The Experts below are selected from a list of 243 Experts worldwide ranked by ideXlab platform
Marshall W. Lightowlers - One of the best experts on this subject based on the ideXlab platform.
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Ultrastructural reconstruction of Taenia ovis Oncospheres from serial sections
International journal for parasitology, 2010Co-Authors: Abdul Jabbar, Z. Swiderski, Daniel Młocicki, Ian Beveridge, Simon Crawford, David Bruce Conn, Malcolm K. Jones, Marshall W. LightowlersAbstract:The cellular organisation of Taenia ovis Oncospheres is interpreted from ultrathin serial sections and transmission electron microscopy following high pressure freezing and freeze-substitution. The surface of a hatched, non-activated T. ovis oncosphere is covered by an oncospheral membrane below which is the tegument bearing microvilli. The basal lamina of the tegument is underlain by broad bands of peripheral somatic musculature. Three pairs of hooks and associated muscles are present in the somatophoric third of the oncosphere. Approximately 19 cells of seven different types were identified which include: (i) a quadri-nucleated syncytium of penetration gland type 1 containing two lateral pairs of cell bodies interconnected by narrow cytoplasmic bridges (PG1); (ii) a quadri-nucleated syncytium of penetration gland type 2 (PG2); (iii) a single-nucleated median mesophoric gland cell; (iv) 10 somatic cells; (v) two germinative cells; (vi) two nerve cells; and (vii) a pair of median somatophoric cells. This study provides a clear understanding of the morphology of T. ovis Oncospheres and forms the basis for further investigations into the biology of taeniid Oncospheres.
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The ultrastructure of taeniid cestode Oncospheres and localization of host-protective antigens.
Parasitology, 2009Co-Authors: Abdul Jabbar, Z. Swiderski, Daniel Młocicki, Ian Beveridge, Marshall W. LightowlersAbstract:Taeniid eggs contain an infective larval form of the parasite, known as the oncosphere, which has been found to be highly susceptible to attack by the host's immune system and this fact has been exploited in the development of highly effective vaccines. Relatively little is known about the structure of taeniid Oncospheres and the localization of host-protective antigens within or on the oncosphere. Here, we briefly review the current state of knowledge of the structure of the oncosphere and present preliminary data on the localization of a host-protective antigen within the Oncospheres of Taenia ovis. The precise localization of the antigens, in the context of a detailed knowledge of the ultrastructure of the parasite, may reveal the immune mechanisms by which the taeniid parasites are killed by vaccine-induced immune responses, which, in turn, may provide clues about how vaccines could be developed against other parasitic helminths.
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IN VITRO ONCOSPHERE-KILLING ASSAYS TO DETERMINE IMMUNITY TO THE LARVAE
2006Co-Authors: Craig T. Kyngdon, Hector H. Garcia, Armando E. Gonzalez, Robert H. Gilman, Charles G. Gauci, Rick A. Rolfe, Jeanette C. Velasquez Guzm, Marilu J. Farttn Salazar, Richard A. Strugnell, Marshall W. LightowlersAbstract:Taeniid cestodes infect humans and livestock, causing considerable morbidity and mortality, as well as economic loss. Substantial progress has been made toward the production of recombinant vaccines against cysticercosis in livestock animals. Further development of these vaccines would be aided if a reliable in vitro test were available to measure host-protective immune responses in vaccinated animals. Here, we describe in vitro oncosphere-killing assays for the quantification of host-protective serum antibodies against Taenia pisiformis, Taenia ovis, Taenia saginata, and Taenia solium in rabbits, sheep, cattle, and pigs, respectively. Activated Oncospheres of T. pisiformis, T. ovisy T. saginata, and T. solium were incubated in vitro in culture medium, test serum, and a source of complement, and oncosphere killing was assessed after 10 days of culture. In vitro oncosphere killing reflected the presence of specific antibody, and the oncosphere-killing assay typically indicated immunity to the homologous parasite that had been determined in vivo. This study describes the first reliable oncosphere-killing assays for T. pisiformis, T. ovis, T. saginata, and T. solium. These assays will be used for further research into the optimization of recombinant vaccines against cysticercosis. Cestodes of the Taeniidae cause substantial human morbidity and mortality worldwide. They also infect livestock, causing considerable economic loss. Highly effective vaccines have been developed against several species of taeniid cestode (John- son et al., 1989; Lightowlers, 1996; Lightowlers, Lawrence et