The Differential and Dynamic Progression of Hepatic Inflammation and Immune Responses During Liver Fibrosis Induced by Schistosoma japonicum or Carbon Tetrachloride in Mice.

Liver fibrosis can result from various causes and could progress to cirrhosis and cancer; however, there are no effective treatments due to that its molecular mechanism is unclear. liver fibrosis model made by Schistosoma japonicum (S. japonicum) infection or Carbon tetrachloride (CCl4) intraperitoneal injection is a conventional model used in liver fibrosis-related studies for mechanism or pharmaceutical research purposes. But the differences in the pathological progression, immune responses and the underlying mechanism between the two liver fibrosis model have not been carefully compared and characterized, which hinders us from correctly understanding and making better use of the two models. In the present study, the pathological changes to the liver, and the cytokines, inflammatory factors, macrophages, and lymphocytes subsets involved were analyzed in the liver fibrosis model of S. japonicum infection or CCl4 intraperitoneal injection. Additionally, the pathological progression, immune responses and the underlying injury mechanism in these two models were compared and characterized. The results showed that the changing trend of interleukin-13 (IL-13), transforming growth factor beta (TGF-ß), inflammatory factors, and M1, M2 macrophages, were consistent with the development trend of fibrosis regardless of whether liver fibrosis was caused by S. japonicum or CCl4. For lymphocyte subsets, the proportions of CD3+ T cells and CD4+ T cells decreased gradually, while proportion of CD8+ T cells peaked at 6 weeks in mice infected with S. japonicum and at 12 weeks in mice injected with CCl4. With prolonged S. japonicum infection time, Th1 (CD4+IFN-γ+) immunity converted to Th2 (CD4+IL-4+)/Th17 (CD4+IL-17+) with weaker regulatory T cell (Treg) (CD4+CD25+FOXP3+) immunity. However, in liver fibrosis caused by CCl4, Th1 cells occupied the dominant position, while proportions of Th2, Th17, and Treg cells decreased gradually. In conclusion, liver fibrosis was a complex pathological process that was regulated by a series of cytokines and immune cells. The pathological progressions and immune responses to S. japonicum or CCl4 induced liver fibrosis were different, possibly because of their different injury mechanisms. The appropriate animal model should be selected according to the needs of different experiments and the pathogenic factors of liver fibrosis in the study.

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.

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.

[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.

[Cloning and function analysis of high mobility group box 1 (HMGB1) protein of Schistosoma japonicum (Mainland strain)].

OBJECTIVE: To clone and express a high mobility group box 1(HMGB1) protein of Schistosomajaponicum (Mainland strain) and analyze its function. METHODS: The DNA fragment of open reading frame encoding Sj HMGB 1 protein was amplified by RT-PCR from the mRNA of S. japonicum worms, then it was subcloned into the expression vector pET28a(+) to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3), and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombinant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding capacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 protein and infected with 45 +/- 2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection, the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue, respectively. The worm and egg reduction rates were calculated respectively. RESULTS: A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR, which was the open reading frame (ORF) encoding SjHMGBlprotein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA, and the recombinant protein immunized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abundantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effective immune protection against S. japonicum. CONCLUSION: The gene encoding HMGB1 from S. japonicum and the soluble recombinant SjHMGB1 protein with natural functional activity are obtained, and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.

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.

Identification of proteins inducing short-lived antibody responses from excreted/secretory products of Schistosoma japonicum adult worms by immunoproteomic analysis.

The excretory/secretory antigens of Schistosoma japonicum (Sj ESAgs) play important roles in host-parasite immune interactions. In this study, the antibody response patterns to Sj ESAgs in sera of individual rabbits at the healthy stage, 2-6 weeks post-infection and 4-16 weeks after treatment were examined. Antigens inducing short-lived antibody responses were selected by comparing differences in immune recognition of proteins in sera across the different stages by Western blotting and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS). The diagnostic value of these short-lived antibody responses for schistosomiasis was investigated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as a major antigen inducing a short-lived antibody response in Sj ESAgs. The antibody response against Sj GAPDH decreased at week 4 and disappeared between weeks 8-12 after effective chemical treatment of rabbits, and this response declined to negative levels in schistosomiasis patients one year after treatment. The intensity of the antibody response against Sj GAPDH was dependent on parasite load in mice. The sensitivity and specificity of IgG antibodies against recombinant Sj GAPDH for schistosomiasis diagnosis were 82.5% and 91.3%. Our findings suggest that Sj GAPDH induces short-lived antibody responses in the host, and detecting IgG against this antigen provides the basis for developing a potential method for diagnosis and evaluating treatment effects for schistosomiasis japonicum. BIOLOGICAL SIGNIFICANCE: Schistosomiasis is one of the world's major public health problems. Developing effective diagnostic methods for detecting schistosome-specific antibodies to effectively identify active infections is part of a critical strategy for blocking transmission of the parasite and eradicating schistosomiasis. The excretory/secretory antigens of S. japonicum (Sj ESAgs) play important roles in host-parasite immune interactions. In our study, we examine the antibody response patterns to Sj ESAgs within individual rabbits at the healthy, schistosome infection and post-treatment stages by Western blotting. Proteins among the Sj ESAgs inducing short-lived antibody responses were identified by Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS), and their potential as immune markers for diagnosis and evaluating therapeutic effects in schistosomiasis was evaluated. Our findings suggest that S. japonicum glyceraldehyde-3-phosphate dehydrogenase (GAPDH) induces short-lived antibody responses in the host, and detecting IgG against this antigen provides the basis for developing a potential method for diagnosis and evaluating treatment effects for schistosomiasis japonicum.

