The egg ribonuclease SjCP1412 accelerates liver fibrosis caused by Schistosoma japonicum infection involving damage-associated molecular patterns (DAMPs).

Schistosomiasis, a parasite infectious disease caused by Schistosoma japonicum, often leads to egg granuloma and fibrosis due to the inflammatory reaction triggered by egg antigens released in the host liver. This study focuses on the role of the egg antigens CP1412 protein of S. japonicum (SjCP1412) with RNase activity in promoting liver fibrosis. In this study, the recombinant egg ribonuclease SjCP1412, which had RNase activity, was successfully prepared. By analysing the serum of the population, it has been proven that the anti-SjCP1412 IgG in the serum of patients with advanced schistosomiasis was moderately correlated with liver fibrosis, and SjCP1412 may be an important antigen associated with liver fibrosis in schistosomiasis. In vitro, the rSjCP1412 protein induced the human liver cancer cell line Hep G2 and liver sinusoidal endothelial cells apoptosis and necrosis and the release of proinflammatory damage-associated molecular patterns (DAMPs). In mice infected with schistosomes, rSjCP1412 immunization or antibody neutralization of SjCP1412 activity significantly reduced cell apoptosis and necroptosis in liver tissue, thereby reducing inflammation and liver fibrosis. In summary, the SjCP1412 protein plays a crucial role in promoting liver fibrosis during schistosomiasis through mediating the liver cells apoptosis and necroptosis to release DAMPs inducing an inflammatory reaction. Blocking SjCP1412 activity could inhibit its proapoptotic and necrotic effects and alleviate hepatic fibrosis. These findings suggest that SjCP1412 may be served as a promising drug target for managing liver fibrosis in schistosomiasis japonica.

Blocking BAFF Alleviates Hepatic Fibrosis in Schistosoma japonicum-Infected Mice.

Schistosomiasis is an immunopathogenic disease characterized by egg granuloma and fibrosis. The hepatic fibrosis of schistosomiasis is caused by the coordinated action of local immune cells, liver-resident cells and related cytokines around the eggs of the liver. B-cell-activating factor (BAFF), expressed in many cells, is an essential factor for promoting the survival, differentiation, and maturation of cells. The overexpression of BAFF is closely related to many autoimmune diseases and fibrosis, but has not been reported to play a role in liver fibrosis caused by schistosomiasis. In the study, we found that, during Schistosoma japonicum (S. japonicum) infection in mice, the level of BAFF and its receptor BAFF-R progressively increased, then decreased with the extension of infection time, which was consistent with the progression of hepatic granuloma and fibrosis. Anti-BAFF treatment attenuated the histopathological damage in the liver of infected mice. The average areas of individual granulomas and liver fibrosis in anti-BAFF treatment mice were significantly lower than those in control mice, respectively. Anti-BAFF treatment increased the IL-10, decreased IL-4, IL-6, IL-17A, TGF-ß, and downregulated the antibody level against S. japonicum antigens. These results suggested that BAFF acts a strong player in the immunopathology of schistosomiasis. Anti-BAFF treatment may influence Th2 and Th17 responses, and reduce the inflammatory reaction and fibrosis of schistosomiasis liver egg granuloma. It is suggested that BAFF might be a prospective target for the development of new methods to treat schistosomiasis liver fibrosis.

Deficiency of PKCλ/ι alleviates the liver pathologic impairment of Schistosoma japonicum infection by thwarting Th2 response.

