Toxoplasma Gondii ME49 Strain Research Articles

The effect of Nigella sativa oil- and wheat germ oil-loaded metal organic frameworks on chronic murine toxoplasmosis

Treatment of chronic toxoplasmosis is challenging as the available drugs are effective only in the acute stage. Therefore, the current study aimed to investigate Nigella sativa oil (NSO) and wheat germ oil (WGO) loaded on copper-benzene tricarboxylic acid metal organic framework (Cu-BTC MOF) for treating chronic toxoplasmosis in a murine model. Eighty mice were divided into 8 groups (G); uninfected untreated negative control (GI), infected untreated positive control (GII), infected and treated with: Spiramycin (GIII), Spiramycin@Cu-BTC (GIV), Cu-BTC (GV), WGO@Cu-BTC (GVI), NSO@Cu-BTC (GVII) and combined WGO+NSO@Cu-BTC (GVIII). The infected groups were orally inoculated with 10 Toxoplasma gondii Me49 strain cysts/mouse. All drugs were orally administered for 14 consecutive days starting 8 weeks post-infection (wpi). The therapeutic efficacy was evaluated by parasitological (survival rate of mice and brain cyst burden) and histopathological (brain, liver, kidney, eye) parameters. At the end of 2-weeks therapy, the highest therapeutic outcome was achieved with GVII and GVIII exhibiting 100% survival, 64.3% and 51.4% reduction of brain cysts, and an apparent amendment of pathological insults. In the next place was GVI with 90% survival, 49.5% reduction of cysts and marked amelioration of pathological lesions. Meanwhile, GIII and GIV showed 80% survival, 42.4% and 41.8% reduction of cysts as well as minimal to moderate alleviation of tissue damage. The lowest effect was obtained with GV resulting in 70% survival and 24.4% reduction of cysts. The current results support the assertion that the new metal-based nanocomposites can be promising remedies of chronic toxoplasmosis particularly if conjugated with natural herbal extracts as NSO and WGO.

A Gene-Based Positive Selection Detection Approach to Identify Vaccine Candidates Using Toxoplasma gondii as a Test Case Protozoan Pathogen.

Over the last two decades, various in silico approaches have been developed and refined that attempt to identify protein and/or peptide vaccines candidates from informative signals encoded in protein sequences of a target pathogen. As to date, no signal has been identified that clearly indicates a protein will effectively contribute to a protective immune response in a host. The premise for this study is that proteins under positive selection from the immune system are more likely suitable vaccine candidates than proteins exposed to other selection pressures. Furthermore, our expectation is that protein sequence regions encoding major histocompatibility complexes (MHC) binding peptides will contain consecutive positive selection sites. Using freely available data and bioinformatic tools, we present a high-throughput approach through a pipeline that predicts positive selection sites, protein subcellular locations, and sequence locations of medium to high T-Cell MHC class I binding peptides. Positive selection sites are estimated from a sequence alignment by comparing rates of synonymous (dS) and non-synonymous (dN) substitutions among protein coding sequences of orthologous genes in a phylogeny. The main pipeline output is a list of protein vaccine candidates predicted to be naturally exposed to the immune system and containing sites under positive selection. Candidates are ranked with respect to the number of consecutive sites located on protein sequence regions encoding MHCI-binding peptides. Results are constrained by the reliability of prediction programs and quality of input data. Protein sequences from Toxoplasma gondii ME49 strain (TGME49) were used as a case study. Surface antigen (SAG), dense granules (GRA), microneme (MIC), and rhoptry (ROP) proteins are considered worthy T. gondii candidates. Given 8263 TGME49 protein sequences processed anonymously, the top 10 predicted candidates were all worthy candidates. In particular, the top ten included ROP5 and ROP18, which are T. gondii virulence determinants. The chance of randomly selecting a ROP protein was 0.2% given 8263 sequences. We conclude that the approach described is a valuable addition to other in silico approaches to identify vaccines candidates worthy of laboratory validation and could be adapted for other apicomplexan parasite species (with appropriate data).

