Autophagy of hepatic stellate cell induced by Clonorchis sinensis.

BACKGROUND: Clonorchis sinensis was a food-borne zoonotic parasite in the worldwide and also an important risk factor of hepatic fibrosis. Excretory/secretion products of C. sinensis (CsESPs) are involved in parasite-host interactions and contribute to the development of hepatic damage. The aim of the present study was to investigate whether CsESPs and CsTP (adult protein) could induce autophagy of hepatic stellate cells (HSCs) and further activate HSCs so as to participate in the pathogenesis of hepatic fibrosis. METHODS AND RESULTS: The human hepatic stellate cell line LX-2 was stimulated by CsESPs and CsTP. CsESPs showed the effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay while CsTP failed. Autophagosomes and autolysosomes were observed after the transmission mRFP-EGFP-LC3 plasmid into the LX-2 cells. CsESPs had more powerful to induce the accumulation of autophagosomes and autolysosomes to enhance autophagic flux compared with CsTP. Western-blotting analysis confirmed that the ratio of LC3-II/I in LX-2 cells was up-regulated after CsESPs treatment for 6 h, which further proved that CsESPs could induce autophagy in LX-2 cells. Meanwhile, q-PCR results showed that the mRNA levels of collagen I, collagen III and α-SMA decreased in LX-2 cells after treatment with autophagy inhibitor chloroquine, whereas they increased when combination with CsESPs. CONCLUSIONS: These results suggested that CsESPs-induced autophagy might be involved in the activation of HSCs, and consequently participate in the pathogenesis of hepatic fibrosis caused by C. sinensis infection.

Identification and characteristics of a cathepsin L-like cysteine protease from Clonorchis sinensis.

Cathepsin L-like protease is an important member of the papain-like cysteine protease and plays numerous indispensable roles in the biology of parasitic organisms. In a previous study, we identified a gene encoding a cathepsin L-like protease of Clonorchis sinensis (CsCPL) that was detected in the cercaria, metacercaria, and adult worm stages by immunolocalization, suggesting that this cysteine protease may be important and involved in the development of C. sinensis. In this study, the mature domain of CsCPL (CsCPL-m) was cloned and expressed in the form of inclusion bodies in Escherichia coli. After refolding, the recombinant CsCPL-m displayed optimal protease activity towards Z-Phe-Arg-AMC substrates but not towards Z-Arg-Arg-AMC, and the activity of the protease was inhibited completely by the cysteine protease-specific inhibitors E-64 and IAA, which further demonstrated that CsCPL belongs to the cathepsin L-like cysteine protease family. Recombinant CsCPL-m exhibited considerable activity at temperatures ranging from 28 to 42 °C, with the highest activity observed at 42 °C. Furthermore, recombinant CsCPL-m exhibited activity across a broad range of pH values (pH 4.0-8.0), with an optimal pH of 5.5. The Km and Vmax of the recombinant CsCPL-m towards Z-Phe-Arg-AMC were determined to be 5.71 × 10-6 M and 0.6 µM/min, respectively, at 37 °C and pH 5.5. The recombinant CsCPL-m could degrade BSA and gelatine, but could not degrade human hemoglobin and human immunoglobulin G. These results implied that CsCPL might participate in the catabolism of host proteins for nutrition during the parasitic life cycle of C. sinensis; thus, CsCPL could be used as a potential vaccine antigen and drug target against C. sinensis infection.

Mitochondrial calcium uptake regulates rapid calcium transients in skeletal muscle during excitation-contraction (E-C) coupling.

Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca(2+) signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca(2+) release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca(2+) removal during excitation-contraction coupling by comparing Ca(2+) transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca(2+) transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ~10% increased amplitude. These regional differences in Ca(2+) transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a fast Ca(2+) chelator that reduces mitochondrial Ca(2+) uptake. Using a mitochondria-targeted Ca(2+) biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca(2+) levels during voltage-induced Ca(2+) release and detected a reduced Ca(2+) uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca(2+) transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca(2+) signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.

41.5-kDa Cathepsin L protease from Clonorchis sinensis: expression, characterization, and serological reactivity of one excretory-secretory antigen.