BACKGROUND: The activation of immune response driven by the eggs of Schistosoma japonicum and the subsequent secretions is the culprit behind granulomatous inflammation and liver fibrosis. Evidence suggests that PKCλ/ι participates in a variety of physiological and pathological processes, including the regulation of metabolism, growth, proliferation and differentiation of cells. However, the role of PKCλ/ι in liver disease caused by Schistosoma japonicum remains unclear. METHODS: In the present study, we observe the pathological changes of egg-induced granulomatous inflammation and fibrosis in the liver of mice infected by Schistosoma japonicum by using conditional PKCλ/ι-knockout mice and wild-type control. Immune cytokines and fibrogenic factors were analyzed by performing flow cytometry and real-time fluorescence quantitative PCR. RESULTS: The results of H&E and Masson staining show that the degree of granulomatous lesions and fibrosis in the liver of the infected PKCλ/ι-knockout mice was significantly reduced compared with those of the infected wild-type mice. The mean area of single granuloma and hepatic fibrosis in the PKCλ/ι-knockout mice was significantly lower than that of the wild-type mice (85,295.10 ± 5399.30 µm2 vs. 1,433,702.04 ± 16,294.01 µm2, P < 0.001; 93,778.20 ± 8949.05 µm2 vs. 163,103.01 ± 11,103.20 µm2, P < 0.001), respectively. Serological analysis showed that the ALT content was significantly reduced in the infected knockout mice compared with infected wild-type mice. RT-PCR analysis showed that IL-4 content in knockout mice was significantly increased after Schistosoma japonicum infection, yet the increase was less than that in infected wild-type mice (P < 0.05). PKCλ/ι deficiency led to reduced expression of fibrosis-related factors, including TGF-ß1, Col-1, Col-3, α-SMA and liver DAMP factor HMGB1. Flow cytometry analysis showed that the increasing percentage of Th2 cells, which mainly secrete IL-4 cytokines in spleen cells, was significantly lower in PKCλ/ι-deficient mice compared with wild-type mice after infection (P < 0.05). CONCLUSIONS: Our data demonstrate that PKCλ/ι deficiency alleviating granulomatous inflammation and fibrosis in the liver of mice with S. japonicum infection by downregulating Th2 immune response is the potential molecular mechanism behind the role of PKCλ/ι in schistosomiasis.

Thymosin Alpha-1 Inhibits Complete Freund's Adjuvant-Induced Pain and Production of Microglia-Mediated Pro-inflammatory Cytokines in Spinal Cord.

Activation of inflammatory responses regulates the transmission of pain pathways through an integrated network in the peripheral and central nervous systems. The immunopotentiator thymosin alpha-1 (Tα1) has recently been reported to have anti-inflammatory and neuroprotective functions in rodents. However, how Tα1 affects inflammatory pain remains unclear. In the present study, intraperitoneal injection of Tα1 attenuated complete Freund's adjuvant (CFA)-induced pain hypersensitivity, and decreased the up-regulation of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in inflamed skin and the spinal cord. We found that CFA-induced peripheral inflammation evoked strong microglial activation, but the effect was reversed by Tα1. Notably, Tα1 reversed the CFA-induced up-regulation of vesicular glutamate transporter (VGLUT) and down-regulated the vesicular γ-aminobutyric acid transporter (VGAT) in the spinal cord. Taken together, these results suggest that Tα1 plays a therapeutic role in inflammatory pain and in the modulation of microglia-induced pro-inflammatory cytokine production in addition to mediation of VGLUT and VGAT expression in the spinal cord.

Identification of Peptide Antagonists to Thioredoxin Glutathione Reductase of Schistosoma japonicum.

Schistosomiasis is one of the world's major public health problems. Praziquantel is currently the only effective drug against schistosomiasis. As resistance of praziquantel has emerged in some endemic areas, development of new antischistosomal agents should be a high priority. In this study, a phage display peptide library was used for screening for peptide antagonists of thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR), which has been identified as an alternative drug target. Three rounds of panning produced four different fusion phages. ELISA proved that all four phages could bind to SjTGR. One peptide, JIPDys1 (aa, WPHNWWPHFKVK), reduced enzyme activity of SjTGR by more than 50%. 2 µM of the synthesized peptide of JIPDys1 inhibited the activity of TrxR, GR, and Grx of SjTGR by 32.5%, 100%, and 100%, respectively. The IC50 values of the synthetic peptide JIPDys1 for TrxR, GR, and Grx were 3.67 µM, 0.11 µM, and 0.97 µM, respectively. Based on computer simulation, it appeared that JIPDys1 binds to the substrate binding sites of glutathione reductase (GR) and glutaredoxin (Grx). Our data show that the peptide, JIPDys1 (aa, WPHNWWPHFKVK), is a promising candidate to develop novel drugs against S. japonicum which acts by binding with SjTGR and reduces enzyme activity of SjTGR.

