from over-reactive immune responses, like allergies and autoimmune diseases. The authors herein demonstrate that the E. multilocularis metacestode releases a protein, EmACT, with significant homology to activin A, a cytokine that might support host TGF-β in its ability to induce the generation of immunosuppressive regulatory T-cells (Treg) in mammals. Like its mammalian counterpart, EmACT was associated with the expansion of TGF-β-induced Treg and stimulated the release of elevated amounts of immunosuppressive IL-10 by CD4+ T-cells. The authors also demonstrate that Treg are locally expanded by the metacestode during an infection of mice. These data confirm an important role of Treg for parasite establishment and growth during AE and suggest a potential role of EmACT in the expansion of these immunosuppressive cells around the parasite.

scholarly journals In VitroandIn VivoActivities of Dicationic Diguanidino Compounds against Echinococcus multilocularis Metacestodes

2013 ◽  
Vol 57 (8) ◽  
pp. 3829-3835 ◽  
Author(s):  
Tatiana Küster ◽  
Nadja Kriegel ◽  
David W. Boykin ◽  
Chad E. Stephens ◽  
Andrew Hemphill
Keyword(s):  
Active Compound ◽  
Echinococcus Multilocularis ◽  
Alveolar Echinococcosis ◽  
Treatment Options ◽  
Efficient Treatment ◽  
Content Type ◽  
Core Group

ABSTRACTAlveolar echinococcosis (AE) is a disease predominantly affecting the liver, with metacestodes (larvae) of the tapewormEchinococcus multilocularisproliferating and exhibiting tumor-like infiltrative growth. For many years, chemotherapeutical treatment against alveolar echinococcosis has relied on the benzimidazoles albendazole and mebendazole, which require long treatment durations and exhibit parasitostatic rather than parasiticidal efficacy. Although benzimidazoles have been and still are beneficial for the patients, there is clearly a demand for alternative and more efficient treatment options. Aromatic dications, more precisely a small panel of di-N-aryl-diguanidino compounds, were screened for efficacy againstE. multilocularismetacestodesin vitro. Only those with a thiophene core group were active against metacestodes, while furans were not. The most active compound, DB1127, was further investigated in terms ofin vivoefficacy in mice experimentally infected withE. multilocularismetacestodes. This diguanidino compound was effective against AE when administered intraperitoneally but not when applied orally. Thus, thiophene-diguanidino derivatives with improved bioavailability when administered orally could lead to treatment options against AE.

scholarly journals In Vitro Metacestodicidal Activities of Genistein and Other Isoflavones against Echinococcus multilocularis and Echinococcus granulosus

2006 ◽  
Vol 50 (11) ◽  
pp. 3770-3778 ◽  
Author(s):  
Arunasalam Naguleswaran ◽  
Martin Spicher ◽  
Nathalie Vonlaufen ◽  
Luis M. Ortega-Mora ◽  
Paul Torgerson ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Echinococcus Granulosus ◽  
Echinococcus Multilocularis ◽  
Structural Integrity ◽  
Treatment Options ◽  
Farm Animals ◽  
Economic Losses ◽  
Benzimidazole Derivatives ◽  
Receptor Binding Site

ABSTRACT Echinococcus multilocularis and Echinococcus granulosus metacestode infections in humans cause alveolar echinococcosis and cystic echinococcosis, respectively, in which metacestode development in visceral organs often results in particular organ failure. Further, cystic hydatidosis in farm animals causes severe economic losses. Although benzimidazole derivatives such as mebendazole and albendazole are being used as therapeutic agents, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of a number of isoflavones against Echinococcus metacestodes and protoscoleces. The most prominent isoflavone, genistein, exhibits significant metacestodicidal activity in vitro. However, genistein binds to the estrogen receptor and can thus induce estrogenic effects, which is a major concern during long-term chemotherapy. We have therefore investigated the activities of a number of synthetic genistein derivatives carrying a modified estrogen receptor binding site. One of these, Rm6423, induced dramatic breakdown of the structural integrity of the metacestode germinal layer of both species within 5 to 7 days of in vitro treatment. Further, examination of the culture medium revealed increased leakage of parasite proteins into the medium during treatment, but zymography demonstrated a decrease in the activity of metalloproteases. Moreover, two of the genistein derivatives, Rm6423 and Rm6426, induced considerable damage in E. granulosus protoscoleces, rendering them nonviable. These findings demonstrate that synthetic isoflavones exhibit distinct in vitro effects on Echinococcus metacestodes and protoscoleces, which could potentially be exploited further for the development of novel chemotherapeutical tools against larval-stage Echinococcus infection.

scholarly journals In Vitro Activities of Itraconazole, Methiazole, and Nitazoxanide versus Echinococcus multilocularis Larvae

2006 ◽  
Vol 50 (9) ◽  
pp. 2966-2970 ◽  
Author(s):  
Stefan Reuter ◽  
Burkhard Manfras ◽  
Marion Merkle ◽  
Georg Härter ◽  
Peter Kern
Keyword(s):  
Echinococcus Multilocularis ◽  
Alveolar Echinococcosis ◽  
Combined Treatment ◽  
Larval Growth ◽  
Animal Experiments ◽  
Drug Discontinuation ◽  
Mongolian Gerbils ◽  
Larval Tissue ◽  
Deadly Disease

