[Component analysis of excretory/secretory protein from Trichinella spiralis adult worm].

Objective: To analyze the component of adult worm excretory/secretory protein(AWESP) from Trichinella spiralis using the shotgun method, and find out the active component underlying its regulatory effect on colitis in humans. Methods: The T. spiralis AWESP was prepared, separated by SDS-PAGE, lysed with trypsin, and analyzed by shotgun LC-MS/MS. The protein components were determined with the Masco software and classified using the Gene Ontology(GO) method in cellular components, molecular functions, and biological processes. Results: The AWESPs isolated by SDS-PAGE had a Mr of 15 000-116 000. A total of 280 proteins were revealed by LC-MS/MS, of which 96 were identified by Masco software, 98 were putative, and the remaining 86 were unclear. Preliminary results showed that 4 proteins had regulatory potential for colitis, including cysteine protease inhibitor, serine protease, 53 000 excretory/secretory antigen, and glutathione-S-transferase. GO enrichment analysis showed that the identified proteins had 104 different molecular functions, involved in 363 biological processes. Conclusion: As revealed by the Masco software, T. spiralis AWESP has complex components and 96 have been identified in this study. Four of them are preliminarily shown to be associated with the anti-colitis effect of T. spiralis.

[In vitro Effect of Hypericin against Toxoplasma gondii Tachyzoites].

Objective: To investigate the killing effect of hypericin on tachyzoites of Toxoplasma gondii RH strain in vitro. Methods: Normal saline （group A） and different concentrations of hypericin (5 µg/ml, group B; 50 µg/ml, group C; 500 µg/ml, group D） were added to T. gondii tachyzoites in 24-well plate（1×10(6)/well）. The tachyzoites were harvested after 2, 4 and 6 h, and underwent the following treatment: trypan blue staining to calculate the dyeing rate, Giemsa staining to observe the morphological and structural alterations of tachyzoites, and transmission electron microscopy to observe the ultrastructure of tachyzoites. In addition, flow cytometry was performed to calculate the survival rate of YFP-carrying Toxoplasma with the same treatment. Results: The trypan blue dyeing rate at 2 h after treatment in groups B, C and D was（11.0±3.6）%, （25.0±6.3）% and（40.0±2.7）% respectively, with a significant difference of group D versus B and C （P<0.01）, and groups C and D versus group A ［（6.0±3.0）%）］. The dyeing rate at 4 h and 6 h in group D was（97.0±2.0）% and （98.0±1.7）%, respectively, both significantly higher than that of groups C ［（30.0±7.2）%, （42.7±5.5）%ï¼½, B ［（20.0±3.0）%, （34.0±6.6）%ï¼½ and A ［（10.0±1.0）%, （19.3±4.9）%］（P<0.01）. Giemsa staining showed gradual end swelling and necrosis of tachyzoites with increased treatment duration and dosage. Transmission electron microscopy showed swelling of worm body, gap between cell membrane and matrix, increase and enlargement of vacuoles inside worm body, disruption of cell membrane, and dissolving of inner structures, with increased treatment duration. Flow cytometry showed significant difference of tachyzoite survival rate at 2, 4 and 6 h after hypericin treatment with that of the control group（P<0.01）. The survival rate of group C at 2 h after hypericin treatment was（7.9±1.9）%, significantly lower than that of groups B ［（38.1±5.5）%ï¼½ and A ［（81.8±6.0）%ï¼½ （P<0.01）. No tachyzoite was found to survive in group D at 2 h and in group C at 4 h. The survival rate of group B at 4 and 6 h after hypericin treatment was（14.3±7.9）% and （1.4±1.8）%, respectively, both significantly lower than that of group A［（73.8±11.3）% and（64.1±14.4）%, respectivelyï¼½ （P<0.01）. Conclusion: Hypericin has a remarkable killing effect on T. gondii tachyzoites, and the efficacy positively correlates with the dose and treatment duration.

[Role of CD8+ T cells in the tumor growth delay induced by Toxoplasma gondii excreted-secreted antigen in B16F10 mouse melanoma model].

OBJECTIVE: To observe the role of CD8+ T cells in the tumor growth delay induced by Toxoplasma gondii excreted-secreted antigens (TgESA) in B16FI0 mouse melanoma model in the early stage. METHODS: TgESA were prepared by incubating T. gondii tachyzoites for 12 h in vitro. 15 C57BL/6 mice were randomly assigned to group A, B, and C (5 mice per group). Each mouse in group B and C was subcutaneously injected in right flank with 2 x 10(5) B16F10 cells. Mice in group C were intraperitoneally injected with TgESA (100 microl per mouse) at 7d after B16F10 cells injection. Mice of group A were only injected with PBS. On the 13th day after melanoma cell injection, the mice were sacrificed and spleen was removed. The percentage of CD8+ T cells in the spleen was analyzed by flow cytometry. CD8+ T cells were isolated from spleen cells by using immunomagnetic beads. The activity of CD8+ T cells against B16F10 melanoma cells was determined by LDH release assay at different effect-to-target cell ratios (2.5:1, 5:1, and 10:1). Other 30 C57BL/6 mice were randomly divided into group E, F, and G. Each mice were injected with 2 x 10(5) B16F10 cells. At the same time, mice in group F and G were simultaneously injected via the tail vein with CD8+ T cells isolated from mice in group B and C. Tumor growth, mortality and survival time of mice were observed and recorded during 35-d observation period. RESULTS: The percentage of CD3+CD8+T cells in the spleen cells of group C [(15.74 +/- 0.28)%] was significantly higher than that of group B [(14.18 +/- 0.27)%] and A [(13.86 +/- 0.13)%] (P < 0.05). At different effect-to-target cell ratios, the activity of CD8+ T cells against B16F10 cells in group C was significantly higher than that of group B (P < 0.05). The average time of tumor formation in group G [(14.9 +/- 1.2) d] was longer than that in group F [(11.9 +/- 0.7) d] and E [(9.4 +/- 1.2) d] (P < 0.05). The tumor size in these groups increased, but there was no obvious difference in the tumor growth rate among the three groups. The tumor size of group G was significantly smaller than the other two groups (P < 0.05). In group E, F and G, mice began to die on the 26th day, the 29th day and the 30th day after tumor inoculation, and the number of survival mice was 3, 5 and 7, respectively, at the 35th day after injection. CONCLUSIONS: TgESA may up-regulate the quantity and function of CD8+ T cell in B16F10 melanoma mouse model, which plays a role of delaying tumor growth in early stage.

