[Real-time fluorescence quantitative polymerase chain reaction detection for Toxoplasma gondii B1 gene in mice urine].

A pair of specific primers and a TaqMan probe were designed based on the sequence of Toxoplasma gondii B1 gene from GenBank database. Total DNA of T. gondii was extracted from fresh mice urine. DNA fragment of B1 gene was amplified by PCR. The PCR product was cloned into pMD18-T vector. Following identification, the positive recombinant plasmid was used as reference template to generate standard curve and melt curve. Sensitivity, reproducibility, linear range and stability of reference plasmids were determined. The sensitivity of this method was 10(4) copies/ml. The coefficient of variation (cv) of intra-assay and inter-assay were 2.42% and 4.18%, respectively. Linear range was (10(3)-10(7)) copies/ml. The specificity was 100%. The reference materials were stable. Real-time FQ-PCR of T. gondii DNA in mice urine has been constructed, which is a convenient, sensitive and reliable method for quantifying T. gondii DNA in mice urine.

[Research progress on Th17 cells].

Th17(T helper 17 cell), a newly discovered subset of T cells is associated with IL-23 and characterized by production of IL-17, the functions of which are distinct from those of Th1, Th2 and Treg subsets. The development of Th17 cells can be promoted by TGF-beta1, IL-6, and IL-23; but inhibited by IFN-gamma, IL-4 and Socs3. It is clear that Th17 cells have protective effects on body by facilitating the pro-inflammatory responses. On the other hand, the role of Th17 cells in the pathophysiology of autoimmune diseases has been described.

[Effect of rmu-IFN-gamma on the splenic T cell subsets and NK cells of pregnant mice infected with Toxoplasma gondii].

OBJECTIVE: To explore the effect of rmu-IFN-gamma on the change of T cell subsets and natural killer cells of pregnant mice infected with T. gondii. METHODS: Early pregnant mice infected with T. gondii were administered with different doses (1 U/g or 10 U/g) of rmu-IFN-gamma for three days before euthanasia. The numbers of splenic CD4+ and CD8+ T cells and natural killer cells were detected by flow cytometry. RESULTS: Compared with the infected mice that were not treated, the level of splenic CD4+ T cells in mice administered with two doses of rmu-IFN-gamma increased on the day 10, 12, 14 of gestation, while the level of CD8+ T cells decreased on the day 10, 14 of gestation. The ratio of CD4+/CD8+ T cells increased significantly on the day 10, 12, 14 of gestation. Survival days of the two administered groups were longer than those of the infected group. CONCLUSION: A proper dose of rmu-IFN-gamma can reverse the decline of the ratio of T cell subsets, improve the proliferation of NK cells, and so increase the level of peripheral cellular immunity of pregnant mice.

In vitro study of the effects of Angiostrongylus cantonensis larvae extracts on apoptosis and dysfunction in the blood-brain barrier (BBB).

It has been hypothesized that blood-brain barrier (BBB) dysfunction in Angiostrongylus cantonensis infection might be due to the apoptosis of the hosts' BBB cells. Here, we evaluated this hypothesis through several methods, all based on an in vitro mouse BBB model consisting of primary culture brain microvascular endothelial cells (BMECs) and brain astrocytic cells (BACs). In the present study, a four-hour percolation and HRP permeability experiment showed that A. cantonensis larvae extracts can increase the permeability of the BBB. Apoptosis among BMECs and BACs after exposure to larvae extracts was monitored by TUNEL and annexin-V-FITC/PI double staining. A. cantonensis larvae extracts were found to induce apoptosis in both BMECs and BACs. For this reason, we concluded that the induction of apoptosis might participate in the BBB dysfunction observed during angiostrongyliasis. Improved fundamental understanding of how A. cantonensis induces apoptosis may lead to new approaches to the treatment or prevention of this parasitic disease.

Induction of protective Th1 immune responses in mice by vaccination with recombinant Toxoplasma gondii nucleoside triphosphate hydrolase-II.

The Toxoplasma gondii nucleoside triphosphate hydrolase (TgNTPase) has apyrase activity, degrading ATP to the di- and mono-phosphate forms and may be used by the parasite to salvage purines from the host cell for survival and replication. To study the immune-protective value of TgNTPase-II, BALB/c mice were immunized with a recombinant form of the antigen rTgNTPase-II combined with alum. All immunized mice produced specific anti-rTgNTPase-II immunoglobulins, with high IgG antibody titers and a mixed IgG1/IgG2a response, with predominance of IgG2a production. The cellular immune response was associated with the production of IFN-γ and IL-2 cytokines and the increase of the percentage of CD8+ T cells. Vaccinated mice displayed significant protection against acute infection with the virulent RH strain (P<0.05 in survival rate) and also chronic infection with PRU cyst (62.9% and 57.6% reduction in brain parasite load for rTgNTPase-II+alum and rTgNTPase-II alone vaccinated groups) compared to the non-vaccinated control group. In conclusion, rTgNTPase-II elicits a strong specific Th1 immune response providing partial protection against both T. gondii acute and chronic infection.