Cysteine proteases (CPs) were associated with the pathogenicity and excystment of Clonorchis sinensis. Most of them were potential antigens for the immunodiagnosis of clonorchiasis. More researches on CPs will let us know more about their functions, and further employ them for the development of more efficient diagnostic reagent and prevention strategies. In the current study, a full-length sequence encoding cathepsin L from C. sinensis (CsCL41.5) was identified from our adult cDNA library. Bioinformatic analysis showed that CsCL41.5 included typical motifs of cathepsin L (ERFNIN and GNFD motifs) and conserved amino acid positions which constituted the active center of the enzyme. The identity of its amino acid sequence with the cathepsin L of Schistosoma japonicum was 49.6 %. Recombinant CsCL41.5 (rCsCL41.5) was highly expressed in the form of inclusion body in Escherichia coli, and soluble rCsCL41.5 was obtained after purification and renaturation. Western blotting analysis indicated that CsCL41.5 is an excretory-secretory antigen of C. sinensis adult. Immunolocalization demonstrated that CsCL41.5 is distributed in the intestine and eggs in the uterus of adult worm, tegument of metacercaria, oral suck, and tail of cercaria. ELISA assays showed that IgG4 was the predominant IgG isotype responding to rCsCL41.5 in sera from clonorchiasis patients. The sensitivity and specificity of specific IgG4 detection with rCsCL41.5 was 62.5 % (15/24) and 81.7 % (49/60), respectively. It was concluded that there were differences in biological function, efficiency of serodiagnosis, and characterization of immune reactivity between CsCL41.5 and other CPs of C. sinensis, combining with previous studies.

RNA Secondary Structurome Revealed Distinct Thermoregulation in Plasmodium falciparum.

The development of Plasmodium parasites, a causative agent of malaria, requests two hosts and the completion of 11 different parasite stages during development. Therefore, an efficient and fast response of parasites to various complex environmental changes, such as ambient temperature, pH, ions, and nutrients, is essential for parasite development and survival. Among many of these environmental changes, temperature is a decisive factor for parasite development and pathogenesis, including the thermoregulation of rRNA expression, gametogenesis, and parasite sequestration in cerebral malaria. However, the exact mechanism of how Plasmodium parasites rapidly respond and adapt to temperature change remains elusive. As a fundamental and pervasive regulator of gene expression, RNA structure can be a specific mechanism for fine tuning various biological processes. For example, dynamic and temperature-dependent changes in RNA secondary structures can control the expression of different gene programs, as shown by RNA thermometers. In this study, we applied the in vitro and in vivo transcriptomic-wide secondary structurome approach icSHAPE to measure parasite RNA structure changes with temperature alteration at single-nucleotide resolution for ring and trophozoite stage parasites. Among 3,000 probed structures at different temperatures, our data showed structural changes in the global transcriptome, such as S-type rRNA, HRPII gene, and the erythrocyte membrane protein family. When the temperature drops from 37°C to 26°C, most of the genes in the trophozoite stage cause significantly more changes to the RNA structure than the genes in the ring stage. A multi-omics analysis of transcriptome data from RNA-seq and RNA structure data from icSHAPE reveals that the specific RNA secondary structure plays a significant role in the regulation of transcript expression for parasites in response to temperature changes. In addition, we identified several RNA thermometers (RNATs) that responded quickly to temperature changes. The possible thermo-responsive RNAs in Plasmodium falciparum were further mapped. To this end, we identified dynamic and temperature-dependent RNA structural changes in the P. falciparum transcriptome and performed a comprehensive characterization of RNA secondary structures over the course of temperature stress in blood stage development. These findings not only contribute to a better understanding of the function of the RNA secondary structure but may also provide novel targets for efficient vaccines or drugs.

[Subcellular mimical localization of the ATP synthase B subunit from Clonorchis sinensis under different conditions of cell cycling].

OBJECTIVE: To investigate the subcellular localization of ATP synthase b subunit from Clonorchis sinensis under different conditions of Hela cell cycling, and the effect of this protein on the expression of its encoding-gene and homologous host genes. METHODS: pEGFP-N1-CsATP-synt_B and the vector pEGFP-N1 were transfected into Hela cells, respectively. Transfected cells were synchronized in G0/G1 by serum starvation, G1/S, S cells by double thymidine block, and G2/M cells by thymidine-Nocodozale block. After synchronization, the subcellular localization of the expressed fusion protein was observed with a laser confocal microscope. The expression level of this fusion protein in cells was detected by flow cytometry (FCM). The expression of CsATP-synt_B and HomoATP-synt_B in different cell cycle phases accessed by RT-PCR. RESULTS: FCM results indicated that in the G0/G1 phase the expression of pEGFP-N1 vector was decreased significantly, while pEGFP-N1-CsATP-synt_B expression showed an upward trend. In the other phases of cell cycle, the protein expression was similar in the above two kinds of plasmids. The intact CsATP-synt_B was expressed in mitochondria in the G0/G1, S, and G2/M phases and nucleus during G1/S phase. After the fusion proteins entered the nucleus, the mRNA expression of CsATP-synt_B and HomoATP-synt_B increased significantly. CONCLUSION: CsATP-synt_B can be expressed in the nucleus during G1/S phase, and regulated by the cell cycle and energy requirements.

