P21 activated kinase-1 (PAK1) in macrophages is required for promotion of Th17 cell response during helminth infection.

CD4+ T cells differentiate into distinct functional effector and inhibitory subsets are facilitated by distinct cytokine cues present at the time of antigen recognition. Maintaining a balance between T helper 17 (Th17) and regulatory T (Treg) cells are critical for the control of the immunopathogenesis of liver diseases. Here, by using the mouse model of helminth Schistosoma japonicum (S japonicum) infection, we show that the hepatic mRNA levels of P21-activated kinase 1 (PAK1), a key regulator of the actin cytoskeleton, adhesion and cell motility, are significantly increased and associated with the development of liver pathology during S japonicum infection. In addition, PAK1-deficient mice are prone to suppression of Th17 cell responses but increased Treg cells. Furthermore, PAK1 enhances macrophage activation through promoting IRF1 nuclear translocation in an NF-κB-dependent pathway, resulting in promoting Th17 cell differentiation through inducing IL-6 production. These findings highlight the importance of PAK1 in macrophages fate determination and suggest that PAK1/IRF1 axis-dependent immunomodulation can ameliorate certain T cell-based immune pathologies.

Schistosoma japonicum infection causes a reprogramming of glycolipid metabolism in the liver.

BACKGROUND: Recent investigations indicate that schistosome infection is closely associated with aberrant glycolipid metabolism. However, the actual glycolipid metabolism gene expression, as well as the possible pathways that regulate glycolipid metabolism in the schistosome-infected liver, has not been extensively explored. METHODS: In this study, we evaluated the dynamic expression of glycolipid metabolism-associated genes and proteins in the livers from mice infected with Schistosoma japonicum at the indicated time points using real-time PCR and immunofluorescence. Then, cultures of macrophages were treated with schistosome soluble egg antigen (SEA) to detect the expression levels of genes associated with glucose and lipid metabolism in order to identify macrophages metabolic characteristics in response to these antigens. Furthermore, SEA-stimulated macrophages were co-cultures with hepatocytes and detected the effects of macrophages on the gene expression of hepatocytes metabolism. RESULTS: The expression of glycolysis-related genes (Ldha, Glut4, Pkm2, Glut1, Pfkfb3, Aldoc, HK2, Pfk) in the liver were upregulated but the gluconeogenesis gene (G6pc) was downregulated during S. japonicum infection. In addition, the mRNA levels of fatty acid (FA) oxidation-related genes (Ucp2, Atp5b, Pparg) in the liver were significantly upregulated; however, the FA synthesis genes (Fas, Acc, Scd1, Srebp1c) and lipid uptake gene (Cd36) were downregulated post-S. japonicum-infection. In consistence with these data, stimulation with SEA in vitro significantly enhanced the gene expression that involved in glycolysis and FA oxidation, but decreased genes related to gluconeogenesis, FA synthesis and lipid uptake in macrophages. The levels of phosphorylated AMPK, AKT and mTORC1 were increased in macrophages after SEA stimulation. Inhibition of phosphorylated AMPK, AKT and mTORC1 promoted SEA-treated macrophages to produce glucose. In addition, suppression of phosphorylated-AMPK, but not phosphorylated-AKT and phosphorylated-mTOR, induced the lipid accumulation in SEA-stimulated macrophages. Furthermore, SEA-treated macrophages significantly reduced the expression of Acc mRNA in hepatocytes in vitro. CONCLUSIONS: These findings reveal S. japonicum infection induces dynamic changes in the expression levels of genes involved in catabolism (glucose uptake, glycolysis and fatty acid oxidation) and suppressing anabolism (glycogen synthesis) in the liver, which could occur via macrophages' metabolic states, particularly those involved in the AMPK, AKT and mTORC1 pathways.

PSMB8 regulates glioma cell migration, proliferation, and apoptosis through modulating ERK1/2 and PI3K/AKT signaling pathways.