al., 1996; Flisser et al., 2004). These vaccines are undergoing further development and optimization with a view toward their practical application in prevention of cysticercosis in pigs, cat- tle, and sheep and against hydatidosis in sheep and other live- niid Oncospheres in vitro were undertaken by Chen (1950), Sil- verman (1955), and Heath and Smyth (1970), who were the first to recognize the potential importance of culture assays in the development of vaccines against metacestodes. Rickard and Outteridge (1974) investigated the mortality of Taenia pisifor- mis Oncospheres when cultured in vitro in the presence of im- mune sera. The low level of mortality that was observed in the previous studies encouraged Heath and Lawrence (1981) to in- clude complement in in vitro cultures involving Echinococcus granulosus Oncospheres. They were successful in demonstrating specific killing of activated E. granulosus Oncospheres in vitro in the presence of sera from sheep infected with E. granulosus and sheep immunized with E. granulosus eggs. The assay has subsequently been used as an integral component of the re- search that led to the development and characterization of the EG95 vaccine against hydatid disease (Heath and Lawrence,
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Echinococcus granulosus: oncosphere-specific transcription of genes encoding a host-protective antigen.
Experimental parasitology, 2004Co-Authors: Conan Chow, Charles G. Gauci, Alan F Cowman, Marshall W. LightowlersAbstract:A recombinant antigen vaccine has been developed which is effective in preventing the hydatid parasite, Echinococcus granulosus from infecting its animal intermediate hosts. The vaccine antigen, designated EG95, is expressed by a cDNA cloned from E. granulosus oncosphere mRNA. The gene encoding EG95 belongs to a small gene family where six of the members are transcribed in the oncosphere. Conditions were established which allowed specific RT-PCR amplification of mRNA for each gene family member and these conditions were applied to determine transcription patterns for each gene family member in gravid adult worms, Oncospheres, and protoscoleces. The four eg95 gene family members which encode an identical EG95 protein, were transcribed only in the oncosphere. In contrast, two gene family members that encode variant EG95 proteins did not have transcription patterns confined to the oncosphere. These findings suggest a common biological role for four genes in the gene family and a separate role for the other, more variant gene family members.
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Molecular cloning of genes encoding oncosphere proteins reveals conservation of modular protein structure in cestode antigens
Molecular and biochemical parasitology, 2003Co-Authors: Charles G. Gauci, Marshall W. LightowlersAbstract:Recombinant oncosphere antigens have been found to be remarkably effective when used as vaccines against cysticercosis and hydatid disease. Comparison of the structural features of these proteins and their associated genes suggest common features between antigens. Here molecular cloning is used to complete comparisons of Taenia solium, Taenia saginata, Taenia ovis, Echinococcus granulosus and Echinococcus multilocularis oncosphere antigens and genes. The exon/intron structure of genes cloned from T. solium and T. ovis genomic DNA (tsol16 and to16, respectively) in this study was found to be highly conserved. Two closely related tsol16 genes were cloned from the T. solium genome. Their corresponding transcripts were cloned from T. solium Oncospheres and a comparison of their deduced amino-acid sequence with that of the protein encoded by to16 indicates that these proteins are the most highly conserved oncosphere proteins identified so far. Cloning of another gene from T. solium (designated tsol18) and comparison with the homologous gene of T. saginata (tsa18) also revealed substantial conservation of gene structure. Comparisons of the genes cloned in this study with genes encoding oncosphere antigens from other taeniid cestodes identified striking conservation of exon structure. The highly conserved regions of the genes encode a putative secretory signal and fibronectin type III domain in each of the oncosphere proteins. The location of exon boundaries in relation to protein features identifies a clear modular structure among all members of these oncosphere antigens. Identification of structural conservation of genes encoding antigenic proteins across several taeniid species suggests that the encoded proteins play important roles in host infection and parasite survival.
Robert H. Gilman - One of the best experts on this subject based on the ideXlab platform.
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Oncospheres of T. solium and T. saginata cultured in HCT-8 and INT-407 cells during 180 days.