A cross-sectional study: Comparing the attitude and knowledge of medical and non-medical students toward 2019 novel coronavirus.

BACKGROUND AND OBJECTIVES: Since December 2019, the rapid epidemic spread of COVID-19 in China has aroused the attention of the government and the public. The purpose of this study is to investigate the attitude and knowledge among medical students and non-medical students toward SARS-CoV-2 infection. METHODS: A web-based survey was disseminated to the students from medical colleges and comprehensive universities via the survey website (www.wjx.cn) and via WeChat. Participation in the study was voluntary with the instruction to click on the website or scan the QR code to complete the anonymous electronic questionnaire from February 5 to 7, 2020. RESULTS: The questionnaire was completed by 588 students from 20 colleges and universities in China. Of the respondents, 66.0% were medical students and 34.0% were non-medical students. 99.6 % of the students held an optimistic attitude toward the COVID-19 epidemic situation. The majority of participants had a good level of knowledge of common symptoms, transmission, and prevention of the disease. In a comparison between non-medical students with medical students, the medical students had a deeper understanding of COVID-19. In this study, we also found that female students had a better understanding of transmission and prevention than male students did. CONCLUSIONS: The majority of students who participated in the questionnaire had a positive attitude and a good perception about COVID-19.

Molecular cloning and analysis of stage and tissue-specific expression of Cathepsin L-like protease from Clonorchis sinensis.

Cathepsin L of parasite plays multiple roles in growth, food uptake, and invasion into host and pathogenesis, which makes it a valuable target for diagnosis, vaccine, and drug. In this study, we identified a cDNA encoding cathepsin L homolog (CsCPL) from the library of Clonorchis sinensis adult by bioinformatics analysis. Sequence encoding proenzyme of CsCPL (removal of signal peptide, CsproCPL) was highly expressed in form of inclusion body in Escherichia coli, and soluble rCsproCPL (about 1 mg/ml) in high purity were obtained after denaturation, purification, and renaturation. Western blot analysis indicated that CsCPL is a component of excretory-secretory products of adult, in mature form of protease. Reverse transcription polymerase chain reaction showed that CsCPL is also expressed in metacercaria and cercaria stage. Immunolocalization demonstrated that CsCPL is deposited at adult intestine, or tegument, and tegumentary cell of metacercaria and cercaria (especially at dorsal tegument of cercaria), indicating different secretory routine roles in adult and larva. The characteristics of CsCPL suggested that it may involve in invasion of cercaria into fish and development to metacercaria, excystment of metacercaria, and protein digestion of adult, which may render it a candidate antigen for fish vaccine and serodiagnosis of human clonorchiasis.

[Tissular and subcellular localization of the ATP synthase B subunit in Clonorchis sinensis].

OBJECTIVE: To illustrate the distribution of ATP synthase b subunit in the tissue of Clonorchis sinensis adult and its subcellular mimical localization in HeLa cells. METHODS: With the antiserum against recombinant CsATP-synt_B protein raised from SD rats as primary antibody, paraffin sections of the adult of C. sinensis were processed by the method of fluorescent immunohistochemistry to observe the distribution of CsATP-synt_B protein in adult worm. According to the prediction by bioinformatics of the definite mitochondrial targeting sequence (MTS) and probable Bipartite nuclear localization signals (NLS_BP)in CsATP-syntB sequence, recombinant pEGFP-N1 plasmids containing the intact and three defective CsATP-synt_B sequence with single defect of MTS or NLS_BP or double defect respectively were constructed. The recombinant plasmids and the control plasmid-pEGFP-N1, pEYFP-Mito and H2B-CFP, were transfected into the HeLa cells by Lipofectamine 2000 reagent and the subcellular location of the GFP fusion protein was observed with confocal microscopy. RESULTS: The CsATP-synt_B protein appeared to distribute all over the adult worm, especially abundant on the acetabulum, ovary, vitellarium and tegument. The intact CsATP-synt_B was definitely expressed in mitochondria and/or nucleus of infected HeLa cells, whereas the MTS-deleted mutant only in cytoplasma and nucleus, the NLS_BP-deleted mutant in mitochondria and cytoplasm, and the double defect mutant only in cytoplasm. CONCLUSION: The distribution of CsATP-synt_B in adult is accord with that of mitochondria, and mainly exits in the organs and the tissues of active energy metabolism. This study first predicted and confirmed that CsATP-synt_B can be expressed in the nucleus.