Glioma has been considered as one of the most aggressive and popular brain tumors of patients. It is essential to explore the mechanism of glioma. In this study, we established PSMB8 as a therapeutic target for glioma treatment. Expression of PSMB8 as well as Ki-67 was higher in glioma tissues demonstrated by western blot and immunohistochemistry. Then, the role of PSMB8 in migration and proliferation of glioma cells was investigated by conducting wound-healing, trans-well assay, cell counting kit (CCK)-8, flow cytometry assay and colony formation analysis. The data showed that interfering PSMB8 may inhibit the migration and proliferation of glioma cells by reducing expression of cyclin A, cyclin B1, cyclin D1, Vimentin, and N-cadherin, and by increasing expression of E-cadherin. Additionally, interfering PSMB8 may induce apoptosis of glioma cells by upregulating caspase-3 expression. Furthermore, these in vitro findings were validated in vivo and the ERK1/2 and PI3k/AKT signaling pathways were involved in PSMB8-triggered migration and proliferation of glioma cells. In an in vivo model, downregulation of PSMB8 suppressed tumor growth. In conclusion, PSMB8 is closely associated with migration, proliferation, and apoptosis of glioma cells, and might be considered as a novel prognostic indicator in patients with gliomas.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori.

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

[Study on immune status of patients with schistosomiasis japonica in Poyang Lake region III Humoral and cellular immune characteristics between Schistosoma japonicum high and low antibody responders].

OBJECTIVE: To explore the immune mechanism of negative results of immune tests of schistosomiasis japonica patients. METHODS: Totally 142 schistosomiasis patients (positive stool examinations) of Poyang Lake region were tested by ELISA method, and the ROC curve was applied to determine the high and low response of the patients. The levels of cellular immunity and cytokines of high and low responders were compared. RESULTS: Totally eight schistosomiasis patients were found as low responders. Besides SWAP-IgA (t = -1.588, P > 0.1), the levels of isotype antibodies were significantly lower in the low responders compared with those in the high responders (t = -14.517 to -2.866, all P < 0.05). In the low responders, the propor- tion of CD3⁺T was increased; and the proportions of CD4⁺T, CD8⁺T, CD4⁺CD25⁺Treg, and the ratio of CD4⁺/CD8⁺ were all de- creased, but all of them were not significant (t = -1.72 to 0.974, all P > 0.05) compared with those in the high responders. The differences of IFN-γ and IL-10 between the high and low responders were both not significant (t = -2.426 to 0.216, all P > 0.05). CONCLUSIONS: There is a significant difference between the high and low responders only in the levels of isotype antibodies. One of the reasons of low response in the immune tests is the much lower antibody level after the antigen-antibody compound is completely formulated.

[Expression characteristics of microRNA in mice with schistosomiasis and praziquantel treatment].

OBJECTIVE: To investigate the expression characteristics of miR-155 and miR-146a in mice with schistosomiasis and praziquantel (PZQ) treatment. METHODS: Totally 40 BABL/c mice were divided into 4 groups: a normal group, a 6W infected group that were infected cutaneously with 10 Schistosoma japonicum cercariae for 6 weeks, a 12W infected group that were infected cutaneously with 10 Schistosoma japonicum cercariae for 12 weeks, and a praziquantel treated group that were infected cutaneously with 10 Schistosomajaponicum cercariae and intragastrically administered with PZQ (300 mg/kg/day) for 1 day in 6 weeks post-infection and continuing surviving 6 weeks. The animals were sacrificed in 6 weeks and 12 weeks post-infection respectively. The left front lobe of each liver was stained with hematoxylin-eosin (HE) to detect pathological lesions. The levels of mRNA expressions of miR-155, miR-146a and pro-inflammatory cytokines (TNF-alpha, IL-1beta and IL-6) in the liver tissue were determined by using quantitative real-time PCR. RESULTS: The levels of mRNA expressions of miR-155, miR-146a and pro-inflammatory cytokines (TNF-alpha, IL-1beta and IL-6) in the 6W infected mice were significantly higher than those of the normal mice and of the 12W infected mice. Compared with the 12W infected mice, the inflammation response of liver egg granuloma in the PZQ-treated mice was ameliorated. Furthermore, there was a marked increase in the levels of mRNA expressions of miR-155, miR-146a and three pro-inflammatory cytokines in the PZQ-treated mice compared to the 12W infected mice. CONCLUSION: miR-155 and miR-146a may play a role in schistosomiasis liver inflammation response and the inflammation regulation of praziquantel treatment.

Immune responses result in misdiagnosis of Schistosoma japonicum by immunodiagnosis kits in egg-positive patients living in a low schistosomiasis transmission area of China.

