[Effect of Schistosoma japonicum Mr 22 600 particulated-antigen on dendritic cells and CD4+CD25+ regulatory T cells].

OBJECTIVE: To study the role of Schistosoma japonicum Mr 22 600 particulated-antigen on dendritic cells (DCs) and CD4+CD25+ regulatory T cells. METHODS: In in vitro experiments, DCs were pulsed with Sepharose 4B coupling rSj22.6/26GST and soluble rSj22.6/26GST, respectively. The surface molecules of DCs were detected by flow cytometry and the function of DCs was detected by mixed lymphocyte reaction. For the reduction of CD4+CD25+ T cells, DCs pulsed with Sepharose 4B coupling rSj22.6/26GST and soluble rSj22.6/26GST, respectively, were co-cultured with CD4+ T cells isolated from the spleen cells. The percentage of CD4+CD25+ Foxp3+ T cells in CD4+ T cells was detected by flow cytometry. In in vivo experiments, BALB/c mice were immunized with Sepharose 4B coupling rSj22.6/26GST, Freund's adjuvant emulsified rSj22.6/26GST, rSj22.6/26GST, Sepharose 4B, Freund's adjuvant and PBS, respectively. The percentage of DCs in draining lymph nodes and the percentage of CD4+CD25+Foxp3+ T cells in spleen cells were detected by flow cytometry. To analyze the inhibitory roles of CD4+CD25+ T cells on CD4+CD25- T cells, CD4+CD25+ T cells were separated from mice immunized with Sepharose 4B coupling rSj22.6/26GST and Freund's adjuvant emulsified rSj22.6/26GST, respectively, and co-cultured with CD4+CD25- T cells. The proliferation of cells was assessed by [3H] thymidine incorporation method. RESULTS: In vitro, the expression rate of the surface molecules of CD40, CD80 and CD86 on the soluble antigen pulsed DCs were (43.5 +/- 6.2)%, (37.7 +/- 0.1)%, and (71.4 +/- 1.4)%, respectively. But on the Sepharose 4B coupling antigen pulsed DCs, they were (31.2 +/- 5.4)%, (32.0 +/- 1.6)%, and (63.8 +/- 1.0)%, respectively, which suggested that the Sepharose 4B coupling rSj22.6/26GST had less stimulating roles on DCs maturation Furthermore, addition of DCs pulsed with Sepharose 4B coupling rSj22.6/26GST caused the expanding of CD4+CD25+ T cells. In vivo, immunization of Sepharose 4B coupling rSj22.6/26GST increased the number of CD4+CD25+ T cells. CD4+CD25+ T cells separated from Sepharose 4B coupling rSj22.6/26GST immunized mice had stronger inhibitory ability (cpm 1 420 +/- 335), compared with that of mice immunized with soluble antigen (cpm 3 558 +/- 147). CONCLUSION: In contrast to the Freund's adjuvant emulsified antigen, immunization with Sepharose 4B coupling rSj22.6/26GST increases the number of CD4+CD25+ T cells, which showed stronger inhibition on the CD4+ CD25- T cell proliferation, and the mechanism of which may be involved in DCs maturation.

[Immunological identification of a synthetic peptide from the paramyosin of Schistosoma japonicum].

