ABSTRACT
Objective To investigate the effect of Toxoplasma gondii Chinese 1 genotype infections on host brain iron metabolism and brain damages. Methods Twenty C57BL/6 mice, each weighing 15 to 17 g, were randomly divided into the control and infection groups, of 10 mice in each group. Each mouse in the infection group was injected intraperitoneally with 4 000 tachyzoites of the TgCtwh3 isolate with Chinese 1 genotype, while each mouse in the control group was injected with an equal amount of sterile phosphate-buffered saline (PBS). All mice were sacrificed 6 day post-infection and brain tissues were sampled. The iron levels were measured in mouse brain specimens using inductively coupled plasma mass spectrometry (ICP-MS). The differentially expressed genes were determined between the experimental and control groups using RNA chips and Gene Ontology (GO) term enrichment analysis of differentially expressed genes was performed. The mRNA expression of Toxoplasma gondii surface antigen 1 (TgSAG1) gene and some Zrt- and Irt-like protein (ZIP) family member coding genes was detected by quantitative real-time PCR (qPCR) assay. The ultrastructure of the hippocampus dentate gyrus in mouse brain specimens was observed using optical and electronic microscopy. The glutathione peroxidase 4 (GPx4) expression was determined using Western blotting, and malondialdehyde (MDA) level was measured using thiobarbituric acid (TBA) test. In addition, the optical density (OD) of vascular endothelial growth factor (VEGF) protein was measured using immunohistochemistry. Results Optical microscopy showed cell necrosis in the hippocampus dentate gyrus of mouse brain specimens in the infection group, and electronic microscopy cytoplasmic vacuolization, nuclear atrophy and necrosis, disruption of cristae mitochondriales and increased autophagosome levels in the mouse brain hippocampus specimens in the infection group. The iron level was significantly greater in mouse brain specimens in the infection group than in the control group [(32.92 ± 0.90) μg/g vs. (37.72 ± 1.10) μg/g; t = 3.397, P < 0.01]. RNA chips revealed 721 up-regulated genes and 276 down-regulated genes in mouse brain specimens between the infection and control groups, and the differentially expressed genes were significantly enriched in metal ion binding ability (molecular function). Elevated expression of metal element transporter ZIP2 mRNA (t = 8.659, P < 0.05), reduced GPx4 expression [(1.046 ± 0.025) vs. (0.720 ± 0.101); t = 3.129, P < 0.01], increased MDA level [(4.37 ± 0.33) nmol/mgprot vs. (5.93 ± 0.54) nmol/mgprot; t = 2.451, P < 0.05], and up-regulated mean OD of VEGF protein [(0.348 3 ± 0.017 8) vs. (0.490 6 ± 0.010 5); t = 6.641, P < 0.01] were found in mouse brain specimens in the infection group than in the control group. Conclusions Chinese 1 genotype T. gondii infection results in iron accumulation in brain tissues, reduced antioxidant ability and elevated levels of oxidative stress in mice, suggesting that T. gondii infection may cause brain damages through affecting iron metabolism in host brain tissues.
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Objective To investigate microRNAs differential expression and polarization of human macrophages in Toxoplasma gondii infection. Methods The microRNAs differential expression of human macrophages in T. gondii infection was analyzed by microarray, and further validated by qRT-PCR. pEGFP-miR-155 was transfected into THP-1 cells by Lipofectamine M2000 and the transfection ratio was detected by flow cytometry. Flow cytometry was used to detect CD86 molecular on the macrophages. qRT-PCR was used to detect iNOS and IL12 mRNA expression. NO and IL12 expression were then evaluated by using ELISA. Results The miR-155 up-regulated more than 4-fold in T. gondii infected macrophages and enhanced as well as post-infection prolong. pEGFP-miR-155 transfection ratio was 82.6%.compared to cells cultured with T. gondii, pEGFP-miR-155 and miR-155-inhibitor, T. gondii and pEGFP-miR-155 inducement enhanced expression of CD86. Additionally, iNOS and IL12 mRNA were enhanced by qRT-PCR (P<0.05). NO and IL12 expression were increased by ELISA. Conclusion T. gondii infection up-regulates the host miR-155 expression to modulate macrophages polarization to M1.
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Objective To investigate microRNAs differential expression and polarization of human macrophages in Toxoplasma gondii infection. Methods The microRNAs differential expression of human macrophages in T. gondii infection was analyzed by microarray, and further validated by qRT-PCR. pEGFP-miR-155 was transfected into THP-1 cells by Lipofectamine M2000 and the transfection ratio was detected by flow cytometry. Flow cytometry was used to detect CD86 molecular on the macrophages. qRT-PCR was used to detect iNOS and IL12 mRNA expression. NO and IL12 expression were then evaluated by using ELISA. Results The miR-155 up-regulated more than 4-fold in T. gondii infected macrophages and enhanced as well as post-infection prolong. pEGFP-miR-155 transfection ratio was 82.6%.compared to cells cultured with T. gondii, pEGFP-miR-155 and miR-155-inhibitor, T. gondii and pEGFP-miR-155 inducement enhanced expression of CD86. Additionally, iNOS and IL12 mRNA were enhanced by qRT-PCR (P<0.05). NO and IL12 expression were increased by ELISA. Conclusion T. gondii infection up-regulates the host miR-155 expression to modulate macrophages polarization to M1.
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Objective To explore the clinical application and significance of polymerase chain reactio n(PCR) in checking human cytomegalovirus (HCMV)in cerebral palsy children. Methods Collecting urine and serum of 56 cerebral palsy (CP) children,using PCR to detec t CMV DNA from urine,isolate CMV from urine,and indirect enzyme-linked immuno so rbent assay(ELISA) detecting CMV IgM、IgG of serum.Results In 56 cases,53.6%cases were CMV DNA positive,there were 9 cases CMV isolation,o bserving CMV characteristic cytopathic effect (CPE) and the positive value of se rum CMV IgM、IgG was 12.5%,37.5% respectively.The positive value in isolation o f the virus and CMV IgM was 100%,10% corresponding with that of CMV DNA.Comp ared the 2 former with the latter,it was significant(P0.05).Conclusions Using PCR can detect CMV DNA from CP children with CMV infection quickly.It can apply in detecting CMV in CP and provide credible evidence for intervention as f ar as early in children with CP. J Appl Clin Pediatr,2005,20(2):157-159