Xuchu Hu

The draft genome of the carcinogenic human liver fluke Clonorchis sinensis.

BackgroundClonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome.ResultsWe combined de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines.ConclusionsThis study provides valuable genomic information about the human liver fluke C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control.

The Carcinogenic Liver Fluke, Clonorchis sinensis: New Assembly, Reannotation and Analysis of the Genome and Characterization of Tissue Transcriptomes

Clonorchis sinensis (C. sinensis), an important food-borne parasite that inhabits the intrahepatic bile duct and causes clonorchiasis, is of interest to both the public health field and the scientific research community. To learn more about the migration, parasitism and pathogenesis of C. sinensis at the molecular level, the present study developed an upgraded genomic assembly and annotation by sequencing paired-end and mate-paired libraries. We also performed transcriptome sequence analyses on multiple C. sinensis tissues (sucker, muscle, ovary and testis). Genes encoding molecules involved in responses to stimuli and muscle-related development were abundantly expressed in the oral sucker. Compared with other species, genes encoding molecules that facilitate the recognition and transport of cholesterol were observed in high copy numbers in the genome and were highly expressed in the oral sucker. Genes encoding transporters for fatty acids, glucose, amino acids and oxygen were also highly expressed, along with other molecules involved in metabolizing these substrates. All genes involved in energy metabolism pathways, including the β-oxidation of fatty acids, the citrate cycle, oxidative phosphorylation, and fumarate reduction, were expressed in the adults. Finally, we also provide valuable insights into the mechanism underlying the process of pathogenesis by characterizing the secretome of C. sinensis. The characterization and elaborate analysis of the upgraded genome and the tissue transcriptomes not only form a detailed and fundamental C. sinensis resource but also provide novel insights into the physiology and pathogenesis of C. sinensis. We anticipate that this work will aid the development of innovative strategies for the prevention and control of clonorchiasis.

Clonorchis sinensis enolase: Identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products

Enolase plays a key role in energy metabolism and development of most organisms. We isolated a gene encoding enolase from Clonorchis sinensis (C. sinensis) adult cDNA library and expressed the recombinant protein in Escherichia coli. C. sinensis enolase (Csenolase) was identified as both an excretory/secretory product and a tegumental component of C. sinensis by western blot analysis. The transcriptional level of Csenolase was examined at adult worm, metacercaria, cercaria and egg of C. sinensis, and results showed that Csenolase is transcribed at the four life stages of C. sinensis while showing a significant higher expression level at the stage of adult worm. Immunohistochemical localization indicated that Csenolase was specifically deposited on the tegument of adult worm and cyst wall of metacercaria. Ligand blot assay revealed a specific characteristic of dose-dependent plasminogen-binding activity of Csenolase and kinetic parameters were explored using 2-phospho-D-glycerate (2-PGA) as the primary substrate by monitoring the conversion of nicotinamide-adenine dinucleotide (NADH) into nicotinamide adenine dinucleotide (NAD). In addition, Csenolase exhibited active enzyme activity in catalytic reactions while the anti-Csenolase serum inhibited the enzyme activity. In vitro incubation experiments revealed that Csenolase might play key roles in the growth of the parasites. In conclusion, Csenolase is an important glycolytic enzyme required for the development of C. sinensis, and may be a potential vaccine candidate and drug target against C. sinensis infection.

Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A2 and investigation of its potential contribution to hepatic fibrosis

A gene encoding a homologue of phospholipase A(2) was identified from the Clonorchis sinensis adult cDNA plasmid library. The deduced amino acid sequence including a signal peptide that has 28-46% identity with secretory phospholipase A(2), group III (group III sPLA(2)) of other species. It also has typical features of group III sPLA(2)s including 10 cysteines, the key residues of the Ca(2+) loop and catalytic site. The recombinant protein encoded by this gene expressed in Escherichia coli showed a product of about 34kDa in SDS-PAGE. Prediction of signal peptide and Western blot analysis indicated the group III secretory phospholipase A(2) of C. sinensis (CsGIIIsPLA(2)) was an excretory-secretory product (ES product). The enzyme activity of the recombinant protein was determined using phosphatidylcholine as substrates. The result revealed that the protein was a Ca(2+)-dependent PLA(2). Both MTT test and cell cycle analysis of LX-2 showed a higher percentage of cells are in proliferation phase. Semi-quantitative RT-PCR experiments demonstrated an up-regulated expression of collagen III in these cells after incubation with the recombinant protein. We also identified that the recombinant CsGIIIsPLA(2) could bind to some membrane proteins on LX-2 cells specifically by immunofluorescence, thus there might be receptors of CsGIIIsPLA(2) on the LX-2 cell membrane. Our results suggest that CsGIIIsPLA(2) might play an important role in the initiation and development of hepatic fibrosis caused by C. sinensis.