2019Co-Authors: Sandra Palma, Nancy Chile, Rogger P Carmen-orozco, Grace Trompeter, Kayla Fishbeck, Virginia Cooper, Laura Rapoport, Edson G Bernal-teran, Beth J Condori, Robert H. GilmanAbstract:Oncospheres of T. solium and T. saginata cultured in HCT-8 and INT-407 cells during 180 days.
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In vitro model of postoncosphere development, and in vivo infection abilities of Taenia solium and Taenia saginata.
Public Library of Science (PLoS), 2019Co-Authors: Sandra Palma, Nancy Chile, Rogger P Carmen-orozco, Grace Trompeter, Kayla Fishbeck, Virginia Cooper, Laura Rapoport, Edson G Bernal-teran, Beth J Condori, Robert H. GilmanAbstract:Taenia solium is known to cause human cysticercosis while T. saginata does not. Comparative in vitro and in vivo studies on the oncosphere and the postoncospheral (PO) forms of T. solium and T. saginata may help to elucidate why cysticercosis can occur from one and not the other. The aim of this study was to use in vitro culture assays and in vivo models to study the differences in the development of the T. solium and T. saginata oncosphere. Furthermore, this study aimed to evaluate the expression of cytokines and metalloproteinases (MMPs) in human peripheral blood mononuclear cells (PBMCs), which were stimulated by these Oncospheres and PO antigens. T. solium and T. saginata activated Oncospheres (AO) were cultured in INT-407 and HCT-8 intestinal cells for 180 days. The T. solium began to die while the T. saginata grew for 180 days and developed to cysticerci in INT-407 cells. Rats were inoculated intracranially with AO and PO forms of either T. saginata or T. solium. Rats infected with T. solium AO and PO forms developed neurocysticercosis (NCC), while those infected with the T. saginata did not. Human PMBCs were stimulated with antigens of AO and PO forms of both species, and the production of cytokines and metalloproteinases (MMPs) was measured. The T. solium AO antigen stimulated a higher production of IL-4, IL-5, IL-13, IFN-γ, and IL-2 cytokines compared to T. saginata AO. In the PO form, the T. saginata PO antigen increased the production of IL-4, IL-5, IL-13, IFN-γ, IL-1β, IL-6, IL-10, TNF-α and IL-12 cytokines compared to T. solium, suggesting that this global immune response stimulated by different forms could permit survival or destruction of the parasite depending of their life-cycle stage. Regarding MMPs, T. solium AO antigen stimulated a higher production of MMP-9 compared to T. saginata AO antigen, which may be responsible for altering the permeability of intestinal cells and facilitating breakdown of the blood-brain barrier during the process of invasion of host tissue
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Carotid Taenia solium Oncosphere Infection: A Novel Porcine Neurocysticercosis Model.
The American journal of tropical medicine and hygiene, 2018Co-Authors: Karen A. Alroy, Robert H. Gilman, Manuela Verastegui, Gianfranco Arroyo, Eloy Gonzales-gustavson, Linda Gallegos, Cesar M. Gavidia, Silvia Rodriguez, Teresa López, Luis A. Gomez-puertaAbstract:Neurocysticercosis (NCC), the infection of the human central nervous system (CNS) with larval cysts of Taenia solium causes widespread neurological morbidity. Animal models are crucial for studying the pathophysiology and treatment of NCC. Some drawbacks of current NCC models include differences in the pathogenesis of the model and wild-type parasite, low rates of infection efficiency and lack of reproducibility. We describe a novel porcine model that recreates infection in the CNS with high efficiency. Activated Oncospheres, either in a high (45,000-50,000) or low (10,000) dose were inoculated in the common carotid artery of 12 pigs by ultrasound-guided catheterization. Following oncosphere injection, either a high (30 mL) or low (1-3 mL) volume of saline flush was also administered. Cyst burden in the CNS was evaluated independently according to oncosphere dose and flush volume. Neurocysticercosis was achieved in 8/12 (66.7%) pigs. Cyst burden in the CNS of pigs was higher in the high versus the low oncosphere dose category (median: 4.5; interquartile ranges [IQR]: 1-8 and median: 1; IQR: 0-4, respectively) and in the high versus the low flush volume category (median 5.5; IQR: 1-8 and median: 1; IQR: 0-2, respectively), although not statistically different. All cysts in the CNS were viable, whereas both viable and degenerated cysts were found in the musculature. Carotid injection of activated Oncospheres in pigs is effective in reproducing NCC. Oncosphere entry into the CNS by way of vasculature mimics wild-type infection, and provides a useful alternative for future investigations on the pathogenesis and antiparasitic treatment of NCC.
