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.

[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.

[Study on immunologic function of thioredoxin glutathione reductase from Schistosoma japonicum].

OBJECTIVE: To study the immunogenicity and the immuno-protection of thioredoxin glutathione reductase from Schistosomajaponicum (SjTGR) against schistosome infection in mice. METHODS: Seventy-five mice were randomly divided into 5 groups, namely, blank group, PBS group, CpG2 immunized group, TGR immunized group and TGR + CpG2 co-immunized group. Each mouse was immunized for 3 times. The mice were tail bled before the first immunization and 2 weeks after the third immunization. The serum antibody levels of total IgG, IgG1 and IgG2a against SjTGR were assayed by ELISA. Two weeks after the third immunization, each mouse was infected with 40 ± 2 S. japonicum cercariae by abdominal skin penetration. Forty-two days later, all the mice were sacrificed to collect schistosome adult worms and liver eggs. The worm and egg reduction rates were calculated respectively. The single splenocyte of mouse was collected 2 weeks after the third immunization, and the expressions of CD44high, CD4+CD44high or CD8+CD44high on splenocytes of mice were examined by flow cytometry. After 72 h incubation with recombinant SjTGR, the levels of IL-2, IL-4, IL-10, and IFN-γ in the single-cell supernatant were determined by using ELISA kit. RESULTS: Two weeks after the third immunization, the titers of serum IgG against SjTGR in mice immunized with SjTGR and co-immunized with SjTGR and CpG2 were higher than 1:200 000. The IgG2a: IgG1 ratio (IgG2a/IgG1) increased slowly with time in both TGR immunized group and TGR + CpG2 co-immunized group. There were obviously higher levels of IFN-γ and IL-2 in the cell supernatant in the TGR immunized group and TGR + CpG2 co-immunized group compared to the blank, PBS and CpG2 groups (P < 0.05). The increased subpopulations of CD44high, CD8+CD44high and CD4+ CD44high cells in the splenocytes from mice immunized by SjTGR and co-immunized by SjTGR and CpG2 were found comparing to the blank, PBS and CpG2 groups (P < 0.05). The TGR immunization and TGR + CpG2 co- immunization caused 9.4% and 10.5% reductions in the number of adult worms and 9.2% and 32.8% reductions in the number of eggs, respectively. CONCLUSIONS: SjTGR displays strong immunogenicity inducing Th1 type immune response in mice. However, it could not produce protective efficacy against S. japonicum infection. CpG2 ODN may be a broadly effective Th1 adjuvant.

[Identification of early diagnostic molecules in soluble egg antigen of Schistosoma japonicum by MASS].

OBJECTIVE: To identify the molecules of soluble egg antigen (SEA) for early diagnosis of Schistosoma japonicum infection by two-dimensional electrophoresis (2-DE), immunoblotting and liquid chromatography with tandem mass spectrometry (LC-MS/MS). METHODS: The 2-DE of SEA was executed through first direction isoelectric focusing (IEF) in immobilized pH gradient gel 3-10 (IPG3-10) and second direction SDS-PAGE. The protein dots of SEA on the SDS-PAGE gel were transferred to nitrocellulose membrane. These nitrocellulose membranes were responded to the sera of healthy, sera of mice at 1 week, 2 weeks and 6 weeks post-infection respectively, then the membrane color was developed with the second antibody of HRP labeled goat anti -mouse IgG conjugate and substrate DAB. The protein dots recognized by sera of mice in the early stage of schistosome infection were identified by LC-MS/MS. RESULTS: After matching and analyzing the Western blot patterns of SEA responding to acute infection sera (1 week and 2 weeks post-infection), chronic infection sera (6 weeks post-infection) and healthy sera by PDQuest 1.0 software, two protein dots were found to be recognized by sera of mice at 1 week, 2 weeks and 6 weeks post-infection, and three protein dots were only recognized by the sera of mice at 6 weeks post-infection, no protein dot was recognized by healthy mouse sera. The data of LC-MS/MS showed that the two protein molecules recognized by the sera of mice with schistosome infection in the early stage were heat shock protein 70 (HSP70) and 78 kDa glucose-regulated protein (Grp78/Bip) respectively. CONCLUSION: The results of this study preliminarily indicate that HSP70 and Grp78 in SEA have early diagnostic value for S. japonicum infection.