Experimental model in rats for study on transmission dynamics and evaluation of Clonorchis sinensis infection immunologically, morphologically, and pathologically

This study aims to gain a better insight into the transmission patterns and immunologic profile of Clonorchis sinensis infection and make a headway on the pathogenesis regarding cholangiocarcinoma and hepatic lesions. Experimental models orally infected by C.sinensis metacercariae were constructed in rats. Immunological assays were performed to measure serum level of IgA, IgE, IgG1, IgG2a, IFN-γ, and IL-4. Infection parameters were assessed by worm recovery rate, eggs per gram faece and worm size. Pathological sections with livers were managed with immunofluorescence, hematoxylin, eosin, and Massons trichrome staining to evaluate the hepatic pathological changes. Interestingly, rats infected with only one C.sinensis metacercariae even gained a high worm recovery rate of 83.3% compared with rats infected with more metacercariae. Serological changes according to different infection doses indicated that immune response presented a tendency to Th2 type by expressing transient high level of IgG1, IL-4, and IgE. Hepatic tissues appeared inflammatory and fibrotic, revealed by different stainings. Intrahepatic bile ducts displayed cholangiectasis and proliferation with excreted/secreted antigen histologically located. C.sinensis, as a fish-borne zoonosis, presented novel transmission patterns which explained high infection rate in endemic areas; infection rate of C.sinensis was frequency-dependent and dose-related. Humoral immunity played a prevalent role in resisting to C.sinensis based on the rat models. C.sinensis infection played an undoubted role in biliary and hepatic diseases.

Identification and Characterization of Paramyosin from Cyst Wall of Metacercariae Implicated Protective Efficacy against Clonorchis sinensis Infection

Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-γ and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization.

Schistosoma japonicum calcium-binding tegumental protein SjTP22.4 immunization confers praziquantel schistosomulumicide and antifecundity effect in mice

A family of platyhelminth tegument-specific proteins comprising of one or two calcium ion binding EF-hand and a dynein light chain-like domain, termed tegumental proteins, are considered as candidates of vaccine. In this study, we cloned and characterized SjTP22.4, a novel membrane-anchored tegumental protein in Schistosoma japonicum with theoretic MW of 22.4. The recombinant SjTP22.4 could be recognized by S. japonicum infected sera. Immunofluorescence revealed that this protein is not only located on the surface of tegument of adult and schistosomulum and cercaria, but also in the parenchymatous tissues and intestinal epithelium. Circular dichroism (CD) measurement demonstrated rSjTP22.4 had Ca(2+)-binding ability. The rSjTP22.4 vaccination without adjuvants produced comparable high level of antibody with that of immunization with adjuvants together indicated it was an antigen of strong antigenicity. The level of IgG1 is much higher than that of IgG2a and IgE is nearly negative in S. japonicum-infected and rSjTP22.4 immunized mice. In cercaria challenge experiment, mice vaccinated with SjTP22.4 showed no reduction in adult burden and egg production, comparing with the control mice, but 41% decrease in egg mature rate and 32% reduction in liver egg granuloma area. However, the SjTP22.4 immunized mice that received praziquantel treatment at 10d post infection caused 26% reduction in adult burden and 53% decrease in egg mature rate, comparing with the control mice only received praziquantel treatment. In conclusion, SjTP22.4 is a valuable vaccine candidate for S. japonicum of anti-pathogenesis and anti-transmission effect and plays a synergetic role in praziquantel to kill schistosomulum.