Characterization of Schistosoma japonicum CP1412 protein as a novel member of the ribonuclease T2 molecule family with immune regulatory function.

BACKGROUND: Schistosome infection typically induces a polarized Th2 type host immune response. As egg antigen molecules play key roles in this immunoregulatory process, clarifying their functions in schistosomiasis would facilitate the development of vaccine and immunotherapeutic methods. Schistosoma japonicum (Sj) CP1412 (GenBank: AY57074.1) has been identified as a new member of the RNase T2 family with immune regulatory functions. METHODS: The expression plasmid Sj CP1412-pET28a was constructed and transformed into bacteria for production of recombinant Sj CP1412 protein (rSj CP1412) via IPTG induction. The RNase activity of Sj CP1412 was predicted by bioinformatic analysis and confirmed by digesting the yeast tRNA with rSj CP1412.C57BL/6j mice were immunized with rSj CP1412, and its immune regulatory effects in vivo and in vitro were investigated. Meanwhile, the relationship between the RNase activity of Sj CP1412 and its immune regulation was observed. RESULTS: Sj CP1412 was confirmed as a novel RNase T2 family protein with RNase activity. Immunoblotting and RT-PCR analyses demonstrated Sj CP1412 as a protein exclusively secreted/excreted from eggs, but not cercariae and adult worms. Stimulating RAW264.7 macrophages with rSj CP1412 raised the expression of CD206, Arg-1 and IL-10, which are related to M2 type macrophage differentiation. Stimulating dendritic cells (DCs) with rSjCP1412 failed to induce their maturation, and the recombinant protein also inhibited LPS-stimulated DC maturation. Depletion of Sj CP1412 from soluble egg antigen (SEA) impaired the ability of SEA to induce M2 type polarization of RAW264.7 macrophages. Immunizing mice with rSj CP1412 induced high antibody titers, increased serum IL-4 and TGF-ß levels and splenic CD4 + CD25 + Foxp3 + T cells, downregulated serum IFN-γ levels and alleviated the egg granuloma pathology of schistosome infection. In vitro stimulation by rSj CP1412 significantly increased CD4 + CD25 + Foxp3 + T cell numbers in splenocytes of healthy mice. The rSj CP1412 protein with RNase activity inactivated by DEPC failed to induce M2 surface marker CD206 expression in RAW264.7 macrophages. CONCLUSIONS: The Sj CP1412 protein expressed specifically in S. japonicum eggs is a novel member of the RNase T2 family. Similar to Omega-1 of Schistosoma mansoni, the Sj CP1412 protein drives polarization of the host Th2 immune response, which is dependent on its RNase activity. These data provide new evidence towards understanding the immune regulatory role of RNase T2 family proteins during schistosome infection.

Oxadiazole-2-oxides may have other functional targets, in addition to SjTGR, through which they cause mortality in Schistosoma japonicum.

BACKGROUND: Schistosomiasis is one of the world's major public health problems. Besides praziquantel (PZQ), there is currently no other effective treatment against schistosomiasis. The development of new antischistosomal agents to curb the emergence of PZQ resistance should be a high priority. Oxadiazole-2-oxides have been identified as potential antischistosomal reagents, with thioredoxin glutathione reductase (TGR) being one of their molecular targets. METHODS: To develop novel treatment reagents against Schistosoma japonicum, 30 novel oxadiazole-2-oxides were synthesised and their antischistosomal activities on juvenile and adult S. japonicum were evaluated in vitro and in vivo. Their inhibitory activities against S. japonicum thioredoxin glutathione reductase (SjTGR) were also analysed. RESULTS: Most of the oxadiazole-2-oxides showed good juvenile and adult S. japonica killing activities in vitro. However, the antischistosomal effects of these compounds were not positively correlated with either their inhibition of SjTGR, or with nitric oxide (NO) release. Compounds 4a, 4b, 7c, 13, 16 and 20 resulted in 87.7%, 83.1%, 87.1%, 84.6%, 90.8% and 69.5%, respectively, mortality in the adult worms, when used to treat infected mice at schistosomula stage. These mortality rates were similar to or higher than that of artemisinin. Furthermore, compounds 4a and 16 resulted in 66.7% and 69.4% reductions in the worm burdens, respectively, when infected mice were treated at the adult worm stage. These treatment effects were similar to PZQ. No differences in activity of the oxadiazole-2-oxides against female and male adult worms were observed. The toxicity of the oxadiazole-2-oxides on mammalian cells appeared to be similar to, or less than, that of PZQ. CONCLUSIONS: The antischistosomal activity of the oxadiazole-2-oxides does not depend on NO production or the inhibition of SjTGR activity. There may be other functional targets of the oxadiazole-2-oxides in S. japonicum. Several of the novel oxadiazole-2-oxides synthesised in this study could be used to develop novel antischistosomal drugs and explore potential molecular targets.