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.

[Epitope identification of monoclonal antibody 5C6 against 14-3-3 protein of Schistosoma japonicum].

OBJECTIVE: To identify the epitope of monoclonal antibody (McAb) 5C6 against 14-3-3 protein of Schistosoma japonicum by phage display peptide library. METHODS: The phage display 12-peptide library was screened with purified McAb 5C6 against 14-3-3 protein of S. japonicum three rounds of bio-panning "adsorption-elution-amplification" to enrich the specific binding phages. The single phage clones selected randomly were amplified, their genomic DNA were extracted and sequenced. The immune response characterization of phages with the same or high homologous foreign inserted DNA sequence was identified by ELISA and Western blotting for further defining the epitope recognized by McAb 5C6. RESULTS: A total of 33 single phage clones were selected and sequenced. Among them, 25 shared the same foreign inserted DNA sequence of 5'-CCACCTAGTAGCAGACCGATTCTCAGTCGAAGGAAA-3', encoding a deduced peptide PPSSRPILSRRK. This peptide was not homologue to Sj14-3-3 protein or any other known native protein in the world. The results of Western blotting showed that this peptide could be recognized by the sera of patients with schistosomiasis, but not by those of healthy persons. CONCLUSION: The mimic antigen epitope of McAb 5C6 against 14-3-3 protein of S. japonicum, which is a conformational epitope, has been defined successfully in this study.

[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.

Monitoring specific antibody responses against the hydrophilic domain of the 23 kDa membrane protein of Schistosoma japonicum for early detection of infection in sentinel mice.

BACKGROUND: Schistosomiasis remains an important public health problem throughout tropical and subtropical countries. Humans are infected through contact with water contaminated with schistosome cercariae. Therefore, issuing early warnings on the risk of infection is an important preventive measure against schistosomiasis. Sentinel mice are used to monitor water body infestations, and identifying appropriate antibody responses to schistosome antigens for early detection of infection would help to improve the efficiency of this system. In this study we explored the potential of detecting antibodies to the hydrophilic domain (HD) of the 23-kDa membrane protein (Sj23HD) and soluble egg antigen (SEA) of Schistosome japonicum for early detection of schistosome infection in sentinel mice. RESULTS: Development of IgM and IgG antibody levels against Sj23HD and SEA in S. japonicum infected mice was evaluated over the course of 42 days post-infection by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The Sj23HD and SEA specific IgM and IgG levels in mice all increased gradually over the course of infection, but IgM and IgG antibodies against Sj23HD presented earlier than those against SEA. Furthermore, the rates of positive antibody responses against Sj23HD were higher than those against SEA in the early stage of schistosome infection, suggesting that the likelihood of detecting early infection using anti-Sj23HD responses would be higher than that with anti-SEA responses. The use of immunoblotting could further improve the early detection of schistosome infection due to its greater sensitivity and specificity compared to ELISA. Additionally, the levels of Sj23HD and SEA specific antibodies positively correlated with the load of cercariae challenge and the duration of schistosome infection. CONCLUSIONS: This study demonstrated that antibody responses to the Sj23HD antigen could be monitored for early detection of schistosome infection in mice, especially by immunoblotting which demonstrated greater sensitivity and specificity than ELISA for detection Sj23HD antibodies.

Detection of the circulating antigen 14-3-3 protein of Schistosoma japonicum by time-resolved fluoroimmunoassay in rabbits.