[Cloning and expression of the F0-ATP synthase B chain of Clonorchis sinensis and immunogenicity identification of the recombinant protein].

OBJECTIVE: To clone and express the Clonorchis sinensis F0-ATP synthase b chain (CsF0-ATP-synt_B) gene and analyze immunogenicity of the recombinant protein. METHODS: The coding region F0-ATP synthase b chain gene with the mitochondrial targeting sequence (MTS) removed was amplified with PCR using the cloned plasmid as template, and the product was cloned into the prokaryotic expression vector pET-28a(+), transformed into E. coli BL21 (DE3) and induced with IPTG. The expressed product was purified by Ni-IDA affinity chromatography,and analyzed by SDS-PAGE for its expression and identified by Western blotting for its immunogenicity. RESULTS: The coding sequence of the F0-ATP synthase b-chain like gene removed off the MTS contains 813 base pairs encoding 271 amino acids with a theoretical molecular weight of 31,171.9. PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-28a (+)-CsF0-ATP-synt_B was constructed successfully, and the resolvable expression was obtained in E.coli BL21. Highly purified recombinant protein was prepared through affinity chromatography. The recombinant protein could be recognized by the immune serum of the SD rat immunized with the recombinant protein. CONCLUSION: The CsF0-ATP-synt_B like gene has been efficiently expressed in prokaryotic expression system with immunogenicity.

Suppressed autophagy flux in skeletal muscle of an amyotrophic lateral sclerosis mouse model during disease progression.

Accumulation of abnormal protein inclusions is implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Autophagy, an intracellular process targeting misfolded proteins and damaged organelles for lysosomal degradation, plays crucial roles in survival and diseased conditions. Efforts were made to understand the role of autophagy in motor neuron degeneration and to target autophagy in motor neuron for ALS treatment. However, results were quite contradictory. Possible autophagy defects in other cell types may also complicate the results. Here, we examined autophagy activity in skeletal muscle of an ALS mouse model G93A. Through overexpression of a fluorescent protein LC3-RFP, we found a basal increase in autophagosome formation in G93A muscle during disease progression when the mice were on a regular diet. As expected, an autophagy induction procedure (starvation plus colchicine) enhanced autophagy flux in skeletal muscle of normal mice. However, in response to the same autophagy induction procedure, G93A muscle showed significant reduction in the autophagy flux. Immunoblot analysis revealed that increased cleaved caspase-3 associated with apoptosis was linked to the cleavage of several key proteins involved in autophagy, including Beclin-1, which is an essential molecule connecting autophagy and apoptosis pathways. Taking together, we provide the evidence that the cytoprotective autophagy pathway is suppressed in G93A skeletal muscle and this suppression may link to the enhanced apoptosis during ALS progression. The abnormal autophagy activity in skeletal muscle likely contributes muscle degeneration and disease progression in ALS.

Defective mitochondrial dynamics is an early event in skeletal muscle of an amyotrophic lateral sclerosis mouse model.