SjTat-TPI facilitates adaptive T-cell responses and reduces hepatic pathology during Schistosoma japonicum infection in BALB/c mice.

BACKGROUND: Schistosomiasis is a kind of parasitic zoonoses which causes serious damage to public health and social development. China is one of the countries most affected by Schistosoma japonicum and an effective vaccine is still needed. In this study, we adopted Tat-mediated protein transduction technology to investigate the impact of different antigen presented approaches on host's immune response and the potential protection against Schistosoma japonicum infection. RESULTS: We successfully constructed the recombinant S. japonicum triosephosphate isomerase, Tat-TPI, as a vaccine candidate. Whether injected with Tat-TPI in foot pad or vaccinated with Tat-TPI in the back subcutaneously for three times, the draining popliteal lymph nodes and spleen both developed a stronger CD8(+)T response (Tc1) in mice. Not only that, but it also helped CD4(+)T cells to produce more IFN-γ than TPI immunisation. In addition, it could boost IgG production, especially IgG1 subclass. Most importantly, Tat-TPI immunisation led to the significant smaller area of a single egg granuloma in the livers as compared with TPI-vaccinated or control groups. However, the anti-infection efficiency induced by Tat-TPI was still restricted. CONCLUSION: This study indicated that immunisation with Tat-fused TPI could contribute to enhance CD4(+)T-cell response and decrease hepatic egg granulomatous area after S. japonicum infection though it did not achieve our expected protection against Schistosoma japonicum infection. The optimal vaccine strategy warrants further research.

[Establishment and diagnostic performance of biotin-avidin complex enzyme linked immunosorbent assay of detecting specific IgG4 of clonorchiasis].

OBJECTIVE: To explore the performance of the biotin-avidin complex enzyme linked immunosorbent assay of detecting specific IgG4 for the diagnosis of clonorchiasis. METHODS: The avidin-biotin complex enzyme linked immunosorbent assay of detecting specific IgG4 (IgG4-ABC-ELISA)against Clonorchis sinensis was established, and used to detect the serum samples of patients with clonorchiosis sinensis, schistosomiasis japonica, paragonimiasis, toxoplasmosis, echinococcosis, cysticercosis and sparganosis mansoni. At the same time, these sera were analyzed by the ELISA of detecting IgG4 (IgG4-ELISA) and ELISA of detecting the total IgG (IgG-ELISA) as controls. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the respective diagnostic performance of the three methods were compared. RESULTS: The IgG4-ABC-ELISA for diagnosis of clonorchiasis was established successfully. The sensitivity and specificity of the IgG4-ABC-ELISA for detecting clonorchiasis were 90.0% and 98.2% respectively, and PPV and NPV were 93.8% and 97.0% respectively. Its diagnostic performance was 96.3%. The sensitivity and specificity of the IgG4-ELISA for detecting clonorchiasis were 86.0% and 98.2% respectively, and PPV and NPV were 93.5% and 95.9% respectively. Its diagnostic performance was 95.4%. The sensitivity and specificity of the IgG-ELISA for detecting clonorchiasis were 94.0% and 88.1% respectively, and PPV and NPV were 70.1% and 98.0% respectively. Its diagnostic performance was 89.4%. The sensitivity of IgG4-ABC-ELISA was higher than that of IgG4-ELISA (P < 0.05), and the specificity of IgG4-ABC-ELISA was higher than that of IgG-ELISA (P < 0.05). CONCLUSIONS: IgG4-ABC-ELISA of detecting specific antibody IgG4 against Clonorchis sinensis has high sensitivity and specificity. Therefore, it has a good application value in the diagnosis of clonorchiasis.