[Study on immune response in BALB/c mice induced by ROP2 protein of Toxoplasma gondii with cimetidine].

OBJECTIVE: To observe the immune response induced by ROP2 protein of Toxoplasma gondii with cimetidine in mice. METHODS: Eighty BALB/c mice were randomly divided into 4 groups: PBS (group A), ROP2 protein (group B), ROP2 protein-Freund's adjuvant (group C) and ROP2 protein-cimetidine (group D). Mice were immunized with PBS, ROP2 protein, ROP2 protein and Freund's adjuvant, ROP2 protein and cimetidine [200 mg/(kg x d)] by subcutaneous injection three times at an interval of 2 weeks, respectively. Experimental mice were immunized with 100 microg ROP2 proteins, which were diluted to a final volume of 200 microl in PBS. On day 13, 27 and 41 after immunization, mice sera were collected for determination of antibody IgG and cytokine IFN-gamma by ELISA. Two weeks after the final immunization, T cells subpopulation was detected by flow cytometry and splenocyte proliferation activity was determined with CCK-8. Another 12 immunized mice in each group were intraperitoneally challenged with 5 x 10(4) tachyzoites of T. gondii and the survival time was observed. RESULTS: Two weeks after final immunization, compared with groups A [(659.750 +/- 239.962) pg/ml] and B [(872.750 +/- 197.011) pg/ml], the level of IFN-gamma significantly increased in groups C [(1 600.750 +/- 480.680) pg/ml] and D [(1494.375 +/- 451.655) pg/ml] (P < 0.01). Similarly, compared with groups A (0.636 +/- 0.108) and B (0.871 +/- 0.089), the level of IgG was also higher in groups C (1.068 +/- 0.111) and D (1.046 +/- 0.147) (P < 0.01). The proliferation of splenocytes rose in group C (0.831 +/- 0.130) after immunization, and similarly in group D (0.762 +/- 0.089), and were both significantly higher than that of groups A (0.504 +/- 0.078) and B (0.592 +/- 0.160) (P < 0.01). Moreover, ratio of CD4+/CD8+ in groups C (0.831 +/- 0.130) and D (0.762 +/- 0.089) were higher than that of groups A (0.504 +/- 0.078) and B (0.592 +/- 0.160) (P < 0.05). After challenge with violent virulence strain of tachyzoites, the median survival time of mice in groups A, B, C, and D were 96, 108, 132, and 132 h, respectively. The mean survival time of mice in groups C and D were longer than that of groups A and B (P < 0.05). There was no significant difference in 5 parameters between C and D: the level of IFN-gamma and IgG, CD4+/CD8+ ratio, splenocyte proliferation, and survival time of mice (P > 0.05). CONCLUSION: Cimetidine can enhance the humoral and cellular immune response induced by ROP2 protein.

[Recombinant expression and immunologic identification of bradyzoite-specific antigen BSR4 of Toxoplasma gondii].

OBJECTIVE: To express the recombinant BSR4 protein of Toxoplasma gondii Prugniaud (PRU) strain and study its immunologic characteristics. METHODS: The recombinant plasmid pET28a(+)-BSR4 was transformed into E. coli BL21, followed by expression of BSR4 induced by IPTG and its purification. The immunoreactivity of the recombinant protein BSR4 was analyzed by Western blotting with the sera from mice infected by PRU strain of T. gondii or normal mice as the first antibody. The 3H-TdR incorporation assay was performed to determine the proliferation of splenocytes in mice infected by PRU strain stimulated by BSR4, and the stimulation index (SI) was calculated. ELISA was used to evaluate the immunoreactivity of BSR4 protein, in which 20 sera from each of acute (anti-T. gondii IgG(-)IgM+) and chronic (IgG+IgM(-)) toxoplasmosis patients, and healthy people were tested. RESULTS: The recombinant BSR4 was induced and expressed. After denaturation, renaturation and purification, the soluble protein (Mr 45 000) was obtained and detected by anti-T. gondii serum with Western blotting. BSR4 in 1, 5, and 25 microg/ml induced the proliferation of splenocytes in mice infected by PRU strain with higher SI (1.13, 0.88, and 1.17) than that of control (0.46, 0.24, and 0.49, respectively, P<0.01). ELISA showed that the recombinant BSR4 specifically reacted with the sera of toxoplasmosis patients (IgG+IgM(-), 20/20), not with that of the acute cases (IgG(-)IgM+, 0/20). CONCLUSION: The recombinant BSR4 shows specific immunogenicity and immunoreactivity.