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Characterization of the carbohydrate components of Taenia solium oncosphere proteins and their role in the antigenicity.
Parasitology research, 2013Co-Authors: Yanina Arana, Hector H. Garcia, Manuela Verastegui, Iskra Tuero, Louis Grandjean, Robert H. GilmanAbstract:This study examines the carbohydrate composition of Taenia solium whole oncosphere antigens (WOAs), in order to improve the understanding of the antigenicity of the T. solium. Better knowledge of oncosphere antigens is crucial to accurately diagnose previous exposure to T. solium eggs and thus predict the development of neurocysticercosis. A set of seven lectins conjugates with wide carbohydrate specificity were used on parasite fixations and somatic extracts. Lectin fluorescence revealed that d-mannose, d-glucose, d-galactose and N-acetyl-d-galactosamine residues were the most abundant constituents of carbohydrate chains on the surface of T. solium oncosphere. Lectin blotting showed that posttranslational modification with N-glycosylation was abundant while little evidence of O-linked carbohydrates was observed. Chemical oxidation and enzymatic deglycosylation in situ were performed to investigate the immunoreactivity of the carbohydrate moieties. Linearizing or removing the carbohydrate moieties from the protein backbones did not diminish the immunoreactivity of these antigens, suggesting that a substantial part of the host immune response against T. solium oncosphere is directed against the peptide epitopes on the parasite antigens. Finally, using carbohydrate probes, we demonstrated for the first time that the presence of several lectins on the surface of the oncosphere was specific to carbohydrates found in intestinal mucus, suggesting a possible role in initial attachment of the parasite to host cells.
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Standardization of a fluorescent-based quantitative adhesion assay to study attachment of Taenia solium oncosphere to epithelial cells in vitro.
Journal of immunological methods, 2011Co-Authors: Nancy Chile, Hector H. Garcia, Armando E. Gonzalez, Robert H. Gilman, Yanina Arana, Sandra Palma, Julio Evangelista, Charles R. Sterling, Manuela VerasteguiAbstract:To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-Oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium Oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-Oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-Oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-Oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of Oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-Oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment.
P.c. Fan - One of the best experts on this subject based on the ideXlab platform.
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infection of normal c3h hen mice with taenia saginata asiatica Oncospheres
Research in Veterinary Science, 2009Co-Authors: Shih-yi Peng, Tzu-hui Chu, I-chuang Wang, Wen-cheng Chung, John Chin Tsaihong, Jason C. Huang, P.c. FanAbstract:Abstract Normal C3H/HeN female mice were used to develop an animal model of Taenia saginata asiatica oncosphere infection. The host cellular immune response in this model was analyzed by a cytokine enzyme-linked immunosorbent assay (cytokine ELISA) and flow cytometry. Tumor-like cysts containing cysticerci were recovered from the inoculation sites of female mice 7 weeks postinfection with the T. saginata asiatica Oncospheres. A sharp increase and sustained elevation in the ability of spleen cells to produce interferon-γ and interleukin (IL)-2 revealed that cellular immunity played an important role during the infection. An immediate increase in the levels of IL-6 at 1 week postinfection indicated the induction of a local acute inflammatory response. However, no significant change in the levels of IL-10 indicated that Th2 cells were not involved in this immune response. The patterns of cell distribution revealed by flow cytometry also supported the same finding. These results suggested that Th1 cells played a major role in the immune response in C3H/HeN mice during the early stages of the oncosphere infection and that the Th2 response was not induced during the stage of cysticercus formation.
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Infection of normal C3H/HeN mice with Taenia saginata asiatica Oncospheres.