Pilot Study on Interferon-γ-producing T Cell Subsets after the Protective Vaccination with Radiation-attenuated Cercaria of Schistosoma japonicum in the Miniature Pig Model.

CLAWN miniature pig has been shown to serve as a suitable host for the experimental infection of Schistosoma japonicum. In this study, we found that radiation-attenuated cercaria (RAC) vaccine gave CLAWN miniature pigs protective immunity against subsequent challenge infection with S. japonicum cercaria. To characterize the protective immune response of the pig model vaccinated by attenuated cercaria, flow cytometric analysis of the reactive T cell subsets was performed. The intracellular interferon (IFN)-γ and the cell surface markers revealed the peripheral blood CD3+ T-lymphocytes produced significant amounts of IFN-γ during the immunization period and after the challenge infection. CD4+ αß-T cells as well as CD4+/CD8α(mid) double positive and/or CD8α(high) αß-T cells were the major IFN-γ-producing CD3+ T cells. On the contrary, γδ T cells did not produce intracellular IFN-γ. Our results suggested that RAC-vaccinated miniature pigs showed effective protective immunity through the activation of αß T cells bearing antigen specific T-cell receptors but not through the activation of γδ T cells.

Synthesis and SAR studies of praziquantel derivatives with activity against Schistosoma japonicum.

The synthesis and structure-activity relationship (SAR) studies of praziquantel derivatives with activity against adult Schistosoma japonicum are described. Several of them showed better worm killing activity than praziquantel and could serve as leads for further optimization.

Characterization of IgG responses of rabbits to Sj14-3-3 protein after experimental infection with Schistosoma japonicum.

An indirect enzyme-linked immunosorbent assay method was developed for detection of IgG against 14-3-3 protein in sera of rabbits. Rabbits infected with 500 cercariae of Schistosoma japonicum were grouped and the characterization of the IgG responses was observed. For the treated group, the IgG could be detected as early as 2-4 weeks post-infection and then their levels rose rapidly and reached a peak at around 6 weeks. After the infected rabbits were treated with praziquantel at 6 weeks post-infection, IgG levels in the sera significantly decreased. While in the untreated group, the IgG levels were constantly very low. For all infected rabbits, 60 % (six of ten) had positive reaction with 14-3-3 protein, and 40 % (four of ten) had high IgG levels. This finding would be more helpful to understand this 14-3-3 protein.

[Progress in research on techniques for prolonging half-life of protein and polypeptide drugs].

Following the advancement in molecular biology and recombinant technology, the protein and polypeptide therapeutics have become an important class of clinical drugs due to their unique superiority. However, the problems of stability and short half-life of proteins and peptides also vastly limit their further use in clinic. The focal points of this paper are to introduce the specific ways and the matters needing attention to increase the biological half-life of the therapeutic protein and polypeptide drugs.

[Expression and characterization of thioredoxin glutathione reductase of Schistosoma japonicum].