OBJECTIVE: To identify the immunologic property of a synthetic peptide Sj97-P22 from paramyosin of Schistosoma japonicum (Sj97). METHODS: Twenty-seven female C57BL/6 mice were divided into 3 groups each with 9 mice, Sj97-P22, control peptide and PBS groups, and each mouse was respectively immunized twice (seven days interval) with 100 microg of Sj97-P22, control peptide or PBS, emulsified with equal value of complete Freund's adjuvant. Seven to ten days after the second immunization, the mouse spleen mononuclear cells were isolated for three-color flow cytometry to detect intracellular cytokines IFN-gamma and IL-4. Then the spleen mononuclear cells were co-cultured with Sj97-P22, control peptide or PBS respectively, and the incorporation rate of 3H-thymidine, as well as the levels of IL-2, IFN-gamma and IL-4 in the cultured cell supernatant, were measured. RESULTS: In CD4+ T cells, the percentage of IFN-gamma-producing cells in Sj97-P22 group [(8.05 +/- 0.54)%] was significantly higher than that of the control peptide group [(4.74 +/- 1.04)%] or PBS group [(6.51 +/- 0.49)%] (P<0.05), while the proportion of IL-4-producing cells was significantly lower in Sj97-P22 group [(0.60 +/- 0.11)%] than that in PBS group [(1.31 +/- 0.27)%] (P<0.05). Also, compared with control peptide or PBS stimulation, Sj97-P22 was able to effectively stimulate the proliferation with the stimulation index (3.12 +/- 1.59) and a higher secretion of IL-2 [(9.13 +/- 1.54) pg/ml] and IFN-gamma [(39.75 +/- 9.69) pg/ml] of spleen mononuclear cells in Sj97-P22-immunized mice (P<0.05). Both Sj97-P22 and control peptide were not effective stimulators to the spleen mononuclear cells from mice of PBS group. CONCLUSION: It is highly possible that Sj97-P22 is a Th1-type epitope specific for C57BL/6 mice.

Protective immunity induced by phage displayed mitochondrial related peptides of Schistosoma japonicum.

New antigens and strategies are necessary for vaccine development against schistosomiasis japonica. Using a pool of 43 high titred anti-SWA sera from individuals residing in an Schistosoma japonicum endemic area of China, we have cloned a S. japonicum gene by cDNA library screening. The recombinant Sj338 protein has 44-46% identity to a mitochondrial precursor receptor protein of humans and rats. Immunization of mice with the recombinant Sj338 conferred 27-32% (p<0.01) reduction in worm burdens following cercarial challenge. In an effort to identify protective epitopes in Sj338 and increase the level of protection, we screened a random 12-mer peptide library constructed in M13 using a polyspecific anti-Sj338 rabbit serum. After five rounds of biopanning, we identified 30 reactive clones consisting of 11 distinct peptide sequences. These clones shared limited primary sequence homology with the recombinant Sj338 protein. Anti-sera raised against these phage clones recognized recombinant Sj338 and SWAP by Western blot. In murine vaccination experiments using whole recombinant phage without adjuvant, four of these clones demonstrated worm reductions of 11.6-25.1% (p=ns - 0.05) compared to M13 vaccinated animals. Animals vaccinated with all four of these phage demonstrated 34.2% (p<0.01) worm reduction compared to controls vaccinated with M13 clone. These data suggest that mimotope peptides are potential vaccine candidates for S. japonicum.

[Preliminary identification of T cell epitopes on 22.6 kDa antigen of Schistosoma japonicum].

OBJECTIVE: To identify the T cell epitopes on 22.6 kDa antigen of Schistosoma japonicum (Sj22.6). METHODS: The primary structure of Sj22.6 molecule was analysed using various predictive algorithms and a panel of 4 peptides were acquired. Their oligonucleotides were designed, synthesized and inserted into the multiple cloning site of plasmid pET-32c(+). The recombinant plasmids were transformed into E. coli BL21 and identified by endonuclease digestion and sequencing. The positive clones containing the recombinant plasmids could express specific fusion proteins (trx-epitope, MW approximately 20 kDa) induced by IPTG. The fusion protein with 6 x His could be coupled with NTA resin specifically, and purified by elusion of the column with buffer containing imidazole. The purified fusion proteins were incubated with splenocytes of C3H mice and then, the proliferation of splenocytes was determined by 3H-TdR incorporation assay. RESULTS: The recombinant plasmids were constructed successfully and the positive clones containing the recombinant plasmids expressed specific fusion proteins. Three of the purified fusion proteins (P4, P5, P6) could stimulate the lymphocyte proliferation. CONCLUSION: Three T cell epitopes on Sj22.6 antigen were identified.

[Purification and immunogenicity identification of the recombinant protein related with specific IgE against Schistosoma japonicum].