Research in veterinary science, 2008Co-Authors: Shih-yi Peng, Tzu-hui Chu, I-chuang Wang, Wen-cheng Chung, John Chin Tsaihong, Jason C. Huang, P.c. FanAbstract:Abstract Normal C3H/HeN female mice were used to develop an animal model of Taenia saginata asiatica oncosphere infection. The host cellular immune response in this model was analyzed by a cytokine enzyme-linked immunosorbent assay (cytokine ELISA) and flow cytometry. Tumor-like cysts containing cysticerci were recovered from the inoculation sites of female mice 7 weeks postinfection with the T. saginata asiatica Oncospheres. A sharp increase and sustained elevation in the ability of spleen cells to produce interferon-γ and interleukin (IL)-2 revealed that cellular immunity played an important role during the infection. An immediate increase in the levels of IL-6 at 1 week postinfection indicated the induction of a local acute inflammatory response. However, no significant change in the levels of IL-10 indicated that Th2 cells were not involved in this immune response. The patterns of cell distribution revealed by flow cytometry also supported the same finding. These results suggested that Th1 cells played a major role in the immune response in C3H/HeN mice during the early stages of the oncosphere infection and that the Th2 response was not induced during the stage of cysticercus formation.
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Cross protection against Taenia taeniaeformis in rats vaccinated with non-viable Oncospheres of Asian Taenia or T. saginata
Journal of helminthology, 1994Co-Authors: Akira Ito, P.c. Fan, W.c. Chung, M. SuzukiAbstract:It was determined to examine whether rats injected with non-viable Oncospheres of Asian Taenia or Taenia saginata became resistant to challenge infection with eggs of Taenia taeniaeformis, since (a) metacestodes of Asian Taenia and T. taeniaeformis develop in the liver of pigs and rats, respectively, and (b) Asian Taenia and T. saginata have human origins. Rats injected intravenously or subcutaneously with complete Freund's adjuvant with non-viable Oncospheres of Asian Taenia showed statistically significant resistance to challenge infection with eggs of T. taeniaeformis, whereas those injected with non-viable Oncospheres of T. saginata did not show any resistance.
Akira Ito - One of the best experts on this subject based on the ideXlab platform.
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Immunization of rodents against Hymenolepis infections using non-viable homologous Oncospheres.
The Kaohsiung journal of medical sciences, 2004Co-Authors: Ping-chin Fan, Wen-cheng Chung, Akira ItoAbstract:Immunity to Taiwan Taenia infection in pigs can be stimulated using homologous or heterologous nonviable Taenia Oncospheres. This study was designed to determine whether homologous non-viable Oncospheres could stimulate immunity to Hymenolepis infection in rodents. Hatched Oncospheres were prepared from eggs of Hymenolepis diminuta, Hymenolepis nana , and Hymenolepis microstoma and kept at −70°C for more than 1 month. A mixture of 500 non-viable Oncospheres of each tapeworm and complete Freund's adjuvant was injected subcutaneously in four groups of Sprague-Dawley rats or ICR mice one to four times at an interval of 1 week; controls were not immunized. After immunization, each rodent was orally inoculated with three fresh active cysticercoids of H. diminuta or H. microstoma or 500 fresh eggs of H. nana . The animals were then necropsied for adult tapeworms. No rats or mice immunized with non-viable Oncospheres of H. diminuta or H. nana were infected by the challenge inoculation. However, 28 of 34 mice immunized with non-viable H. microstoma Oncospheres were infected after inoculation with cysticercoids. This study demonstrated complete protection against infection by homologous parasites in rats or mice immunized with non-viable Oncospheres of H. diminuta and H. nana , respectively. Repeated immunization may not be required if resistance is stimulated in rodent hosts.
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Protective antibodies against Taenia taeniaeformis in rats infected with eggs or injected with non-viable Oncospheres or recombinant antigens of Oncospheres.
Parasite immunology, 1994Co-Authors: Akira Ito, Kazuhito Asano, Ken-ichi OkamotoAbstract:Antibody responses against Taenia taeniaeformis in rats infected with eggs or injected with non-viable Oncospheres or recombinant antigens of Oncospheres were analysed by passive transfer of serum and Western blotting. When recipient rats were injected with 1 ml serum from donors infected with eggs (infected serum), they all showed complete resistance to oral egg challenge, whereas those injected with 1 ml serum from donors injected with either Oncospheres or recombinant antigens (vaccinated serum) showed no resistance. IgG and IgG subclass responses detected by Western blotting revealed that antibody responses to oncosphere antigens in infected serum thoroughly differed from those in vaccinated serum. It is suggested that IgG2 alpha responses in infected serum should be used for screening of epitopes for candidate vaccine.
