[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.

[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.

Pulmonary cryptococcosis presenting as wandering multiple bilateral shadows and hilar and mediastinal lymph node enlargement in the lungs.

We present a case of pulmonary cryptococcosis presenting as wandering multiple bilateral shadows and hilar and mediastinal lymph node enlargement in which the fluconazole treatment suppressed the symptoms. This case illustrates the complex nature of immunological responses in the lungs and highlights the need to consider the existence of cryptococcal allergies.

[Expression of DNMT gene in bone marrow of patients with acute myelogenous leukemia and its significance].

This study was aimed to explore the expression and significance of DNMT1 gene in bone marrow of patients with acute myelogenous leukemia (AML). The expression of DNMT1 gene was detected by real-time PCR in 30 healthy people and 126 AML patients. The results showed that the expression level of DNMT1 gene was lower in the 30 healthy people and was higher in AML patients. There was a marked decline in the expression level of DNMT1 gene after complete remission (CR) as compared with the initial treatment. The expression level of DNMT1 gene did not correlated with age, sex and the clinical characteristics at initial diagnosis such as white blood cell count and chromosomal karyotype in AML patients. The CR rate in AML patients with low expression level of DNMT1 gene was lower than that in those with high expression level. It is concluded that bone marrow DNMT1 gene level may play an important role in AML pathogenesis and can serve as an index in evaluating AML prognosis.

[beta-elemene enhances aclarubicin-induced apoptotic effect in HL-60 cells and its mechanism.].

OBJECTIVE: To explore the effects of beta-elemene combined with aclarubicin on the induction of HL-60 cell apoptosis and its mechanisms in antileukemia therapy. METHODS: HL-60 cells were treated for 20 hours with different dose of aclarubicin (0.05, 0.10, 0.25 microg/ml) or with different concentrations of beta-elemene (10, 20, 40 microg/ml) in the presence or absence of aclarubicin (0.10 microg/m). The apoptotic rate was analyzed by flow cytometry (FCM), the productions of PGE2 in culture supernatants was detected by competitive ELISA and the expressions of COX-2 and NF-kappaB activity in HL-60 cells by Western blot. RESULTS: Lower concentration of aclarubicin (0.05, 0.10 microg/ml) didn't affect apoptotic rate, and COX-2, NF-kappa B and PGE2 expression on HL-60 cells. Combined treatment of beta-elemene and aclarubicin (0.10 microg/ml) enhanced the apoptotic effect and down-regulated COX-2, NF-kappaB and PGE2 expressions. There was a positive correlation between the effects and beta-elemene concentrations. CONCLUSION: beta-elemene enhances aclarubicin-mediated apoptotic effect, down-regulation of COX-2 and their inducing products PGE2 in HL-60 cells by suppressing activitation of NF-kappaB.

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.

Gene expression profile of CD4+ T cells reveals an interferon signaling suppression associated with progression of experimental Schistosoma japonicum infection.

To understand the natural history of immune responses centering CD4+ T cells at genetic level during experimental infection with Schistosoma japonicum (S. japonicum), the mRNA profiles of CD4+ T cells from spleens of mice at 0, 3, 6, and 13 weeks after the onset of the infection, were compared using mouse oliogonucleotide microarrays (Affymetrix GeneChip U74A). Of about 12,000 mouse probe sets in a microarray, nearly 10% encoded a variety of immune regulators, including many cytokine and chemokine genes, immunoglobulin-related genes, and genes related to apoptosis and the stress response. These changed in transcript representation as the schistosome infection progressed, and a key finding, which was validated by semi-quantitative PCR, was that a significant portion of the genes which were down-regulated as infection progressed coded for interferon (IFN)-inducible molecules, including GTPases, transcription factors and chemokines. The results thus showed that there is a characteristic change in IFN-inducible gene expression over the course of the schistosome infection, and it is suggested that the IFN-gamma-regulated GTPase family may be involved in IFN-mediated resistance against S. japonicum.