OBJECTIVE: To purify the specific IgE antibody-related recombinant protein of Schistosoma japonicum and to identify its immunogenicity. METHODS: The recombinant plasmid Sj43B/pGEX-6p-1 was expressed in E. coli BL 21. The inclusion body of the fusion protein was washed by TNMFX buffer and separated by FPLC. After renaturation, the fusion protein was used to vaccinate the mice. The specific IgG and IgE antibodies were detected by dot-ELISA and Western blotting analysis, respectively. RESULTS: Most of the proteins mixed with the inclusion body of the recombinant protein could be eliminated by washing with TNMFX buffer. The purified recombinant fusion protein could be obtained by FPLC separation. The experiment on mice immunized with the fusion protein showed that the specific IgE antibody was generated against the target part of the fusion protein, but not the specific IgG antibody. CONCLUSION: The fusion protein expressed by the recombinant plasmid Sj43B/pGEX-6p-1 could induce specific IgE response of the immunized mice.

[Identification of epitope of 22.6 kDa antigen of Schistosoma japonicum with phage-displayed random peptide library].

OBJECTIVE: To identify the epitopes of 22.6 kDa antigen of Schistosoma japonicum (Sj22.6). METHODS: A 12-mer library displayed on pIII of fd phage was used to screen the epitopes of Sj22.6 with the purified multiple clone IgG antibody against the Sj22.6 antigen. Five rounds of biopanning were carried out and fourteen clones from the fifth round biopanning were randomly selected and identified by Western blotting. The mice were immunized with the obtained positive clones and the antibody titers of sera from the mice were detected. The clones which could stimulate the mice to produce anti-Sj22.6 antibodies were sequenced and their amino acid sequences were compared with that of Sj22.6. RESULTS: Six clones selected from the fourteen ones could stimulate the mice to produce anti-Sj22.6 antibodies analysed by Western blotting. The amino acid sequence of one epitope showed high homology with Sj22.6. CONCLUSION: Four epitopes of Sj22.6 were obtained. One may be a structure epitope and the other three were mimic epitopes.

[Studies on the characteristic of interferon-gamma mediating resistance in mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the molecular characteristic of interferon-gamma mediating protective immunity against schistosomiasis japonica in mice. METHODS: CD4+ T cells were isolated from spleens of mice infected with Schistosoma japonicum at different time-points. The cDNA microarray technique combined with RT-PCR was used to explore IFN-gamma inducible GTPase family gene expression profile of CD4+ T cell. IGTP, a representative IFN-gamma, inducible GTPase having vital anti-infection activity, was amplified from spleen of BALB/c mice using RT-PCR, then cloned into pGEM(r)-T easy vector for sequencing. RESULTS: IFN-gamma inducible GTPase family had the similar characteristic over the course of S. japonicum infection. The gene expression of these members were up-regulated or had little change at 3 wk post-infection, then down-modulated from 6 wk to 13 wk post-infection, which was also confirmed by RT-PCR. As for IGTP, two inserts were identified after sequencing. One was 142 bp shorter than another, but the fragment was lost due to low annealing temperature. CONCLUSION: There is a dramatic inhibition of IFN-gamma pathway and IFN-gamma-dependent anti-infective immunity during the infection of S. japonicum.

Murine CD8(+)T cell cytotoxicity against schistosomula induced by inoculation of schistosomal 22.6/26GST coupled Sepharose 4B beads.

Schistosomasis is a world-wide parasitic disease. Although chemotherapy is the main treatment method for schistosomasis currently, it cannot prevent schistosome reinfection. Up to now no effective vaccine is available to prevent schistosomiasis. Dendritic cells (DCs) are one of the key players in the cellular immune response and play an important role in antigen presentation as antigen-presenting cells. Here we reported a novel large particulate antigen, in which Sepharose 4B beads were coated with Sj22.6/26GST. Our results showed that this particulate antigen could be cross-presented by DCs to CD8(+)T cells. Furthermore, CD8(+)T cells stimulated by particulate antigen directly exerted cytotoxicity against Schistosoma japonicum schistosomula. We also demonstrated that S. japonicum schistosomula acquired the MHC class I molecules from host blood serum and presented the molecules at the larval surface. While it may help them escape from the host immune surveillance, these MHC I-antigen complexes presented on the surface render schistosomula the potential targets of the CD8(+)T cell cytotoxicity induced by particulate antigen-based vaccine. Finally we evaluated the protective immunity of this particulate vaccine in a mouse infection challenge model. Our data clearly showed that the particulate vaccine induced a partial reduction in both worm burdens and egg loads. Taken together, these results suggest that this large particulate vaccine could be a potential vaccine for the prevention of schistosome infection.