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In vitro hatching of Oncospheres of Taenia taeniaeformis using eggs isolated from fresh, frozen, formalin-fixed and ethanol-fixed segments
Journal of helminthology, 1994Co-Authors: T. Negita, Akira ItoAbstract:In order to establish a simple means of handling eggs of taeniid cestodes under non-biohazardous conditions, gravid segments of Taenia taeniaeformis were fixed in ethanol or formalin or were frozen. In vitro hatching of Oncospheres was carried out by the 0.5% sodium hypochlorite method using eggs isolated from the nonviable and viable segments. No Oncospheres hatched from formalized eggs, whereas almost all Oncospheres hatched from all other eggs but the number of Oncospheres recovered was highly variable. Oncospheres hatched from eggs isolated from frozen segments were highly fragile. The highest figures for in vitro hatching of Oncospheres were recorded when eggs isolated either from segments fixed in 70% ethanol or from viable segments were used.
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Cross protection against Taenia taeniaeformis in rats vaccinated with non-viable Oncospheres of Asian Taenia or T. saginata
Journal of helminthology, 1994Co-Authors: Akira Ito, P.c. Fan, W.c. Chung, M. SuzukiAbstract:It was determined to examine whether rats injected with non-viable Oncospheres of Asian Taenia or Taenia saginata became resistant to challenge infection with eggs of Taenia taeniaeformis, since (a) metacestodes of Asian Taenia and T. taeniaeformis develop in the liver of pigs and rats, respectively, and (b) Asian Taenia and T. saginata have human origins. Rats injected intravenously or subcutaneously with complete Freund's adjuvant with non-viable Oncospheres of Asian Taenia showed statistically significant resistance to challenge infection with eggs of T. taeniaeformis, whereas those injected with non-viable Oncospheres of T. saginata did not show any resistance.
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Vaccination with hatched but non-activated, non-viable Oncospheres of Taenia taeniaeformis in rats
Journal of helminthology, 1993Co-Authors: Akira Ito, A. HashimotoAbstract:The usefulness of hatched but non-activated Oncospheres as a candidate vaccine was evaluated using a Taenia taeniaeformis/rat system, since preparation of these Oncospheres in vitro is known to be very simple. The findings were: (1) rats vaccinated with non-viable Oncospheres became completely resistant to challenge infection; (2) intra-venous injection was the most effective to induce complete resistance; (3) a single oncosphere was sufficient to induce complete resistance in infected rats, whereas approximately 50 and 500 non-viable Oncospheres were required to evoke strong and complete resistance, respectively, in vaccinated rats. The usefulness of non-viable Oncospheres without adjuvant is discussed.
Hector H. Garcia - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the carbohydrate components of Taenia solium oncosphere proteins and their role in the antigenicity.
Parasitology research, 2013Co-Authors: Yanina Arana, Hector H. Garcia, Manuela Verastegui, Iskra Tuero, Louis Grandjean, Robert H. GilmanAbstract:This study examines the carbohydrate composition of Taenia solium whole oncosphere antigens (WOAs), in order to improve the understanding of the antigenicity of the T. solium. Better knowledge of oncosphere antigens is crucial to accurately diagnose previous exposure to T. solium eggs and thus predict the development of neurocysticercosis. A set of seven lectins conjugates with wide carbohydrate specificity were used on parasite fixations and somatic extracts. Lectin fluorescence revealed that d-mannose, d-glucose, d-galactose and N-acetyl-d-galactosamine residues were the most abundant constituents of carbohydrate chains on the surface of T. solium oncosphere. Lectin blotting showed that posttranslational modification with N-glycosylation was abundant while little evidence of O-linked carbohydrates was observed. Chemical oxidation and enzymatic deglycosylation in situ were performed to investigate the immunoreactivity of the carbohydrate moieties. Linearizing or removing the carbohydrate moieties from the protein backbones did not diminish the immunoreactivity of these antigens, suggesting that a substantial part of the host immune response against T. solium oncosphere is directed against the peptide epitopes on the parasite antigens. Finally, using carbohydrate probes, we demonstrated for the first time that the presence of several lectins on the surface of the oncosphere was specific to carbohydrates found in intestinal mucus, suggesting a possible role in initial attachment of the parasite to host cells.