OBJECTIVE: To prepare the recombinant thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR) with biological activity. METHODS: The open reading frame DNA sequence of SjTGR was fused with a bacterial-type selenosysteine insertion sequence (SECIS) element by PCR to form a chimeric gene. The chimeric gene was subcloned into expression plasmid pET-41a to construct a recombinant plasmid SjTGR-pET-41a. Then the recombinant plasmid SjTGR-pET-41a was co-transformed into E. coli BL21 with plasmid pSU ABC. The SjTGR protein was expressed by inducing with IPTG. The recombinant SjTGR was purified from expression products by affinity chromatography with an adenosine 2', 5'- diphosphate agarose column. The polyclonal anti-serum against recombinant SjTGR was obtained by immunizing mice with purified SjTGR. The native TGR in S. japonicum was evidenced by using Western blotting. Thiorendoxin reductase (TrxR) activity, glutathione reductase (GR) activity and gluaredoxin (Grx) activity of recombinant TGR were analyzed according to the biochemical method. RESULTS: The chimeric gene of SjTGR with a bacterial-type selenosysteine insertion sequence (SECIS) element was constructed successfully. The bacteria containing the recombinant plasmid SjTGR-pET-41a could express the soluble SjTGR by inducing with IPTG at static growth stage for 24 h at 24 degrees C. The expressed products of plasmid pSU ABC could promote the integration of selenocysteine and increase the yield of selenoprotein. The result of Western blotting showed that the polyclonal antiserum against recombinant SjTGR could recognize the native TGR in S. japonicum adult worms. The enzymatic assay indicated that SjTGR was a multifunctional enzyme with activities of TrxR, GR and Grx. CONCLUSION: The recombinant SjTGR with biological activity is expressed successfully, which lays the foundation for further study on the function and applied values of SjTGR.

Application of DNA-based diagnostics in detection of schistosomal DNA in early infection and after drug treatment.

BACKGROUND: Research is now focused on identification of sensitive and specific diagnostic tests for early identification of schistosomal infection and evaluation of chemotherapy in field situations in China. RESULTS: This study compared loop-mediated isothermal amplification (LAMP) with conventional PCR as DNA-based diagnostic techniques for the early detection of schistosomal DNA and the evaluation of chemotherapy. The results showed that both PCR and LAMP assays targeting a 301 base pair (bp) sequence of the highly repetitive retrotransposon, SjR2, amplified DNA from schistosomes but were unable to distinguish between schistosome species. LAMP and conventional PCR were shown to amplify the target sequence of the SjR2-pCR2.1 recombinant plasmid template with limits of detection of 10⁻4 ng and 10⁻² ng, respectively, thus demonstrating the superior sensitivity of the LAMP method. Schistosoma japonicum DNA was detected in all serum samples obtained from the three experimental groups at 1 week post-infection by LAMP assay, while the rate of detection by conventional PCR ranged from 50% to 66%. The potential application of PCR and LAMP assays for the evaluation of artesunate and praziquantel chemotherapy was investigated. PCR was shown to be less sensitive for detection of schistosomal DNA in drug-treated rabbit sera than the LAMP method. CONCLUSIONS: The data presented here indicate that LAMP is suitable for the detection of early infection in the groups primarily infected with Schistosoma japonicum, such as migrants, travellers, military personnel and the younger age groups. However, it is less suitable for evaluation of the efficacy of chemotherapy in the early stages because of its high sensitivity.

Effect of praziquantel prolonged administration on granuloma formation around Schistosoma japonicum eggs in lung of sensitized mice.