Gene profile of chemokines on hepatic stellate cells of schistosome-infected mice and antifibrotic roles of CXCL9/10 on liver non-parenchymal cells.

Hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis caused by schistosomiasis. Chemokines were widely expressed and involved in cellular activation, proliferation and migration in inflammatory and infectious diseases. However, little is known about the expressions of chemokines on HSCs in the schistosoma infection. In addition, the roles of chemokines in pathogenesis of liver fibrosis are not totally clear. In our study, we used microarray to analyze the temporal gene expressions of primary HSCs isolated from mice with both acute and chronic schistosomiasis. Our microarray data showed that most of the chemokines expressed on HSCs were upregulated at 3 weeks post-infection (p.i) when the egg granulomatous response was not obviously evoked in the liver. However, some of them like CXCL9, CXCL10 and CXCL11 were subsequently decreased at 6 weeks p.i when the granulomatous response reached the peak. In the chronic stage, most of the differentially expressed chemokines maintained persistent high-abundances. Furthermore, several chemokines including CCR2, CCR5, CCR7, CXCR3, CXCR4, CCL2, CCL5, CCL21, CXCL9 and CXCL10 were expressed by HCSs and the abundances of them were changed following the praziquantel treatment in the chronic stage, indicating that chemokines were possibly necessary for the persistence of the chronic stage. In vitro experiments, hepatic non-parenchymal cells, primary HSCs and human HSCs line LX-2 were stimulated by chemokines. The results showed that CXCL9 and CXCL10, but not CXCL11 or CXCL4, significantly inhibited the gene expressions of Col1α1, Col3α1 and α-SMA, indicating the potential anti-fibrosis effect of CXCL9 and CXCL10 in schistosomiasis. More interestingly, soluble egg antigen (SEA) of Schistosoma japonicum was able to inhibit transcriptional expressions of some chemokines by LX-2 cells, suggesting that SEA was capable of regulating the expression pattern of chemokine family and modulating the hepatic immune microenvironment in schistosomiasis.

New insight into the antifibrotic effects of praziquantel on mice in infection with Schistosoma japonicum.

BACKGROUND: Schistosomiasis is a parasitic disease infecting more than 200 million people in the world. Although chemotherapy targeting on killing schistosomes is one of the main strategies in the disease control, there are few effective ways of dealing with liver fibrosis caused by the parasite infection in the chronic and advanced stages of schistosomiasis. For this reason, new strategies and prospective drugs, which exert antifibrotic effects, are urgently required. METHODS AND FINDINGS: The antifibrotic effects of praziquantel were assessed in the murine models of schistosomiasis japonica. Murine fibrosis models were established by cutaneous infection with 14 ± 2 Schistosoma japonicum cercariae. Then, the mice of both chronic (8 weeks post-infection) and advanced (15 weeks post-infection) schistosomiasis were treated by gavage of praziquantel (250 mg/kg, once daily for 3 days) to eliminate worms, and followed by praziquantel anti-fibrosis treatment (300 mg/kg, twice daily for 30 days). The fibrosis-related parameters assessed were areas of collagen deposition, content of hydroxyproline and mRNA expressions of Col1α1, Col3α1, α-SMA, TGF-ß, MMP9, TIMP1, IL-4, IL-10, IL-13 and IFN-γ of liver. Spleen weight index, alanine aminotransferase activity and liver portal venous pressure were also measured. The results showed that anti-fibrosis treatment improved liver fibrosis, splenomegaly, hepatic function, as well as liver portal hypertension. In order to confirm the anti-fibrotic properties of praziquantel, we established a CCL(4)-induced model and revealed that CCL(4)-induced liver fibrosis was inhibited by PZQ treatment for 30 days. Furthermore, we analyzed the effects of praziquantel on mouse primary hepatic stellate cells (HSCs). It is indicated that mRNA expressions of Col1α1, Col3α1, α-SMA, TGF-ß, MMP9 and TIMP1 of HSCs were all inhibited after praziquantel anti-parasite treatments. CONCLUSIONS: The significant amelioration of hepatic fibrosis by praziquantel treatment validates it as a promising drug of anti-fibrosis and offers potential of a new chemotherapy for hepatic fibrosis resulting from schistosomiasis.