BACKGROUND: In recent field surveys, we failed to detect the presence of specific antibody against Schistosoma japonicum in some egg-positive patients by commonly used immunodiagnostic kits. To find out whether low levels of specific antibody truly exist among egg-positive individuals and elucidate the underlying immune mechanisms, we carried out a cross-sectional epidemiologic study in a S. japonicum low transmission endemic area of Poyang Lake region, China and compared the humoral and cellular immune characteristics between S. japonicum high and low antibody responders. METHODS: Kato-Katz thick smear assay was used to determine the schistosomiasis status of 3,384 participants residing in two Poyang Lake region villages, Jiangxi, China. Among the 142 stool egg-positive participants, we identified low and high S. japonicum antibody responders with soluble egg antigen (SEA) and adult worm antigen (AWA) specific IgG levels by adopting ROC curve analysis. To compare the humoral and cellular immune responses between high and low S. japonicum antibody responders, serum specific antibody levels as well as the percentage of T lymphocyte subpopulation in PMBC, and cell stimulated cytokines (IFN- gamma and interlukin-10) were detected. RESULTS: Eight S. japonicum egg-positive participants were defined as low antibody responders. Although the percentage of CD3⁺T cells in low responders was slightly higher and the percentage of CD4⁺ T cells, CD8⁺ T cells, the ratio of CD4⁺/CD8⁺ and CD4⁺ CD25⁺ Treg cells were lower than those in high responders, the differences between the two groups were not significant (P > 0.05). AWA -stimulated interlukin-10 level was significantly higher in high responders, while other cytokines did not show differences between two groups. For antibody profiles, except AWA specific IgA, significant differences of each antibody isotype between low and high responders were detected (P < 0.05). CONCLUSIONS: Our study confirmed that there are S. japonicum antibody low responders among schistosome egg-positive residents in S. japonicum low-transmission areas in China. Thus, mis-diagnosis using immune-diagnosis kits do exist. Significant differences of responding antibody levels between low and high responders were detected, while no major cellular response changes were observed.

Cross-strait parasitological research priorities arrived at by historical tracking and advanced dialogue.

To further enhance dialogue and promote cross-strait cooperation in the prevention and control of parasitic diseases, this paper reviewed the progress and current challenges in the cross-strait control and research of parasitic infections, based on three cross-strait meetings on parasitological research in the last decade. The major outcome of the 3rd Meeting of Cross-Strait Parasitological Research held in April 2013 was identifying the research priorities for parasitological research.

Schistosoma japonicum soluble egg antigens attenuate IFN-γ-induced MHC class II expression in RAW 264.7 macrophages.

Innate immune response plays the key role in initiating and guiding the immune response. Elucidating the innate immune related molecular events involved in the interaction between the parasite and the host will aid in the development of an effective vaccine and anti-schistosome pharmaceuticals. In this study, we examined the regulatory effect of Schistosoma japonicum soluble egg antigen (SEA) on MHC class II expression in macrophage cell line RAW 264.7. We demonstrated that SEA possesses the ability to down-regulate IFN-γ-induced MHC class II expression in RAW 264.7 cells. The production of IL-10 and IL-6 in RAW 264.7 cells, induced by SEA, was responsible for mediating the down-regulation of MHC class II. Our findings suggest that in RAW 264.7 cells (1) IFN-γ provides a condition for lower concentrations of SEA to attenuate MHC class II expression; (2) SEA attenuated IFN-γ-induced MHC class II expression and the IL-10 and IL-6 production is mediated at least partly by the interaction of SEA with TLR4; and (3) SEA attenuated IFN-γ-induced MHC class II expression at the transcriptional level.

[Antibody responses to leucine aminopeptidase in Schistosoma japonicum infection].

OBJECTIVE: To investigate the antibody response to leucine aminopeptidase in the different stages of Schistosoma japonicum infection. METHODS: The expression product of SjLAP was identified by Western blot and further purified by using nickel column. The IgG levels in the response to SjLAP in murine and porcine sera were detected by ELISA at different time points after the infection of S. japonicum. RESULTS: SjLAP expressed by E. coli was recognized by anti-his monoclonal antibody and S. japonicum-infected mice sera by Western blot. The results of ELISA showed that IgG responses to SjLAP rose gradually and reached the peak at 4 weeks post-infection for pigs (P1) and 6 weeks post-infection for mice (P2). With the appearance of a large number of eggs in the tissue, SjLAP-specific IgG levels were significantly down-regulated ( P1 = 0. 0004, P2 = 0. 0001). CONCLUSION: SjLAP originated from the adult worm might become a potential target for early diagnosis of schistosomiasis japonica.

[Development of multiplex microbead immunoassay for detection of specific antibodies against Cryptosporidium parvum].