Cloning and expression of 21.1-kDa tegumental protein of Clonorchis sinensis and human antibody response to it as a trematode-nematode pan-specific serodiagnosis antigen.

A complete cDNA encoding a 21.1-kDa tegumental protein (CsTP21.1) was recognized from Clonorchis sinensis adult full-length cDNA plasmid library by bioinformatics analysis. Recombinant CsTP21.1 was highly expressed in Escherichia coli, purified by affinity chromatography, and identified by Western blotting. Immunohistochemistry demonstrated that CsTP21.1 is localized in the tegument of the adult worm. The rCsTP21.1-specific IgG1, IgG2, and IgG4 subclasses could be detected in the sera of clonorchiasis patients by ELISA, but their sensitivity was much lower than that of total IgG. The sensitivity and specificity of IgG in 66 serum samples of clonorchiasis patients were 100% and 95.5%, and the sensitivity was independent of worm loads; the cross-reaction rates in 86, 24, and 31 serum samples from patients infected with Fasciola hepatica, Schistosoma japonicum, and nematode were 98.8%, 83.3%, 93.3%, respectively, whereas no cross-reactions with Toxoplasma gondii and sparganum. This study demonstrated that CsTP21.1 is a trematode–nematode pan-specific antigen that is valuable in the development of a universal immunodiagnostic kit for human trematode and nematode infections.

Dexamethasone Down-Regulates the Expression of microRNA-155 in the Livers of Septic Mice

To investigate the expression of microRNA-155 (miRNA-155) in the livers of mice with lipopolysaccharide (LPS)-induced sepsis and to determine the role of dexamethasone (DXM) in the regulation of miRNA-155 expression, we pretreated mice with or without DXM prior to LPS exposure. Our study demonstrated that the expression of miRNA-155 and inflammatory factors increased in the liver tissues of mice with LPS-induced sepsis and that DXM down-regulated their expression in a dose-dependent manner. Moreover, DXM alone inhibited the expression of miRNA-155 to below the baseline level, but did not impact the expression of inflammatory factors, suggesting that the down-regulation of miRNA-155 by DXM may partially, but not completely, depend on the suppression of pro-inflammatory cytokines by DXM. Our data indicate that the overexpression of miRNA-155 in the livers of mice with LPS-induced sepsis may play an important role in the pathological processes of sepsis and that the down-regulation of miRNA-155 by DXM may be a novel mechanism regulating inflammation and immunity.

Clinical efficacy and immunological impact of tacrolimus in Chinese patients with generalized myasthenia gravis

In this multicenter, open-label pilot study, the efficacy, safety, and immunological impact of tacrolimus in Chinese patients with generalized myasthenia gravis are assessed. Forty-seven generalized myasthenia gravis (MG) patients were enrolled into this study and given 3mg/day tacrolimus for 24 weeks. The primary efficacy measurements used to monitor response to tacrolimus in MG patients were the Osserman grade, the quantitative MG score (QMGS) recommended by the MGFA, the MG-specific manual muscle testing (MMT) score, and the MG-related activities of daily living (MG-ADL) scale. Also, reduction in steroid doses was used to monitor the effect of tacrolimus. Clinical evaluations were conducted at weeks 4, 8, 12, 16, 20, and 24, while immunological parameters were measured at weeks 4, 12, and 24. Measurements of the Osserman grade, QMGS, MMT, and MG-ADL all suggested improvement in patient health by the fourth week of treatment. Steroid dosage was reduced during the course of the study in 74.2% of the forty-three patients who completed the study. There were thirty-one reported adverse events in the study. Only one was considered serious. We found that tacrolimus reduced levels of the IFN-γ, IL-2, IL-10, and IL-13 cytokines and induced the proliferation of tolerogenic plasmacytoid dendritic cells after treatment. Tacrolimus did not change the population of T cell subtypes but did steadily reduce the population of BAFF-R(+) CD19(+) B cells over the course of the study. Our results show that tacrolimus improves the clinical condition of MG patients and is well tolerated. The decrease in IL-13 and reduction of BAFF-R(+) CD19(+) B cells may be related to the therapeutic effect of tacrolimus.