[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.

[Toxicity of several drugs against Schistosoma japonicum adult worms in vitro].

OBJECTIVE: To observe the toxicity of auranofin, cisplatin, adriamycin, compounds 4N, H, B, O against Schistosoma japonicum adult worms in vitro and their inhibition on thioredoxin glutathione reductase (TGR). METHODS: The drugs mentioned above with different concentrations were added into RPMI 1640 medium with Schistosoma japonicum adult worms, which had been cultured for 30 - 60 min. The activity, morphological changes and death situation of the worms were observed after 1, 6, 24, 48 h and 72 h, respectively, then the worms were transferred to fresh medium without drugs to observe whether their activity would be recovered, and 50% lethal dose (LD50) of the drugs against adult worms was determined. The TrxR and GR activities of thioredoxin glutathione reductase of Schistosoma japonicum in homogenized supernatant of adult worms processed by drugs were tested following the DTNB reduction and NADPH oxidation methods. RESULTS: The mortality rates of 5 microg/ml of auranofin treating for 24 h, 20 microg/ml of 4N treating for 72 h, 60 microg/ml of H treating for 72 h, and 80 microg/ml of cisplatin treating for 72 h on adult worms were 100%, 60%, 66.7% and 100%, respectively, and there were statistically significant differences compared with the negative control group. LD50(s) of auranofin, 4N, H and cisplatin were 2.56, 17.59, 54.14 microg/ml and 52.87 microg/ml, respectively, but no toxic effects of other drugs on schistosome worms were found. The toxic effects of auranofin, 4N, cisplatin and H on adult worms were irreversible. Auranofin and cisplatin inhibited TGR activity of Schistosoma japonicum, but other drugs had no similar effect. 5 - 30 microg/ml of auranofin, 20 - 30 microg/ml of 4N, 70 - 150 g/ml of cisplatin, and 60 - 220 microg/ml of H caused the morphological changes of the worms after treating for 24 h. CONCLUSIONS: Auranofin, cisplatin and compounds 4N and H have toxicity on Schistosoma japonicum adult worms in vitro, and the schistosomicidal effect of auranofin and cisplatin may be related to the inhibition of TGR activity.

[Evaluation of early diagnostic value of 6 antigens from Schistosoma japonicum in mice].

OBJECTIVE: To find out the candidate antigen for immunoreagent, which could be used to diagnose Schistosoma japonicum infection early in mice. METHODS: The mice were infected with cereariae of S. japonicum Chinese mainland strain. The sera of mice before and after infection at different time were collected. The recombinant fusion protein (GST-HD) of the large hydrophilic domain (HD) of 23 kDa membrane protein of S. japonicum with the Glutathione-S-transferase (GST) of S. japonicum, soluble eggs antigen (SEA), TSP2 hydrophilic domain of S. japonicum (TSP2HD), IL4-inducing principle of S. mansoni eggs (IPSE), fusion protein GST-SjMP10 (SjMP-10), and recombinant S. japonicum (Chinese strain) signaling protein 14-3-3 (Sj14-3-3) were used as diagnostic antigens, the specific IgG and IgM antibodies were measured respectively by enzyme linked immunosorbent assay (ELISA). The antigens with the value of diagnosing schistosomiasis early were screened by analyzing the changes of the levels of specific IgG (or IgM) antibodies and the positive rates of specific antibodies in the sera of mice before and post infection at different time. Moreover, the antigen's value of early diagnosis was further validated by Immunoblot. RESULTS: On the 18th, 21st and 28th day post infection, the positive rates of specific antibody IgM against GST-HD were 60%, 70% and 100%, respectively; the positive rates of specific antibody IgG against GST-HD were 40%, 60% and 90%, respectively. The positive rates of antibody IgM against SEA were 50%, 60% and 90%, respectively; the positive rates of antibody IgG against SEA were 20%, 50% and 70%, respectively. The positive rates of IgM against TSP2HD were 30%, 40% and 50%, respectively; the rates of IgG against TSP2HD were 20%, 30% and 70%, respectively. The positive rates of IgM against IPSE were 20%, 30% and 50%, respectively; the positive rates of IgG against IPSE were 20%, 30% and 60%, respectively. The positive rates of IgM against SjMP-10 were 10%, 20% and 20%, respectively; the rates of IgG against SjMP-10 were 10%, 20% and 30%, respectively. The positive rates of IgM against Sj14-3-3 were 0, 10% and 20%, respectively; the rates of IgG against Sj14-3-3 were 0, 10% and 30%, respectively. The sensitivities of GST-HD and SEA for diagnosing schistosome infection early in mice were significantly higher than those of Sj14-3-3, IPSE, TSP, and MP-10. The sensitivity of IgM was higher than that of IgG. In Western blotting, the about 73 kDa protein band of SEA was recognized by sera of mice one week post infection and showed stronger reaction as the infected time went on. Moreover, the bands (33 kDa) of GST-HD were earliest recognized by the mouse sera on the 10th day post-infection, the bands showed strong reaction with the mouse sera of 5-week post-infection. CONCLUSIONS: The GST-HD fusion protein and the protein of which molecular weight is about 73 kDa of SEA have the early diagnostic value for schistosomiasis, and the sensitivity of Immunoblot is higher than that of ELISA.