OBJECTIVE: To develop a multiplex microbead immunoassay for detection of specific antibodies against Cryptosporidium parvum using recombinant proteins CP23, SA35 and SA40. METHODS: By using purified recombinant proteins CP23, SA35 and SA40 as detected antigens, and bovine serum albumin (BSA) as internal control, the four proteins aforementioned were coupled with micro beads and MIA was developed. Then, the efficiency of the coupled proteins was tested, the difference between the single MIA method and the multiple MIA method was compared, and the difference between plates was also compared. RESULTS: The purified proteins and BSA were coupled with microbeads successfully, and the MIA method was developed. Finally, the sensitivity and specificity of MIA method were confirmed. CONCLUSIONS: The multiplicate MIA method could be used to detect multiple antibodies after Cryptosporidium parvum infection, and the specificity and sensitivity of MIA are very high. The multiplicate MIA method can be one of the tools used in epidemiological survey.

High prevalence of Schistosoma japonicum infection in water buffaloes in the Philippines assessed by real-time polymerase chain reaction.

Difficulty in controlling human Schistosoma japonicum infection is partly attributed to the presence of non-human definitive hosts. Water buffaloes are a major reservoir for transmission of S. japonicum to humans in China. However, in the Philippines, reports based on microscopic examination of buffalo stool identified a low prevalence of S. japonicum, and mathematical models using these data concluded that water buffaloes are not a major reservoir for transmission of S. japonicum to humans. We collected stool from 81 buffaloes in Macanip, Leyte, the Philippines, and assayed for S. japonicum infection by the Danish Bilharziasis Laboratory technique, the Kato-Katz technique, miracidia hatching, and a highly validated real-time polymerase chain reaction. The prevalence defined by each assay was 3.7%, 3.7%, 0%, and 51.5% respectively. Our results demonstrate that microscopic-based techniques dramatically underestimate the prevalence of S. japonicum infection in water buffaloes in the Philippines and warrant reexamination of the role of bovines in transmission of S. japonicum to humans in the Philippines.

[A historical reflect on the disciplinary development of human parasitology].

The authors, in this paper, has briefly looked back the developmental history of human parasitology, which, as an independent discipline, was established in late 19th century and early 20th century. In the process, it underwent an early height of development, then met with setback and relative decline. Since 70s-80s of last century, the introduction and application of new theory of modern biology, especially advanced biotechniques to parasitology has led to a striking development in many fields of the discipline, leading to deeper understanding of parasite-host interplay as well as providing new ideals and tools for disease control. The authors also stressed that nowadays the discipline is still relatively isolated from the mainstream of modern biologic research and is still neglected by scientific community and medical education in the world, though it still is one of the major problems in public health, particularly in developing world including China. To argue the currently neglected situation of parasitology, especially in medical education, the authors emphasized the continuing requirements for the discipline and reflected on the developmental strategy of parasitology to meet the coming challenges and opportunities for further development.

Routine Kato-Katz technique underestimates the prevalence of Schistosoma japonicum: a case study in an endemic area of the People's Republic of China.

There is an evidence that the Kato-Katz technique lacks sensitivity and may hence be an unsuitable method for the assessment of the 'real infection status' in community with low-intensity infections. In this study, six Kato-Katz thick smears (examination of two stool samples with three thick smears each) were used as the diagnostic 'gold' standard for estimating the prevalence of Schistosoma japonicum infection and the results were compared with results based on fewer Kato-Katz thick smear readings. A total of 1055 individuals in 2005 and 725 in 2006 from an endemic village were recruited for the study. The observed prevalence increased gradually with the number of Kato-Katz thick smears examined, and hence the rate of underestimation decreased accordingly. The prevalence based on single Kato-Katz thick smear readings was significantly lower than that obtained using five or six thick smears. The rate of underestimation based on using two and three Kato-Katz thick smears, a typical diagnostic effort in the national schistosomiasis control programme, was about 36.0% (28.4-48.9%) and 25.0% (15.9-40.7%). The number of Kato-Katz thick smears required to secure detection of a S. japonicum infection varies with the infection intensity level. Indeed, examination of a single thick smear was sufficient when the geometric mean of the fecal content of eggs per gram (EPG) was 250 or higher in infected individuals, while six Kato-Katz thick smears were required when the EPG score was lower than 10. In conclusion, our results confirm that the prevalence of S. japonicum infection in a community is generally considerably "underestimated". Moreover, our findings provide a benchmark for the proper application of the Kato-Katz technique and the rational evaluation of the epidemic situation, as well as a scientific basis for constructing a mathematic diagnostic model.

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.

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.

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.

[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.

[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.