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Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro
Guimar?es, Erick Vaz;Carvalho, Laís de;Barbosa, Helene Santos;
Memórias do Instituto Oswaldo Cruz , 2009, DOI: 10.1590/S0074-02762009000200007
Abstract: infection by the protozoan parasite toxoplasma gondii is widely prevalent in humans and animals. to prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. in this context, the use of skeletal muscle cells (skmcs) as a cellular model opens up new approaches to investigate t. gondii-host cell interactions. immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of t. gondii in in vitro cultures of skmcs occurs after 96 h of infection. ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. the present study demonstrates the potential use of primary cultures of skmcs to evaluate different molecular aspects of t. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.
In vitro Anti-Toxoplasma gondii Activity of Root Extract/Fractions of Eurycoma longifolia Jack
Nowroji Kavitha, Rahmah Noordin, Kit-Lam Chan, Sasidharan Sreenivasan
BMC Complementary and Alternative Medicine , 2012, DOI: 10.1186/1472-6882-12-91
Abstract: In vitro toxoplasmacidal evaluation was performed using Vero cells as host for T. gondii. Light microscopy technique was used to study in situ antiparasitic activity.Significant anti-T. gondii activity was observed with clindamycin (EC50?=?0.016 μg/ml), follow by TAF 355 (EC50?=?0.369 μg/ml) and TAF 401 (EC50?=?0.882 μg/ml). Light microscopy revealed that most Vero cells were infected after 3 h of exposure to T. gondii. After 36 h of exposure to the E. longifolia fraction, the host Vero cells showed no visible intracellular parasite and no remarkable morphological changes.Our study demonstrated that TAF 355 and TAF401 fractions may be the sources of new anti-T. gondii compounds.
In vitro multiplication of Toxoplasma gondii and Trypanosoma cruzi in mouse, rat, and hamster astrocytes
Troyo,Adriana; Chinchilla,Misael;
Revista de Biología Tropical , 2003,
Abstract: the infection and multiplication of toxoplasma gondii and trypanosoma cruzi were compared in primary cultures of white rat, mouse and hamster astrocytes. these cells were cultured on cover slides and infected with t. gondii tachyzoites or t. cruzi blood trypomastigotes. results show that hamster astrocytes are more susceptible to the multiplication of both parasites than rat and mouse cells. there was no statistical difference between the t. gondii infection in rat and mouse astrocytes (p<0.05), and this suggests an important role of other mechanisms or cells in the white rat natural resistance to this parasite. because the hamster astrocytes are less resistant to these parasites multiplication and not necessarily to the invasion, any difference observed could be due to an intracellular effect: hamster brain astrocytes favor survival and multiplication of these parasites
In vitro multiplication of Toxoplasma gondii and Trypanosoma cruzi in mouse, rat, and hamster astrocytes
Adriana Troyo,Misael Chinchilla
Revista de Biología Tropical , 2003,
Abstract: The infection and multiplication of Toxoplasma gondii and Trypanosoma cruzi were compared in primary cultures of white rat, mouse and hamster astrocytes. These cells were cultured on cover slides and infected with T. gondii tachyzoites or T. cruzi blood trypomastigotes. Results show that hamster astrocytes are more susceptible to the multiplication of both parasites than rat and mouse cells. There was no statistical difference between the T. gondii infection in rat and mouse astrocytes (p<0.05), and this suggests an important role of other mechanisms or cells in the white rat natural resistance to this parasite. Because the hamster astrocytes are less resistant to these parasites multiplication and not necessarily to the invasion, any difference observed could be due to an intracellular effect: hamster brain astrocytes favor survival and multiplication of these parasites Se comparó la infección y multiplicación de Toxoplasma gondii y Trypanosoma cruzi en cultivos celulares primarios de astrocitos, a partir de células de cerebro de rata blanca, ratón y hámster. Las células fueron cultivadas en cubreobjetos e infectadas con taquizoitos de T. gondii o tripomastigotos sanguíneos de T. cruzi. Los resultados muestran que los astrocitos de hámster son más susceptibles que los de rata y ratón a la multiplicación de ambos parásitos. No se encontró diferencia estadísticamente significativa entre la infección por T. gondii en los astrocitos de ratón y rata (p<0.05), lo que sugiere la importancia de otros mecanismos o células en la resistencia natural de la rata blanca a esta parásito. Al ser los astrositos de hámster menos resistentes a la multiplicación de estos parásitos y no necesariamente a la invasión, cualquier diferencia observada podría deberse a un fenómeno intracelular: los astrocitos de hámster favorecen la permanencia y multiplicación de estos parásitos