[Application of F-ELISA for immunodiagnosis of schistosomiasis japonica].

OBJECTIVE: To optimize the experimental conditions of fast enzyme-linked immunosorbent assay (F-ELISA) and evaluate its performance for immunodiagnosis of schistosomiasis japonica. METHODS: HRP-SPA was used at different concentrations in the assay to determine the best HRP-SPA concentration by comparing the detection accuracy. The working conditions of F-ELISA were optimized by examining the ratio of absorbance values of positive and negative controls with different incubation time of serum samples and HRP-SPA. To evaluate the performance of F-ELISA in diagnosis of schistosomiasis, the detection accuracy of F-ELISA was compared with routine ELISA and dipstick dye immunoassay (DDIA) by testing serum samples from both healthy subjects and those clinically diagnosed with schistosomiasis and clonorchiasis in parallel. RESULTS: In F-ELISA, the best working concentration of HRP-SPA was 1:2 000 while the optimized incubation condition for serum samples and HRP-SPA was 60 min at 37 degrees C. Using F-ELISA, we identified 93.8% to be schistosomiasis positive among chronic schistosomiasis subjects, 4% within the clonorchiasis group, and a false positive rate of 1.5% in healthy individuals. This diagnostic accuracy for F-ELISA was similar to routine ELISA and DDIA. CONCLUSIONS: F-ELISA combines the 2-steps of routine ELISA into a single step making the assay process faster and simpler without sacrificing the diagnostic accuracy. With further investigations, F-ELISA as an easy and practical method is promising to be applied for schistosomiasis diagnosis in the field.

[Preparation and characteristics of Schistosoma japonicum signaling protein 14-3-3].

OBJECTIVE: To prepare soluble recombinant signal transduction protein 14-3-3 of Schistosoma japonicum (rSj14-3-3) and investigate its immunologic features. METHODS: The cDNA fragment of signal transduction protein 14-3-3 gene was prepared from Schistosoma japonicum adult worm mRNA, and was subcloned to the downstream of glutathione S transferase gene of expression vector pGEX-4T-3 to construct a recombinant expression plasmid 14-3-3/pGEX-4T-3. The recombinant plasmids were transferred into E. coli BL21, the transforments containing recombinant plasmid were induced by IPTG, and the expression situation of fusion protein GST-14-3-3 was observed by SDS-PAGE. The pure recombinant 14-3-3 protein was prepared by digesting the fusion protein GST-rSj14-3-3 with thrombin and affinity chromatography. The specific antibody against rSj14-3-3 protein was prepared by an immunized rabbit with rSj14-3-3 protein. The immunogenicity and immune reactivity of rSj14-3-3 protein were analyzed by Western blotting. RESULTS: The gene encoding signal transduction protein 14-3-3 of Schistosoma japonicum Jiangsu strain was cloned successfully, the homology of open read frame sequence to the registrated sequence of Sj14-3-3 protein in genbank was 99.08%. A 55 kilo Dalton fusion protein GST-rSj14-3-3 was expressed by transferring the recombinant plasmid 14-3-3/pGEX-4T-3 into E. coli BL21 and induced with IPTG. The high titer antibody against rSj14-3-3 was produced by the immunized rabbit with rSj14-3-3 ,and could recognize the nature and recombinant rSj14-3-3 proteins. CONCLUSIONS: The soluble rSj14-3-3 protein has been prepared successfully in this study, and it has good immunogenicity and reactivity.

[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.

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.

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.