The Toxoplasma gondii ME-49 strain upregulates levels of A20 that inhibit NF-\u03baB activation and promotes apoptosis in human leukaemia T-cell lines

BackgroundAcute T-lymphocyte leukaemia is a form of haematological malignancy with abnormal activation of NF-κB pathway, which results in high expression of A20 and ABIN1, which constitute a negative feedback mechanism for the regulation of NF-κB activation. Clinical studies have found that acute T-lymphocyte leukaemia patients are susceptible to Toxoplasma gondii infection; however, the effect of T. gondii on the proliferation and apoptosis of human leukaemia T-cells remains unclear. Here, we used the T. gondii ME-49 strain to infect human leukaemia T-cell lines Jurkat and Molt-4, to explore the effect of T. gondii on proliferation and apoptosis, which is mediated by NF-κB in human leukaemia T-cells.MethodsThe Tunel assay was used to detect cell apoptosis. Cell Counting Kit-8 was used to detect cell proliferation viability. The apoptosis level and the expression level of NF-κB related proteins in human leukaemia T-cells were detected by flow cytometry and Western blotting.ResultsWestern blotting analyses revealed that the T. gondii ME-49 strain increased the expression of A20 and decreased both ABIN1 expression and NF-κB p65 phosphorylation. By constructing a lentiviral-mediated shRNA to knockdown the A20 gene in Jurkat T-cells and Molt-4 T-cells, the apoptosis levels of the two cell lines decreased after T. gondii ME-49 infection, and levels of NF-κB p65 phosphorylation and ABIN1 were higher than in the non-konckdown group. After knockingdown ABIN1 gene expression by constructing the lentiviral-mediated shRNA and transfecting the recombinant expression plasmid containing the ABIN1 gene into two cell lines, apoptosis levels and cleaved caspase-8 expression increased or decreased in response to T. gondii ME-49 infection, respectively.ConclusionsOur data suggest that ABIN1 protects human leukaemia T-cells by allowing them to resist the apoptosis induced by T. gondii ME-49 and that the T. gondii ME-49 strain induces the apoptosis of human leukaemia T-cells via A20-mediated downregulation of ABIN1 expression.

Prostaglandin E2 inhibits the proinflammatory phenotype induced by TGF-β on monocyte-derived dendritic cells

Prostaglandin E2 inhibits the proinflammatory phenotype induced by TGF-β on monocyte-derived dendritic cells

Experimental infection with the Toxoplasma gondii ME-49 strain in the Brazilian BR-1 mini pig is a suitable animal model for human toxoplasmosis.

Toxoplasma gondii causes toxoplasmosis, a worldwide disease. Experimentation with pigs is necessary for the development of new therapeutic approaches to human diseases. BR-1 mini pigs were intramuscularly infected with T. gondii with tachyzoites (RH strain) or orally infected with cysts (ME-49 strain). Haematology and serum biochemistry were analysed and buffy coat cells were inoculated in mice to determine tachyzoite circulation. No alterations were observed in erythrocyte and platelet values; however, band neutrophils increased seven days after infection with ME-49. Serology of the mice inoculated with pig blood leucocytes revealed circulating ME-49 or RH strain tachyzoites in the pigs' peripheral blood at two and seven or nine days post-infection. The tachyzoites were also directly observed in blood smears from the infected pigs outside and inside leucocytes for longer periods. Alanine-aminotransferase was high at days 21 and 32 in the RH infected pigs. After 90 days, the pigs were euthanised and their tissue samples were processed and inoculated into mice. The mice serology revealed the presence of parasites in the hearts, ileums and mesenteric lymph nodes of the pigs. Additionally, cysts in the mice were only observed after pig heart tissue inoculation. The infected pigs presented similar human outcomes with relatively low pathogenicity and the BR-1 mini pig model infected with ME-49 is suitable to monitor experimental toxoplasmosis.

AVALIAÇÃO DO EFEITO DE MEDICAMENTOS IMUNOSSUPRESSORES NA REATIVAÇÃO DA TOXOPLASMOSE CRÔNICA MURINA

This study evaluated the potential for reactivation of chronic toxoplasmosis in an immunocompromised murine model. To this end, BALB/c mice were orally infected with 20 cysts of Toxoplasma gondii ME-49 strain. After 60 days, the treatment with orally administered immunosuppressive drugs, such as azathioprine and dexamethasone, and cortisone acetate (administered by subcutaneous route) alone or in combination, started. The treatment was maintained for 28 days and afterwards the animals were euthanized and had their blood and organs removed for serological, histopathological and clinical examinations. When the drugs were associated to cortisone acetate, the clinical manifestations presented by the mice suggested reactivation due to epidermal lesions in 62.5% of the mice when compared to the control group. The results from this study suggest that with the murine model used, the chronic toxoplasmosis reactivation is greatly influenced by the immunosuppressive treatment proposed. In these cases, the clinic manifestations indicative of chronic toxoplasmosis reactivation may be present even before its detection by serological and histopathological methods.