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Standardization of a fluorescent-based quantitative adhesion assay to study attachment of Taenia solium oncosphere to epithelial cells in vitro.
Journal of immunological methods, 2011Co-Authors: Nancy Chile, Hector H. Garcia, Armando E. Gonzalez, Robert H. Gilman, Yanina Arana, Sandra Palma, Julio Evangelista, Charles R. Sterling, Manuela VerasteguiAbstract:To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-Oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium Oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-Oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-Oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-Oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of Oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-Oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment.
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The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs
Veterinary Immunology and Immunopathology, 2011Co-Authors: Mirko Zimic, Robert H. Gilman, Manuela Verastegui, Nancy Chile, Monica Pajuelo, Andres H. Gutierrez, Luis D. Rueda, Myra Flores, Armando Gonzalez, Hector H. GarciaAbstract:Abstract Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium Oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata Oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by Oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium Oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection.
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proteomic study of activated taenia solium Oncospheres
Molecular and Biochemical Parasitology, 2010Co-Authors: Saul J Santivanez, Armando E. Gonzalez, Robert H. Gilman, Manuela Verastegui, Ana Hernandezgonzalez, Nancy Chile, Ana Oleaga, Yanina Arana, Sandra Palma, Hector H. GarciaAbstract:Taenia solium cysticerci are a major cause of human seizures and epilepsy in the world. In the gastrointestinal tract of infected individuals, taeniid eggs release the Oncospheres, which are then activated by intestinal stimuli, getting ready to penetrate the gut wall and reach distant locations where they transform in cysticerci. Information about oncospheral molecules is scarce, and elucidation of the oncosphere proteome could help understanding the host-parasite relationship during the first steps of infection. In this study, using liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis, we could identify a set of oncospheral proteins involved in adhesion, protein folding, detoxification and proteolysis, among others. In addition, we have characterized one of the identified molecules, the parasite 14-3-3, by immunoblot and immunolocalization. The identification of these oncospheral proteins represents the first step to elucidate their specific roles in the biology of the host-parasite relationship.
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Taenia solium Oncosphere Adhesion to Intestinal Epithelial and Chinese Hamster Ovary Cells In Vitro
Infection and immunity, 2007Co-Authors: Manuela Verastegui, Robert H. Gilman, Nancy Chile, Yanina Arana, Dylan Barber, Jeanette Velásquez, Marilu Farfán, Jon C. Kosek, Margaret Kosek, Hector H. GarciaAbstract:The specific mechanisms underlying Taenia solium oncosphere adherence and penetration in the host have not been studied previously. We developed an in vitro adhesion model assay to evaluate the mechanisms of T. solium oncosphere adherence to the host cells. The following substrates were used: porcine intestinal mucosal scrapings (PIMS), porcine small intestinal mucosal explants (PSIME), Chinese hamster ovary cells (CHO cells), epithelial cells from ileocecal colorectal adenocarcinoma (HCT-8 cells), and epithelial cells from colorectal adenocarcinoma (Caco-2 cells). CHO cells were used to compare oncosphere adherence to fixed and viable cells, to determine the optimum time of oncosphere incubation, to determine the role of sera and monolayer cell maturation, and to determine the effect of temperature on oncosphere adherence. Light microscopy, scanning microscopy, and transmission microscopy were used to observe morphological characteristics of adhered Oncospheres. This study showed in vitro adherence of activated T. solium Oncospheres to PIMS, PSIME, monolayer CHO cells, Caco-2 cells, and HCT-8 cells. The reproducibility of T. solium oncosphere adherence was most easily measured with CHO cells. Adherence was enhanced by serum-binding medium with >5% fetal bovine serum, which resulted in a significantly greater number of Oncospheres adhering than the number adhering when serum at a concentration less than 2.5% was used (P < 0.05). Oncosphere adherence decreased with incubation of cells at 4°C compared with the adherence at 37°C. Our studies also demonstrated that T. solium Oncospheres attach to cells with elongated microvillus processes and that the Oncospheres expel external secretory vesicles that have the same oncosphere processes.