Schistosomiasis remains a major public health problem and it is an immune disease. The schistosome egg is the primary parasite factor responsible for the overt disease. The eggs release the soluble antigen, which induces intensive tissue reaction, a granulomatous reaction to the eggs. If granuloma formation could be suppressed, overt disease might not develop. Praziquantel is an effective antischistosomal drug especially for adult worms. However, whether praziquantel has a suppressing effect on granuloma formation around schistosome eggs directly remains unclear. The purpose of the present study was to investigate the effect of praziquantel, especially administered persistently, on granuloma formation around Schistosoma japonicum eggs in the lung of sensitized mice. Thirty-six mice were divided into three groups averagely. Group A was a control group. First, the mice were injected with schistosomal eggs hypodermically in abdomen, and 10 days later injected with schistosomal eggs intravenously via a tail vein. Group B was a praziquantel short administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 300 mg/kg for 3 days, from 1 day before the intravenous injection of the eggs. Group C was a praziquantel prolonged administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 150 mg/kg for 5 days weekly until the mice were sacrificed. Three mice of each group were sacrificed on days 7, 14, 28, and 56, respectively after the intravenous injection of the eggs, and the lung tissues were fixed with formalin and the slices were HE stained. The granulomas containing eggs in their centers were selected, and 25-30 granulomas from the animals of each group were measured at each time period. The mean areas of egg granulomas of each group were calculated, and the neutrophilic granulocytes, eosinocytes, lymphocytes, fibroblasts, and macrophages within the egg granulomas were counted. The mean numbers of them of each group were calculated. All the data of each group were analyzed and compared statistically. On day 56 after the intravenous injection of the eggs, the mean area of schistosomal egg granulomas in group B was (227.4 ± 728.0) × 10(3) µm(2), less than that of [(297.9 ± 153.3) × 10(3) µm(2)] in group A, and the suppression rate was 23.7% (P < 0.05). On days 7, 14, 28, and 56, the mean areas of schistosomal egg granulomas in group C were (575.8 ± 155.6) × 10(3) µm(2), (310.5 ± 854.0) × 10(3) µm(2), (267.7 ± 513.3) × 10(3) µm(2), and (214.9 ± 446.4) × 10(3) µm(2), respectively, significantly less than those of [(692.7 ± 232.6) × 10(3) µm(2), (439.4 ± 165.0) × 10(3) µm(2), (385.7 ± 129.3) × 10(3) µm(2), and (297.9 ± 153.3) × 10(3) µm(2)] in group A. The suppression rates were 16.9%, 29.3%, 30.6%, and 27.9%, respectively (P values <0.05). On day 56, the mean numbers of neutrophilic granulocytes were 11.4 ± 5.0 in group A and 5.2 ± 3.1 in group C, respectively, with the suppression rate of 54.4% in group C (P < 0.05). On day 56, the mean numbers of eosinocytes within the egg granulomas were 2.3 ± 2.0, 0.1 ± 0.3, and 0.3 ± 0.6 in groups A, B, and C, respectively, with the suppression rate of 95.7% in group B and 87.0% in group C (P values <0.05). On day 56, the mean numbers of macrophages within egg granulomas were 14.3 ± 6.9 in group C, compared with 18.6 ± 8.2 in group A, the suppression rate was 23.1% (P < 0.05). On day 56, the mean numbers of fibroblasts within the egg granulomas were 6.6 ± 4.4 and 5.8 ± 2.6 in groups B and C, respectively, and compared with 14.3 ± 7.8 in group A, the increasing extents decreased by 53.8% and 59.4%, respectively (P values <0.05). Therefore, the administration of praziquantel, especially the prolonged administration, can suppress the formation of schistosomal egg granulomas, including reduction in the areas of granulomas and suppression of the inflammatory cells and the hyperplasia of fibroblasts within granulomas.

Kinetics of circulating antigen 14-3-3 in sera of rabbits firstly infected with Schistosoma japonicum and treated with/without praziquantel.

A sandwich ELISA was developed for the detection of circulating antigen 14-3-3 in the sera of rabbits. Rabbits that were infected with 500 cercariae of Schistosoma japonicum were grouped and the kinetics of 14-3-3 was observed. For the treated group, the 14-3-3 protein could be detected as early as 2-4 weeks postinfection and then its levels rose rapidly and reached a peak at around 6 weeks. The 14-3-3 levels in the sera significantly decreased after the infected rabbits were treated with praziquantel at 6 weeks postinfection and declined to the initial level about 8 weeks posttreatment. While in the untreated group, 14-3-3 levels reached a peak in 8 weeks postinfection and then remained at plateau level for about 6 weeks. Our findings showed that detection of S. japonicum 14-3-3 has an important value for diagnosis of acute infection of S. japonicum and evaluation of chemotherapy.

Proteome approach for identification of schistosomiasis japonica vaccine candidate antigen.