Experimental study of scoring model for liver cirrhosis diseases

Objective To investigate the effect of Toxoplasma gondii ME49 strain on the apoptosis of mouse placental trophoblastic cells in vitro.Methods Mouse placental trophoblastic cells (concentration of 5 × 106 /ml) were cultured in the different cell culture vessels.The cells were treated for 8 h with different concentrations of Toxoplasma gondii ME49 strain (the concentration of tachyzoites was 2 × 106 /ml,4 × 106 /ml,and 8 × 106 /ml,respectively).FCM was used to examine the apoptosis rates of the placental trophoblastic cells stained with the fluorescent dye of Annexin Ⅴ-FITC/PI;fluorescence microscopy was used to observe the changes of cellular morphology,and Western blotting analysis was used to detect the protein levels of Bax and Bcl-2.Results The trophoblastic cells infected with Toxoplasma gondii ME49 strain showed a higher apoptosis compared to the normal cells(P 0.05),and the apoptosis rates increased with the concentration of tachyzoites in the infected groups.The highest apoptosis rate was 28.37% which was found 8 h after culture with 8 ×106 /ml tachyzoites.Fluorescence microscope observed that the apoptosis of trophoblastic cells increased with the increase of Toxoplasma gondii.Western blotting analysis showed that the relative expression levels of Bax and Bcl-2 were 1.24 ±0.05,1.37 ± 0.03,1.78 ± 0.04,and 1.15 ± 0.03,1.09 ± 0.05,0.97 ± 0.01,respectively,which were significantly different from those of the control group (1.17 ±0.06,1.23 ± 0.02,P 0.05).Conclusion Infection with Toxoplasma gondii ME49 strain can promote the apoptosis of mouse placental trophoblastic cells in vitro through up-regulating Bax expression and down-regulating Bcl-2 expression.

Toxoplasma gondii: The role of IFN-gamma, TNFRp55 and iNOS in inflammatory changes during infection

In order to examine the role of IFN-γ, TNFRp55 and iNOS in inflammatory reaction during toxoplasmosis, IFN-γ −/−, TNFRp55 −/− and iNOS −/− mice were experimentally infected with Toxoplasma gondii ME-49 strain. The organs of the mice were evaluated for histology and immunohistochemistry in detection of tissue parasitism and iNOS positive cells. IFN-γ −/− mice presented mild inflammation in peripheral organs associated with a high parasitism and mortality in the acute phase of infection. In contrast, the peripheral organs of WT, TNFRp55 −/− and iNOS −/− mice, presented a significant inflammatory reaction and low tissue parasitism in the same period of infection. The inflammatory lesions and tissue parasitism were increased and more severe in the Central Nervous System (CNS) of TNFRp55 −/− and iNOS −/− with a progression of infection, when compared to WT mice. In these knockout animals, the inflammatory changes were associated with low levels or no expression of iNOS in TNFRp55 −/− and iNOS −/− mice, respectively.

Toxoplasma gondii: localization of purine nucleoside phosphorylase activity in vitro and in vivo by electron microscopy

Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) activity was revealed by enzyme histochemistry in Toxoplasma gondii ME49 strain isolated from murine cerebral cysts and from in vitro cultivation. The activity of the enzyme was revealed by an insoluble electron-opaque precipitate of lead phosphate at the site of the reaction. In bradyzoites and tachyzoites of T. gondii, the enzyme activity could be observed only in the cytoplasm. In bradyzoites, one or two foci of important PNP activity were detected near the nucleus. In tachyzoites, an important PNP activity underlined the plasma membrane. For both bradyzoites and tachyzoites, localization neither in the nucleus nor in cytoplasmic organelles could be detected.

Ocular toxoplasmosis in mice: comparison of two routes of infection

This paper aims to test the influence of route of infection (intravitreal and instillation) on the course of ocular toxoplasmosis in mice, using the Toxoplasma gondii Me-49 strain. All mice inoculated intravitreally or by instillation presented the same pattern of infection. Using either route, parasites were observed in the retinal vessel with the formation of a glial reaction in the inner plexiforme layer and discontinuity of the pigmented epithelium of the retina 7 days after infection. However, when the intravitreal route was used a more intense inflammatory infiltrate was observed in the retina. The results suggest that inoculation route remarkably influences the inflammatory pattern in ocular toxoplasmosis and that the instillation route should be preferentially used in experimental infections in the murine ocular model of infection by T. gondii, specially with small animals where there is extensive needle damage, which is not observed in the instillation route.