Novel Triazine JPC-2067-B Inhibits Toxoplasma gondii In Vitro and In Vivo  [PDF]
Ernest J. Mui,Guy A. Schiehser,Wilbur K. Milhous,Honghue Hsu,Craig W. Roberts,Michael Kirisits,Stephen Muench,David Rice,J. P. Dubey,Joseph W. Fowble,Pradipsinh K. Rathod,Sherry F. Queener,Susan R. Liu,David P. Jacobus,Rima McLeod
PLOS Neglected Tropical Diseases , 2008, DOI: 10.1371/journal.pntd.0000190
Abstract: Background and Methodology Toxoplasma gondii causes substantial morbidity, mortality, and costs for healthcare in the developed and developing world. Current medicines are not well tolerated and cause hypersensitivity reactions. The dihydrotriazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3′(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), which inhibits dihydrofolate reductase (DHFR), is highly effective against Plasmodium falciparum, Plasmodium vivax, and apicomplexans related to T. gondii. JPC-2067-B is the primary metabolite of the orally active biguanide JPC-2056 1-(3′-(2-chloro-4-trifluoromethoxyphenyl?oxy)propyloxy)- 5-isopropylbiguanide, which is being advanced to clinical trials for malaria. Efficacy of the prodrug JPC-2056 and the active metabolite JPC-2067-B against T. gondii and T. gondii DHFR as well as toxicity toward mammalian cells were tested. Principal Findings and Conclusions Herein, we found that JPC-2067-B is highly effective against T. gondii. We demonstrate that JPC-2067-B inhibits T. gondii growth in culture (IC50 20 nM), inhibits the purified enzyme (IC50 6.5 nM), is more efficacious than pyrimethamine, and is cidal in vitro. JPC-2067-B administered parenterally and the orally administered pro-drug (JPC-2056) are also effective against T. gondii tachyzoites in vivo. A molecular model of T. gondii DHFR-TS complexed with JPC-2067-B was developed. We found that the three main parasite clonal types and isolates from South and Central America, the United States, Canada, China, and Sri Lanka have the same amino acid sequences preserving key binding sites for the triazine. Significance JPC-2056/JPC-2067-B have potential to be more effective and possibly less toxic treatments for toxoplasmosis than currently available medicines.
Auranofin Is Highly Efficacious against Toxoplasma gondii In Vitro and in an In Vivo Experimental Model of Acute Toxoplasmosis  [PDF]
Rosa M. Andrade ,Juan D. Chaparro,Edmund Capparelli,Sharon L. Reed
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0002973
Abstract: Background The mainstay of toxoplasmosis treatment targets the folate biosynthetic pathways and has not changed for the last 50 years. The activity of these chemotherapeutic agents is restricted to one lifecycle stage of Toxoplasma gondii, they have significant toxicity, and the impending threat of emerging resistance to these agents makes the discovery of new therapies a priority. We now demonstrate that auranofin, an orally administered gold containing compound that was FDA approved for treatment of rheumatoid arthritis, has activity against Toxoplasma gondii in vitro (IC50 = 0.28 μM) and in vivo (1 mg/kg). Methods/Principal Findings Replication within human foreskin fibroblasts of RH tachyzoites was inhibited by auranofin. At 0.4 μM, auranofin inhibited replication, as measured by percent infected fibroblasts at 24 hrs, (10.94% vs. 24.66% of controls; p = 0.0003) with no effect on parasite invasion (16.95% vs. 12.91% p = 0.4331). After 18 hrs, 62% of extracellular parasites treated with auranofin were non-viable compared to control using an ATP viability assay (p = 0.0003). In vivo, a previously standardized chicken embryo model of acute toxoplasmosis was used. Fourteen day old chicken embryos were injected through the chorioallantoic vein with 1×104 tachyzoites of the virulent RH strain. The treatment group received one dose of auranofin at the time of inoculation (1 mg/kg estimated body weight). On day 5, auranofin-treated chicken embryos were 100% protected against death (p = 0.0002) and had a significantly reduced parasite load as determined by histopathology, immunohistochemistry and by the number of parasites quantified by real-time PCR. Conclusions These results reveal in vitro and in vivo activity of auranofin against T. gondii, suggesting that it may be an effective alternative treatment for toxoplasmosis.