BACKGROUND: Schistosomiasis remains a major public health concern that afflicts millions of people worldwide. Low levels of Schistosoma infection require more sensitive diagnostic methods. In this study, a time-resolved fluoroimmunoassay (TRFIA) was developed for detecting the signal transduction protein 14-3-3, a circulating antigen of Schistosoma japonicum. RESULTS: The detection limit of 14-3-3-TRFIA was 0.78 ng/ml, with a linear measurement range from 0.78 to 800 ng/ml. The average intra-assay and inter-assay variability of this TRFIA was 8.9% and 12.2% respectively, and the mean recovery rate ranged from 92.1% to 115.5%. Within the first 21 days post-infection in rabbits, the positive rates of the 14-3-3-TRFIA were distinctly higher compared to ELISA. All these findings illustrate that 14-3-3-TRFIA has a higher detection efficacy and is a good early diagnostic method for active Schistosoma infection. CONCLUSIONS: A sandwich TRFIA for detecting the circulating antigen 14-3-3 of S. japonicum has been developed, and has demonstrated to be a good potential diagnostic method for schistosomiasis.

[Expression and characterization of recombinant Sj23HD-HSA fusion protein in Saccharomyces cerevisiae].

OBJECTIVES: To prepare the fusion protein of large hydrophilic domain of 23 kDa membrane protein of Schistosoma japonicum and the mature peptide of human serum albumin (Sj23HD-HSA) and investigate its immunoreactivity. METHODS: A fusion protein gene encoding Sj23HD-HSA fusion protein was prepared by overlapping PCR, which was confirmed by TA cloning and DNA sequencing. The fusion gene of Sj23HD-HSA was directionally subcloned into yeast expression plasmid pWX530 to construct a recombinant plasmid Sj23HD-HSA/pWX530. The transformants of Saccharomyces cerevisiae containing the recombinant plasmid Sj23HD-HSA/pWX530 were screened on leu deficient SD medium after yeast competent cells were transformed with recombinant plasmid. The excretive Sj23HD-HSA protein was expressed by culturing the yeast transformants at 30 degrees C for 1 week, and the protein component of culture supernatant was analyzed by SDS-PAGE. Sj23HD-HSA fusion protein was purified through Ion Exchange Chromatography. The immunoreactivity of recombinant Sj23HD-HSA fusion protein was determined by Western blotting with sera of schistosomiasis, clonorchiasis and healthy. RESULTS: The gene encoding the Sj23HD-HSA fusion protein was constructed successfully, which was confirmed by DNA sequencing. The yeast transformants containing plasmid Sj23HD-HSA/pWX530 could express the excretive Sj23HD-HSA fusion protein without inducing. The results of Western blotting indicated Sj23HD-HSA could be recognized by the sera of schistosomiasis, but could not be recognized by the sera of clonorchiasis and healthy respectively. CONCLUSIONS: Sj23HD-HSA fusion protein with good immune reactivity is prepared successfully, which will be a potential antigen for schistosomiasis immunodiagnosis.

Homocysteine modulates the proteolytic potential of human arterial smooth muscle cells through a reactive oxygen species dependant mechanism.

Pathological levels of homocysteine induce a dramatic degradation of arterial elastic structures. This severe metalloproteinase-dependant elastolysis affects elastic structures all over the media suggesting that smooth muscle cells (SMC) may participate to this process induced by homocysteine. Therefore, we investigated the effect of physiological (10 microM) and pathological (50, 100, and 500 microM) concentrations of homocysteine on the metalloproteinase-dependant proteolytic potential of human arterial SMC in culture. Pathological levels of homocysteine increased concomitantly the secretion of latent MMP-2 and TIMP-2 while the secretion of other elastolytic matrix metalloproteinases (MMPs) and expression of MT1-MMP were not altered. The increased secretion of latent MMP-2 induced by homocysteine was associated with an increased production of reactive oxygen species (ROS). Moreover, the increased secretion of latent MMP-2 induced by homocysteine was inhibited by antioxidant superoxide dismutase alone or in combination with catalase. These results suggest that SMC could participate, through an oxidative stress dependant secretion of elastolytic MMP-2, to the metalloproteinase-dependant degradation of arterial elastic structures induced by homocysteine.