Th1-type epitopes-based cocktail PDDV attenuates hepatic fibrosis in C57BL/6 mice with chronic Schistosoma japonicum infection.

Schistosomiasis is one of the world's major public health problems in terms of morbidity and mortality, which is characterized by a marked egg-induced CD4(+) T-cell programmed granulomatous inflammation and cumulative fibrosis. Here PDDV (peptide-DNA dual vaccine), a widely used non-viral gene delivery system, was applied. The cocktail PDDV, based on four Th1-type epitope peptides identified from Schistosoma japonicum vaccine candidates and CpG ODN1826, could induce dominant Th1-type response in C57BL/6J mice (P<0.05). The histopathological staging and collagen assessment for fibrosis showed that the cocktail PDDV presented an obvious down-regulation effect on hepatic fibrosis caused by chronic S. japonicum infection (P<0.05), and IFN-gamma, IL-4 and IL-13 mRNAs in liver detected by RT-PCR also showed that the cocktail PDDV represented the ability to up-regulate Th1-type responses, which paralleled with a decrease expression of alpha-SMA (P<0.05) and the up-regulated MMP9/TIMP1 balance (P<0.05) when compared to the control groups. Therefore, it is indicated that the cocktail PDDV can significantly attenuate hepatic fibrosis, in parallel with the decreased HSCs activation and the up-regulated MMP9/TIMP1 balance in favor of matrix degradation, which may be partially dependent on the increased Th1 response to restore the Th1/Th2 balance.

Evaluation of Kato-Katz examination method in three areas with low-level endemicity of schistosomiasis japonica in China: A Bayesian modeling approach.

The Kato-Katz technique is widely used to determine faecal egg counts of intestinal schistosomiasis. Although numerous studies have reported considerable underestimation of the 'true' infection prevalence while using this method, little is known regarding how many infections are missed as a function of the overall endemicity of intestinal schistosomiasis. In the present study, we used a Bayesian modeling approach to assess how much the Kato-Katz technique underestimates the prevalence of Schistosoma japonicum in three low endemic areas, characterized by different levels of infection. We found that up to 83% of S. japonicum infections were missed in an average when only a single Kato-Katz thick smear was examined. We further analyzed inter- and intra-specimen variation using agreement analysis. The results revealed a clear trend of higher agreement with infection intensity. In addition, our data also confirmed that intra-specimen consistency was better than that of inter-specimen. Our results suggest that a single Kato-Katz thick smear could only detect a certain small part of infections in areas with low endemicity; the disagreement of Kato-Katz results are mainly driven by day-to-day variation of eggs in stool; and light intensities are characterized by very high underestimation rates. There is a pressing need to develop more sensitive diagnostic tools for accurate detection of light infection intensities of schistosome infections.

Dynamics of CD4+CD25+ T cells in spleens and mesenteric lymph nodes of mice infected with Schistosoma japonicum.

CD4+CD25+ T cells play a major role in modulating immune response, but few reports have been published about schistosomiasis. Here, we investigated the changes in CD4+CD25+ T cell populations in spleens and mesenteric lymph nodes of mice infected with Schistosoma japonicum. The proportions of CD4+CD25+ T cells in total CD4+ T cells were analyzed by flow cytometry. CD25 and Foxp3 expression was measured by real-time quantitative polymerase chain reaction. The suppressive activities of CD4+CD25+ T cells were detected by in vitro proliferation of splenocytes. Evidence showed that the percentage of CD4+CD25+ T cells was the same as controls 3 weeks post-infection. At the acute stage of infection, the percentage decreased significantly. However, at the chronic stage of infection, it rebounded to normal levels or even higher. The expression of the CD25 and Foxp3 showed gradual increase along with the infection progress. In vitro experiment also showed the strong suppressive effect of CD4+CD25+ T cells, isolated during the chronic stage, on proliferation of the CD25- splenocytes. This is the first time that the dynamics of CD4+CD25+ T cell populations was demonstrated in mice infected with schistosomiasis. In conclusion, our data indicated that CD4+CD25+ cells might be involved in the immune modulation during S. japonicum infection, which enhances current knowledge of the mechanisms of the immuno-downregulation and re-infection in schistosomiasis.

