Population Pharmacokinetics and Pharmacodynamics of Piperacillin/Tazobactam in Patients with Nosocomial Infections.

OBJECTIVE: The study was to establish a population pharmacokinetic (PPK) model of piperacillin (PIP) and tazobactam (TAZ) that explain pharmacokinetic variability and to propose optimized dosage regimens in patients with nosocomial infections. METHODS: In total, 310 PIP and 280 TAZ concentration-time points were collected at steady state over multiple dosing intervals from 50 patients who received PIP/TAZ infused within 30 min or over 3 h. Drug analysis was performed by high-performance liquid chromatography (HPLC). Nonlinear mixed effects modeling was employed to develop PPK model and 1000 Monte Carlo simulation was used to predict the probability of target attainment (PTA) with a target time of non-protein-bound concentration above MIC > 50 % of the dosing interval. RESULTS: A model with one-compartment model had the best predictive performance for the PPK model. The population estimates of PIP were 13.8 L/h (31.1 %) for clearance (CL) and 21.7 L (38 %) for volume of distribution (V). The population estimates of TAZ were 9.3 L/h (29.1 %) for CL and 16 L (35.3 %) for V. Influence of creatinine clearance (CLcr) and body weight were identified as important covariates for PIP/TAZ CL and V, respectively. A 30-min infusion of 4 g every 6 h achieved robust (≥90 %) PTAs for MIC ≤ 16 mg/L. As an alternative mode of administration, a 3-h infusion of 4 g every 6 h achieved robust PTAs for Pseudomonas aeruginosa and Klebsiella pneumoniae. CONCLUSIONS: Prolonged infusions achieved better PTAs compared with shorter infusions at similar daily doses. This benefit was most pronounced for MICs between 16 and 40 mg/L.

N-palmitoylethanolamine and N-acetylethanolamine are effective in asteatotic eczema: results of a randomized, double-blind, controlled study in 60 patients.

BACKGROUND: Asteatotic eczema (AE) is characterized by itchy, dry, rough, and scaling skin. The treatments for AE are mainly emollients, usually containing urea, lactic acid, or a lactate salt. N-palmitoylethanolamine (PEA) and N-acetylethanolamine (AEA) are both endogenous lipids used as novel therapeutic tools in the treatment of many skin diseases. The purpose of this study was to compare a PEA/AEA emollient with a traditional emollient in the treatment of AE. METHODS: A monocentric, randomized, double-blind, comparative trial was conducted in 60 AE patients to evaluate and compare the efficacy of the two emollients. The level of skin dryness among the subjects ranged from mild to moderate. The subjects' skin barrier function and the current perception threshold were tested for 28 days by clinical scoring and bioengineering technology. RESULTS: The results showed that, although some aspects were improved in both groups, the group using the emollient containing PEA/AEA presented a better skin surface change in capacitance. However, the most impressive finding was the ability of the PEA/AEA emollient to increase the 5 Hz current perception threshold to a normal level after 7 days, with a significant difference between values at baseline and after 14 days. A current perception threshold of 5 Hz was positively and significantly correlated with skin surface hydration and negatively correlated with transepidermal water loss in the PEA/AEA emollient group. CONCLUSION: Compared with traditional emollients, regular application of a topical PEA/AEA emollient could improve both passive and active skin functions simultaneously.

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.

Population pharmacokinetics of digoxin in elderly patients.

This study was aimed at determining the population pharmacokinetics of digoxin and identifying factors that explain pharmacokinetic variability in elderly patients. The data of 142 elderly patients and 448 samples were collected after repetitive oral digoxin. Blood samples were drawn at various times after administration. Population pharmacokinetic analysis was performed using nonlinear mixed effects modelling program (NONMEM). A one-compartment model with first-order absorption and elimination was selected as the base model. The influence of demographic characteristics, biochemical and haematological indices as well as other commonly used co-medications were explored. The typical values with interindividual variability for apparent clearance (CL/F) and apparent volume of distribution (V/F) were 8.9 L h(-1) (43.2 %) and 420 L (65.8 %), respectively. The residual variability was 31.6 %. CL/F decreased significantly with renal function, total body weight, calcium channel blockers or spironolactone co-therapy and symptom with congestive heart failure. The median parameter estimates from a nonparametric bootstrap procedure were comparable and within 5 % of the estimates from NONMEM. These results provide important information for clinicians to optimize digoxin regimens in elderly patients.

[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.

[Characterization and preliminary application of six monoclonal antibodies against recombinant signal protein 14-3-3 of Schistosoma japonicum].

OBJECTIVES: To identify the properties of six monoclonal antibodies (McAbs) against recombinant signal protein 14-3-3 of Schistosoma japonicum, and investigate their value in the diagnosis of schistosomiasis. METHODS: The subclasses, titers, affinity-constants, detection limits and specificities of six McAbs were identified by ELISA and Western blotting. Dot Enzyme-linked Immunosorbent Assay (Dot-ELISA) for detecting the 14-3-3 protein in the sera of rabbits infected with Schistosoma japonicum was established, then it was used to observe the dynamics of 14-3-3 protein in sera of rabbits infected with Schistosoma japonicum before and after treatment. The diagnostic value of Dot-ELISA was investigated through detecting a group of sera samples of rabbits infected with Schistosoma japonicum. RESULTS: The types of six McAbs against recombinant signal protein 14-3-3 of Schistosoma japonicum of 3F1, 3F7, 5C6, 5D1, 5G9 and 1G6 were all IgG, their subclasses were IgG1, IgG2a, IgG2b, IgG1, IgG1 and IgG1, their antibody-titers in ascites were 1:6.4 x 10(5), 1:8.0 x 10(5) , 1:6.4 x 10(5), 1:3.2 x 10(5), 1:4.8 x 10(5) and 1:2.0 x 10(4), their affinity-constants were 8.82 x 10(8), 4.93 x 10(8), 1.56 x 10(8), 5.12 x 10(8), 1.41 x 10(8) moL/L and 2.30 x 10(7) mol/L, their detection limits in dot-ELISA were 1, 10, 100, 10, 10, 100 ng, respectively. All the six McAbs recognized the recombinant signal protein 14-3-3 of Schistosoma japonicum and the natural signal protein 14-3-3 in SEA, ESA and AWA, and did not react with the protein of E. coli and Clonorchis sinensis in Western blotting. 3F1 and 5D1 were selected to establish the Dot- ELISA for detecting the sera of rabbits infected with Schistosoma japonicum before and after treatment at different time points, it was found that the concentration of 14-3-3 protein in sera of rabbits was increased gradually with time and decreased gradually after the infected rabbits treated with praziquantel. Forty-two sera samples of rabbits infected with Schistosoma japonicum were detected by this Dot-ELISA, 41 samples were positive, the sensitivity was 97.6%, and all of the ten healthy rabbits' sera were negative, the specificity was 100%. CONCLUSIONS: All the six McAbs could recognize natural signal protein 14-3-3. It has been proved preliminarily that the Dot-ELISA based on 3F1 and 5D1 is valuable for the diagnosis of active infection of Schistosoma japonicum and accessing the chemotherapeutic effect of schistosomiasis.

[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.

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.

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.

[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.