Mitochondria are dynamic organelles that constantly undergo fusion and fission to maintain their normal functionality. Impairment of mitochondrial dynamics is implicated in various neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is an adult-onset neuromuscular degenerative disorder characterized by motor neuron death and muscle atrophy. ALS onset and progression clearly involve motor neuron degeneration but accumulating evidence suggests primary muscle pathology may also be involved. Here, we examined mitochondrial dynamics in live skeletal muscle of an ALS mouse model (G93A) harboring a superoxide dismutase mutation (SOD1(G93A)). Using confocal microscopy combined with overexpression of mitochondria-targeted photoactivatable fluorescent proteins, we discovered abnormal mitochondrial dynamics in skeletal muscle of young G93A mice before disease onset. We further demonstrated that similar abnormalities in mitochondrial dynamics were induced by overexpression of mutant SOD1(G93A) in skeletal muscle of normal mice, indicating the SOD1 mutation drives ALS-like muscle pathology in the absence of motor neuron degeneration. Mutant SOD1(G93A) forms aggregates inside muscle mitochondria and leads to fragmentation of the mitochondrial network as well as mitochondrial depolarization. Partial depolarization of mitochondrial membrane potential in normal muscle by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused abnormalities in mitochondrial dynamics similar to that in the SOD1(G93A) model muscle. A specific mitochondrial fission inhibitor (Mdivi-1) reversed the SOD1(G93A) action on mitochondrial dynamics, indicating SOD1(G93A) likely promotes mitochondrial fission process. Our results suggest that accumulation of mutant SOD1(G93A) inside mitochondria, depolarization of mitochondrial membrane potential and abnormal mitochondrial dynamics are causally linked and cause intrinsic muscle pathology, which occurs early in the course of ALS and may actively promote ALS progression.

[Investigation on spread and hazards of human parasites in Qushui Village, Suixi County, Zhanjiang City].

OBJECTIVE: To understand the transmission of human parasites in Qushui Village, Yangqing Town, Suixi County, Zhanjing City, Guangdong Province. METHODS: The direct stool smear, floatation, Kato-Katz technique, and hookworm larva culture were used for the parasite infections. The questionnaire survey was applied for the hazards of parasites. The dissections on rats and snails were used for Angiostrongylus cantonensis infection. RESULTS: Five parasites were found and the total infection rate was 10.75%. The infection rates of hookworm (Necator americanus), Ascaris lumbricoides and Trichuris trichiura were 6.07%, 1.87% and 1.87%, respectively, and the infection rates of Enterobius vermicularis and Tyroglyrhus farinae were both 0.47%. The infections were not correlated with the career and age but preferred to males. The densities of infections were slight. The rate of dermatitis caused by hookworm larvae was 69.23%. The infection rates of Angiostrongylus cantonensis were 16.66%, 13.04% and 10.00%, respectively in rats, Achatina fulica and Ampularum crossean. CONCLUSION: The main species of human parasites are nematodes, with hookworm predominately, in Qushui Village, Suixi County. This area is the natural foci of Angiostrongylus cantonensis.

Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A(2) 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.

[Excretory/secretory antigens from Clonorchis sinensis induces hepatic fibrosis in rats].

OBJECTIVE: To investigate the role of excretory/secretory antigens from Clonorchis sinensis (CsESAs) in hepatic fibrosis induced by C. sinensis infection in rats and explore the possible mechanism. METHODS: CsESAs was collected from adult C. sinensis cultured in sterile condition for 12 h and injected intraperitoneally in Wistar rats. Masson staining was used to observe the changes in the hepatic collagen fiber after the injection. HE staining and immunofluorescence staining were performed to detect the expression of alpha-smooth muscle actin (alpha-SMA) to examine the proliferation and the activity of hepatic stellate cells. The specific antibody titer of CsESAs was determined using enzyme-linked immunosorbent assay to investigate the role of the antigen-antibody complex in the development of hepatic fibrosis. RESULTS: After intraperitoneal injection of CsESAs, obvious hepatic fibrosis and hepatic stellate cell proliferation and activation were observed in the rat livers. The severity of the hepatic fibrosis was associated with the dose of CsESAs injected, whereas the titer of the specific antibody against CsESAs showed no direct relation to the hepatic fibrosis. CONCLUSION: Intraperitoneal injection of CsESAs can cause hepatic stellate cell activation and hepatic fibrosis in rats, but the antigen-antibody complex does not seem to play the key role in the activation of the hepatic stellate cells.

[The effects of Lysophospholipase from Clonorchis sinensis on the hepatic stellate cells and oval cells of rat].