Characterization of CD4+ T cell responses in mice infected with Schistosoma japonicum.

To better understand the interaction between Schistosoma japonicum and its murine host, we characterized the immune response of CD4+ T cells generated during an experimental S. japonicum infection based on different key aspects, from gene expression to cell behavior. Mouse oligonucleotide microarrays were used to compare gene expression profiles of CD4+ T cells from spleens of mice at 0, 3, 6 and 13 weeks post-infection. Flow cytometry analysis was used to determine type 1 and type 2 cytokine-secreting CD4+ T cells, to test apoptosis of CD4+ T cells and to count CD4+CD25+ T cells, a kind of regulatory subpopulation of CD4+ T cells. The percentage of interleukin-4-producing CD4+ T cells was found to be much higher than that of gamma-interferon-producing cells, especially after stimulation with S. japonicum egg antigen, which was consistent with type 1 and type 2 cytokine gene expression in the genechip. Microarray data also showed that S. japonicum induced the increased expression of Th2 response-related genes, whereas some transcripts related to the Th1 responsive pathway were depressed. Flow cytometry analysis showed a marked increase in the apoptotic CD4+ T cells from 6 weeks post-infection and in the ratio of CD4+CD25+ to CD4+ T cells in infected mice after 13 weeks. We therefore concluded that experimental infection of mice with S. japonicum resulted in a Th2-skewed immune response, which was to a great extent monitored by the immune regulatory network, including cytokine cross-modulation, cell apoptosis and the subpopulation of regulatory cells.

Gene transcription profile in mice vaccinated with ultraviolet-attenuated cercariae of Schistosoma japonicum reveals molecules contributing to elevated IFN-gamma levels.

Vaccination with ultraviolet-attenuated cercariae of Schistosoma japonicum induced protective immunity against challenge infection in experimental animal models. Our preliminary study on the transcription levels of IFN-gamma and IL-4 in splenic CD4+ T cells revealed that attenuated cercariae elicited predominantly a Th1 response in mice at the early stage, whereas normal cercariae stimulated primarily Th2-dependent responses. Further analysis on the gene profile of the skin-draining lymph nodes demonstrated that the levels of IFN-gamma were significantly higher in vaccinated mice than those in infected mice at day 4, 7 and 14 post-vaccination or post-infection. However, for IL-12 and IL-4, the potent inducers of Th1 and Th2 responses, respectively, as well as IL-10, there were no differences over the course of the experiment between the infected and vaccinated mice. To explore the underlying factors that may potentially contribute to elevated IFN-gamma in vaccinated mice, the mRNA profiles of the skin-draining lymph nodes at day 4 post-exposure were compared using oligonucleotide microarrays. Within the 847 probe sets with increased signal values, we focused on chemokines, cytokines and relevant receptors, which were validated by semi-quantitative RT-PCR. A comprehensive understanding of the immune mechanisms of attenuated cercariae-induced protection may contribute to developing efficient vaccination strategies against S. japonicum, especially during the early stage of infection.

Identification of immunodominant Th1-type T cell epitopes from Schistosoma japonicum 28 kDa glutathione-S-transferase, a vaccine candidate.