Experimental vaccination with radiation-attenuated cercariae (RAC) confers possible practical levels of resistance to challenge infection by humoral and by cellular mechanism. Here, we aimed to identify possible vaccine antigens by using specific IgG antibody from RAC vaccinated miniature pig. Two milligrams of soluble egg antigen (SEA) or schistosomal worm antigen preparation (SWAP) was fractionated using two dimensional liquid chromatography (proteome PF 2D) consisted of high performance chromatofocusing (HPCF) and high resolution reversed phase chromatography (HPRP). Of the 42 HPCF fractions of SEA or SWAP, 26 (61.9%) or 15 (35.7%) showed positive dot blot reaction with RAC vaccinated serum respectively. The dot blot positive fractions were applied to the second HPRP column. One hundred and seven out of 26 x 96 of SEA fractions and 18 out of 15 x 96 SWAP fractions reacted with RAC vaccinated serum. From the positive fractions we chose 17 of SEA and 10 of SWAP that had no reactivity with normal cercariae infected (NCI) sera and had single peak of 214 nm; and automated N-terminal amino acid sequence based on in situ Edman Reaction was conducted. Four sequences were obtained and applied to the homology search in NCBI database. A total of eight candidate genes were listed up and their cDNA clones from schistosomula stage were obtained. Two of the recombinant proteins (AAW27472.1 and AXX25883.1) showed strong reactivity with the RAC vaccinated serum but marginal with NCI serum. This protocol using proteome PF 2D could be applicable in identifying immunoreactive proteins from crude extract for the development of vaccines or for diagnostics.

Inactivation of a two-component signal transduction system, SaeRS, eliminates adherence and attenuates virulence of Staphylococcus aureus.

Staphylococcus aureus is a major human and animal pathogen. During infection, this organism not only is able to attach to and enter host cells by using its cell surface-associated factors but also exports toxins to induce apoptosis and kill invaded cells. In this study, we identified the regulon of a two-component signal transduction system, SaeRS, and demonstrated that the SaeRS system is required for S. aureus to cause infection both in vitro and in vivo. Using microarray and real-time reverse transcriptase PCR analyses, we found that SaeRS regulates the expression of genes involved in adhesion and invasion (such as those encoding fibronectin-binding proteins and fibrinogen-binding proteins) and genes encoding alpha-, beta-, and gamma-hemolysins. Surprisingly, we found that SaeRS represses the Agr regulatory system since the mutation of saeS up-regulates agrA expression, which was confirmed by using an agr promoter-reporter fusion system. More importantly, we demonstrated that inactivation of the SaeRS system significantly decreases the bacterium-induced apoptosis and/or death of lung epithelial cells (A549) and attenuates virulence in a murine infection model. Moreover, we found that inactivation of the SaeRS system eliminates staphylococcal adhesion and internalization of lung epithelial cells. We also found that both a novel hypothetical protein (the SA1000 protein) and a bifunctional protein (Efb), which binds to extracellular fibrinogen and complement factor C3, might partially contribute to bacterial adhesion to and invasion of epithelial cells. Our results indicate that activation of the SaeRS system may be required for S. aureus to adhere to and invade epithelial cells.

[Fusion expression and antigenicity analysis of miracidial antigen from eggs of Schistosoma japonicum].

OBJECTIVES: To express the miracidial antigen from eggs of Schistosoma japonicum (Chinese mainland strain) (SjMP10), and investigate the role of the miracidial antigen during the hepatic granuloma formation of schistosomiasis. METHODS: A pair of specific primers was designed and synthesized according to the nucleotide sequence of the open reading frame of the miracidial antigen gene. The open reading frame of the miracidial antigen gene was amplified, digested by restrictive enzyme (BamHI, SolI), and cloned directly into the expression plasmid pGEX-4T-3 to construct the recombinant plasmid. The recombinant plasmids were transformed into E. coli BL21(DE3), and induced by IPTG to express the fusion protein of GST-SjMP10. The expressed fusion protein was purified by electric elution method, and its antigenicity was examined by Western blotting and lymphocyte proliferation test. RESULTS: The gene of miracidial antigen was cloned into the expression plasmid pGEX-4T-3. After induced by IPTG, the recombinant expressed a fusion protein of GST-SjMP10, with a molecular weight of 39 000 approximately. The purified fusion protein showed proper antigenicity that could be recognized by the sera of rabbits heavily infected by Schistosoma japonicum and could stimulate the proliferation of splenic lymphocytes of infected BALB/c mice. CONCLUSION: The miracidial antigen from eggs of Schistosoma japonicum was expressed successfully.