In Vitro Infection of Human Nervous Cells by Two Strains of Toxoplasma gondii: A Kinetic Analysis of Immune Mediators and Parasite Multiplication  [PDF]
Nour Mammari, Philippe Vignoles, Mohamad Adnan Halabi, Marie Laure Darde, Bertrand Courtioux
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0098491
Abstract: The severity of toxoplasmic infection depends mainly on the immune status of the host, but also on the Toxoplasma gondii strains, which differ by their virulence profile. The relationship between the human host and T. gondii has not yet been elucidated because few studies have been conducted on human models. The immune mechanisms involved in the persistence of T. gondii in the brains of immunocompetent subjects and during the reactivation of latent infections are still unclear. In this study, we analyzed the kinetics of immune mediators in human nervous cells in vitro, infected with two strains of T. gondii. Human neuroblast cell line (SH SY5Y), microglial (CMH5) and endothelial cells (Hbmec) were infected separately by RH (type I) or PRU (type II) strains for 8 h, 14 h, 24 h and 48 h (ratio 1 cell: 2 tachyzoites). Pro-inflammatory protein expression was different between the two strains and among different human nervous cells. The cytokines IL-6, IL-8 and the chemokines MCP-1 and GROα, and SERPIN E1 were significantly increased in CMH5 and SH SY5Y at 24 h pi. At this point of infection, the parasite burden declined in microglial cells and neurons, but remained high in endothelial cells. This differential effect on the early parasite multiplication may be correlated with a higher production of immune mediators by neurons and microglial cells compared to endothelial cells. Regarding strain differences, PRU strain, but not RH strain, stimulates all cells to produce pro-inflammatory growth factors, G-CSF and GM-CSF. These proteins could increase the inflammatory effect of this type II strain. These results suggest that the different protein expression profiles depend on the parasitic strain and on the human nervous cell type, and that this could be at the origin of diverse brain lesions caused by T. gondii.
Ultrastructural study of the TG180 murine sarcoma cell invasion by Toxoplasma gondii: comparison between in vivo and in vitro cell cultures
Barbosa, Hugo Marcelo Ribeiro;Silva, Marcos;Ferro, Eloisa Amália Vieira;Mineo, José Roberto;
Memórias do Instituto Oswaldo Cruz , 2000, DOI: 10.1590/S0074-02762000000200023
Abstract: infection of non-adherent tg180 murine sarcoma cells with toxoplasma gondii was compared, at the ultrastructural level, in both in vivo and in vitro conditions. suspensions of 3.0 x 106 tg180 cells infected in vitro with 1.0 x 106 parasites of the rh strain were harvested between the first and 6th day post-infection and processed for transmission electron microscopy. in vivo infection was made by intraperitoneal inoculation in mice of 1.0 x 106 tg180 cells, that were co-inoculated with a parasite suspension at the same cell concentration. cells were harvested 10, 20, 30 min and 24, 48 h post-inoculation and processed for transmission electron microscopy at the same conditions of the in vitro culture. it was observed tg180 murine sarcoma cells with intense and equivalent intracellular parasitism in both conditions. host cells with parasitophorous vacuoles containing up to 16 parasites, as well as parasites undergoing mitoses or presenting a bradyzoite-like morphology, were frequently seen in both culture methods.
Ultrastructural study of the TG180 murine sarcoma cell invasion by Toxoplasma gondii: comparison between in vivo and in vitro cell cultures  [cached]
Barbosa Hugo Marcelo Ribeiro,Silva Marcos,Ferro Eloisa Amália Vieira,Mineo José Roberto
Memórias do Instituto Oswaldo Cruz , 2000,
Abstract: Infection of non-adherent TG180 murine sarcoma cells with Toxoplasma gondii was compared, at the ultrastructural level, in both in vivo and in vitro conditions. Suspensions of 3.0 x 10(6) TG180 cells infected in vitro with 1.0 x 10(6) parasites of the RH strain were harvested between the first and 6th day post-infection and processed for transmission electron microscopy. In vivo infection was made by intraperitoneal inoculation in mice of 1.0 x 10(6) TG180 cells, that were co-inoculated with a parasite suspension at the same cell concentration. Cells were harvested 10, 20, 30 min and 24, 48 h post-inoculation and processed for transmission electron microscopy at the same conditions of the in vitro culture. It was observed TG180 murine sarcoma cells with intense and equivalent intracellular parasitism in both conditions. Host cells with parasitophorous vacuoles containing up to 16 parasites, as well as parasites undergoing mitoses or presenting a bradyzoite-like morphology, were frequently seen in both culture methods.
Anti-parasitic action and elimination of intracellular Toxoplasma gondii in the presence of novel thiosemicarbazone and its 4-thiazolidinone derivatives
Carvalho, C.S.;de Melo, E.J.T.;
Brazilian Journal of Medical and Biological Research , 2010, DOI: 10.1590/S0100-879X2009005000038
Abstract: toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. in the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of toxoplasma. the compounds were tested in infected vero cell culture using concentration screens (0.1 to 20 mm). the final concentration of 1 mm was chosen for biological assay. the following results were obtained: 1) these new derivatives decreased t. gondii infection with an in vitro parasite ic50% of 0.2-0.7 mm, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) the main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) the death and elimination of t. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mm concentration for 24 h of treatment.
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