Th1-type cytokines produced by the stimulation of Th1-type epitopes derived from defined schistosome-associated antigens are correlated with the development of resistance to the parasite infection. Schistosoma mansoni 28 kDa glutathione-S-transferase (Sm28GST), a major detoxification enzyme, has been recognized as a vaccine candidate and a phase II clinical trial has been carried out. Sheep immunized with recombinant Schistosoma japonicum 28GST (Sj28GST) have shown immune protection against the parasite infection. In the present study, six candidate peptides (P1, P2, P3, P4, P7 and P8) from Sj28GST were predicted, using software, to be T cell epitopes, and peptides P5 and P6 were designed by extending five amino acids at the N-terminal and C-terminal of P1, respectively. The peptide 190-211 aa in Sj28GST corresponding to the Th1-type epitope (190-211 aa) identified from Sm28GST was selected and named P9. The nine candidate peptides were synthesized or produced as the fusion protein with thioredoxin in the pET32c(+)/BL21(DE3) system. Their capacity to induce a Th1-type response in vitro was measured using lymphocyte proliferation, cytokine detection experiments and flow cytometry. The results showed that P6 (73-86 aa) generated the strongest stimulation effect on T cells among the nine candidate peptides, and drove the highest level of IFN-gamma and IL-2. Therefore, P6 is a functional Th1-type T cell epitope that is different from that in Sm28GST, and will be useful for the development of effective vaccines which can trigger acquired immunity against S. japonicum. Moreover, our strategy of identifying the Th1-type epitope by a combination of software prediction and experimental confirmation provides a convenient and cost-saving alternative approach to previous methods.

[Prediction of T-cell epitopes from schistosoma japonicum 28 kDa glutathione-S-transferase and identification of their Th1 type T-cell epitopes].

AIM: To predict T-cell epitopes of recombinant schistosoma japonicum 28 kDa glutathione-S-transferase(GST) with software and identify the Th1 type T-cell epitopes by experiments. METHODS: T-cell epitopes of recombinant schistosoma japonicum 28kDa GST were predicted with software and several epitopic candidates were screened from them according to their scores. Some of the epitopic candidates were synthesized and the other epitope peptides fused with thioredoxin(Trx) were expressed in E.coli BL21(DE3) and purified by Ni(+) column affinity chromatography. C57BL/6 (H-2(b)) mice's were immunized via peritoneal infection with ultraviolet ray irradiated-cercariae and then boosted with recombinant schistosoma japonicum 28 kDa GST. The immunized mice splenocytes were prepared, cultured and stimulated with synthesized epitope peptides and epitope peptide fusion proteins, respectively. Stimulation activity of synthesized epitope peptides and epitope peptides fusion proteins were assayed by lymphocyte proliferation assay. Levels of IFN-gamma and IL-2 were measured by ELISA. CD4(+) T cells and T cells secreting IFN-gamma and IL-4 were detected by flow cytometry. RESULTS: Epitope P6(73-86aa) among 9 epitopic candidates could generate the strongest stimulation effect on splenocytes, stimulate secretion of higher levels of IFN-gamma and IL-2, and induce more IFN-gamma(+) and IL-4 (+) T cells. CONCLUSION: The recombinant 28 kDa GST possesses functional Th1 type T-cell epitope.

[Detection of Schistosomia japonicum 5D gene by polymerase chain reaction and genechip technique].

OBJECTIVE: In order to develop the diagnostic genechip for specific detection of Schistosoma japonicum (Chinese mainland strain). METHODS: Probe and primers were designed based on the Schistosoma japonicum 5D gene encoding an immunogenic miracidial antigen. The probe for the conservative and specific gene sequence was spotted onto the specially treated glass slides by pin-based spotting robot Pixsys 5500 and was employed to make genechips. A polymerase chain reaction (PCR) protocol was designed to effectively amplify the 5D gene fragment containing the probe sequence from cercaria, egg, adult worm and infected Oncomelania DNA as well as other flukes DNA, respectively. After 35 cycles by PCR, the products were then labeled with fluorescent Cy3-labeled primer, using dissymmetrical PCR. The labeled PCR products of the target genes were hybridized to the diagnostic genechips for detection of Schistosoma japonicum and a fluorescent scanner (ScanArray 3000) was used to observe and record the hybridization signals. RESULTS: The result obtained from the study showed that a 262 bp DNA fragment was amplified from cercaria, egg and adult worm with the designed primers and enable the genechip be applied to detect a single cercaria, egg and adult worm. When the genechip was used to detect Clonorchis sinensis, Fasciolopsis busk, and Paragonimus westermani DNA, the results showed negative, indicating that the genechip had good specificity. CONCLUSION: The genchip technique for detection of Schistosoma japonicum was established successfully and having the characteristics of high sensitivity and specificity.