ABSTRACT Albendazole (ABZ) and mebendazole are the only drugs licensed for treatment of human alveolar echinococcosis. In order to augment the armamentarium against this deadly disease, we tested a series of drugs for their efficacy against Echinococcus multilocularis larvae. E. multilocularis larvae grown intraperitoneally in Mongolian gerbils were transferred into tissue culture. Vesicles budded from the tissue blocks and after 6 weeks, drugs were added, and the effect on the vesicles was observed. We tested the following drugs at various concentrations: ABZ, artemether, caspofungin, itraconazole (ITZ), ivermectin, methiazole (MTZ), miltefosine, nitazoxanide (NTZ), rifampin, and trimethoprim-sulfamethoxazole. ABZ, ITZ, MTZ, and NTZ effectively destroyed parasite vesicles in this in vitro culture system. At high NTZ doses of 10 μg/ml, disintegration of all vesicles was observed after 7 days and was significantly more rapid than with ABZ at equal concentrations (21 days). After drug discontinuation, regrowth of vesicles occurred between 7 and 14 days for all four drugs, indicating a parasitostatic effect. Combination treatment with NTZ-ABZ at concentrations between 1 and 10 μg/ml for either 3 weeks, 3 months, or 6 months yielded no vesicle regrowth during 8 months after drug discontinuation. The treated larval tissue was injected intraperitoneally into gerbils, and no regrowth of larval tissue was observed, suggesting a parasitocidal effect after combined treatment. ITZ, MTZ, and NTZ are potent inhibitors of larval growth, although they proved to be parasitostatic only. The combination of NTZ plus ABZ was parasitocidal in vitro. Animal experiments are warranted for studies of dose, toxicity, and drug interactions.

scholarly journals Effect of Amphotericin B on Larval Growth of Echinococcus multilocularis

2003 ◽  
Vol 47 (2) ◽  
pp. 620-625 ◽  
Author(s):  
Stefan Reuter ◽  
Marion Merkle ◽  
Klaus Brehm ◽  
Peter Kern ◽  
Burkhard Manfras
Keyword(s):  
Amphotericin B ◽  
Echinococcus Multilocularis ◽  
Alveolar Echinococcosis ◽  
Larval Growth ◽  
Mongolian Gerbils ◽  
Effective Treatment Option ◽  
Destructive Effect ◽  
Promising Alternative ◽  
Vitro Effect

ABSTRACT Alveolar echinococcosis is caused by the parasitic cestode Echinococcus multilocularis. Benzimidazoles, namely, mebendazole and albendazole, are the only drugs available for the treatment of inoperable alveolar echinococcosis. At present, no therapeutic alternative is available for patients with progressive disease under treatment or for patients who are unable to tolerate the side effects of the benzimidazoles. In addition, benzimidazoles are only parasitostatic for E. multilocularis. Thus, new therapeutic options are of paramount importance. In the present study we examined the in vitro effect of amphotericin B on E. multilocularis larvae. E. multilocularis metacestodes grown in the peritoneal cavities of Mongolian gerbils were transferred into a culture system. Vesicles budded from the tissue blocks and increased in number and size during the first 5 weeks. After 6 weeks drugs were added and deleterious effects on the vesicles were observed macroscopically and microscopically. By use of this in vitro tissue culture model we demonstrated that amphotericin B effectively inhibits the growth of E. multilocularis metacestodes. This destructive effect was significantly more rapid with amphotericin B than with the benzimidazoles. Cyclic treatment was effective in suppressing parasite growth. However, amphotericin B appears to be parasitostatic for E. multilocularis larvae, and regrowth occurs even after extended periods. In summary, amphotericin B constitutes the first promising alternative for the treatment of alveolar echinococcosis in cases of intolerance or resistance to benzimidazoles. It holds promise as an effective treatment option for otherwise fatal courses of disease.

Maytansine-Induced Mitotic Arrest in Human Lymphoblasts

1977 ◽  
Vol 35 ◽  
pp. 460-461
Author(s):  
Tai-Te Chao ◽  
John Sullivan ◽  
Awtar Krishan
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Transmission Electron Microscopy ◽  
Tubulin Polymerization ◽  
Vinca Alkaloids ◽  
Antimitotic Activity ◽  
Transmission Electron ◽  
Flow Microfluorometry ◽  
Brain Tubulin

Maytansine, a novel ansa macrolide (1), has potent anti-tumor and antimitotic activity (2, 3). It blocks cell cycle traverse in mitosis with resultant accumulation of metaphase cells (4). Inhibition of brain tubulin polymerization in vitro by maytansine has also been reported (3). The C-mitotic effect of this drug is similar to that of the well known Vinca- alkaloids, vinblastine and vincristine. This study was carried out to examine the effects of maytansine on the cell cycle traverse and the fine struc- I ture of human lymphoblasts.Log-phase cultures of CCRF-CEM human lymphoblasts were exposed to maytansine concentrations from 10-6 M to 10-10 M for 18 hrs. Aliquots of cells were removed for cell cycle analysis by flow microfluorometry (FMF) (5) and also processed for transmission electron microscopy (TEM). FMF analysis of cells treated with 10-8 M maytansine showed a reduction in the number of G1 cells and a corresponding build-up of cells with G2/M DNA content.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

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