AIM: To clarify the effects of the recombinant protein of Lysophospholipase from Clonorchis sinensis (CsLysoPLA) on the hepatic stellate cells (HSC) and oval cells of rat. METHODS: Binding of the recombinant CslysoPLA protein to the membrane of HSC and oval cells was identified by immunofluorescent staining. The HSC and oval cells were cultured and treated with the recombinant protein at different doses, and proliferation was quantified by MTT method. Cell cycle analysis was performed by flow cytometry. RESULTS: The recombinant CslysoPLA protein could bind to the membrane of HSC and oval cells. Compared to control, 2 mg/L and 20 mg/L the recombinant protein could promote HSC and oval cells growth (P<0.05), whereas 200 mg/L the recombinant protein could induce the cells necrosis, which associated with overt plasma membrane disruption. Oval cell number in G(2) phase of the recombinant protein 20 mg/L treated group was higher than that of control group. CONCLUSION: In vitro, the recombinant protein could induce HSC and oval cells proliferation at low concentrations (2 mg/L and 20 mg/L), whereas it also could induce the cells necrosis at high concentration (200 mg/L). These results suggested that CslysoPLA might play a role in the pathogenicity of C. sinensis.

Immunogenicity of recombinant Bacillus subtilis spores expressing Clonorchis sinensis tegumental protein.

Clonorchis sinensis, which causes clonorchiasis, is of major socioeconomic importance in China. In this study, we report the use of CotC, a major component of the Bacillus subtilis spore coat, as a fusion partner for the expression of C. sinensis TP20.8 (Tegumental Protein 20.8 kDa) on the spore coat. Western blotting was used to identify TP20.8 surface expression on spores. Recombinant spores displaying the TP20.8 antigen were used for oral immunization and were shown to generate mucosal response in rats. TP20.8-specific secretory IgA in feces reached significant levels 2 weeks after oral dosing. This report shows that surface display of recombinant C. sinensis TP20.8 on B. subtilis spores was immunogenic and B. subtilis spores can be used as a mucosal immunization vehicle for parasite prevention and control.

Oral administration of a Bacillus subtilis spore-based vaccine expressing Clonorchis sinensis tegumental protein 22.3 kDa confers protection against Clonorchis sinensis.

Human clonorchiasis, caused by the infection of Clonorchis sinensis, is endemic in China. In this study, a full-length cDNA sequence encoding C. sinensis tegumental protein 22.3 kDa (CsTP22.3) was identified from adult cDNA library. Recombinant CsTP22.3 was expressed and purified from Escherichia coli BL21. CsTP22.3 was immunolocalized at the tegument of adult C. sinensis by using anti-recombinant CsTP22.3 sera. ELISA was performed using rCsTP22.3 protein to investigate the IgG response to CsTP22.3. Sera from clonorchiasis patients had clear IgG response to CsTP22.3. We used the CotC, a major component of the Bacillus subtilis spore coat, as a fusion partner for the expression of C. sinensis TP22.3 on the spore coat. Western blotting and immunofluorescence analyses were used to identify TP22.3 surface expression on spores. Recombinant spores displaying the TP22.3 antigen were used for oral immunization and were shown to generate mucosal response in rats. TP22.3-specific secretory IgA in faeces reached significant levels 2 weeks after oral dosing. In addition, the recombinant spores induced a significant level of protection (44.7%, p<0.05) in rats challenged with C. sinensis metacercariae. This report shows that C. sinensis TP22.3 expressed on B. subtilis spores was immunogenic and oral administration of TP22.3 spore can provide protection against C. sinensis.

Immunological cross-reactivity analysis on recombinant histamine-releasing factors from Schistosoma japonicum, Clonorchis sinensis, and Wistar rat.

Studies have demonstrated a declined incidence of allergic disorders in the population with helminthic infection. Though several hypotheses have been proposed to explain how helminthic infection protected people against allergies, the underlying mechanisms remain poorly understood. A human histamine-releasing factor (HRF) has been proved to be closely related to the development of allergic disorders and the homologues are ubiquitously expressed in all eukaryotic organisms including parasites. To study the role of this HRF in the relationship between parasitic infection and allergic diseases with experimental model of rats, the cDNA of the homologues of the human HRF from Wistar rat, Schistosoma japonicum, and Clonorchis sinensis containing a coding region of 519, 510, and 510 bp, respectively, were cloned. In addition, the cross-reactivity between recombinant rat HRF (rRHRF) and recombinant S. japonicum HRF (rSjHRF) as well as that between rRHRF and recombinant C. sinensis HRF (rCsHRF) was identified with ELISA and Western blotting. Based on their detected cross-reactivities, a hypothesis was put forward that the anti-parasitic HRFs antibodies could inhibit the effects of host HRF and those of parasitic HRFs and thus decreased the host sensitivities to allergens.