OBJECTIVETo characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis.

METHODSThe full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein.

RESULTSThe recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1: 51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis.

CONCLUSIONThe pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis- associated hepatic fibrosis.

Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; immunology ; Calmodulin ; immunology ; Clonorchiasis ; immunology ; Clonorchis sinensis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Library ; Immunoglobulin G ; blood ; Inflammation ; Liver Cirrhosis ; parasitology ; Male ; Mice ; Rats ; Recombinant Proteins ; immunology

Dendritic cells (DCs), which are regarded as the most potent antigen-presenting cells, are involved in innate and adaptive immunity. Upon uptake of pathogens, DCs express cell surface markers and secrete cytokines. In this study, we analyzed production of cytokines and found that interleukin (IL)-10 and transforming growth factor (TGF)-beta production significantly increased in bone marrow-derived DCs and a mouse DC line, DC2.4, after treatment with crude antigen (CA) from liver fluke, Clonorchis sinensis. However, expression patterns of several activation molecules did not change. In addition, following treatment of DC2.4 cells with antigen from the lung fluke, Paragonimus westermani, production of IL-10 and TGF-beta significantly increased compared with groups treated with other parasite antigens, Spirometra erinacei plerocercoid CA and Echinococcus granulosus hydatid cystic fluid. We also found that treatment of DC2.4 cells with C. sinensis CA resulted in rapid and significant phosphorylation of extracellular signal-regulated kinase 1/2, a mitogen-activated protein kinase. Following treatment of DC2.4 cells with C. sinensis CA, treatment with an inhibitor specific to an extracellular signal-regulated kinase inhibited production of IL-10 and TGF-beta. Our results suggest that CA from C. sinensis has a role in the anti-inflammatory function of DC cells by inducing IL-10 and TGF-beta through activation of extracellular signal-regulated kinase 1/2.
Animals ; Antigens, Helminth/*pharmacology ; Cells, Cultured ; Clonorchis sinensis/*immunology ; Dendritic Cells/drug effects/*metabolism ; Interleukin-10/genetics/*metabolism ; MAP Kinase Signaling System ; Mice ; Transforming Growth Factor beta/genetics/*metabolism

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.
Animals ; Antibodies, Helminth/immunology ; Antibodies, Monoclonal/*immunology ; Antigens, Helminth/*immunology ; Clonorchis sinensis/immunology ; Cross Reactions/immunology ; Fasciola hepatica/immunology ; Female ; Helminth Proteins/*immunology ; Heterophyidae/*immunology ; Immunologic Tests ; Mice ; Mice, Inbred BALB C ; Paragonimus westermani/immunology ; Trematode Infections/*diagnosis/immunology

The main causes of biliary obstruction are stones and cancers. Fascioliasis is a very rare case which causes biliary obstruction. Fascioliasis is a zoonosis caused by Fasciola hepatica which infects herbivores like sheep and cattle. F. hepatica lives in the biliary system or the liver parenchyma of a host. In Korea, the occurrence of this infection in human is very rare and only few cases have been reported. A 32-year-old male presented with upper abdominal pain and jaundice. His laboratory finding revealed elevated liver transaminases. Abdomen CT scan showed mild left intrahepatic bile duct dilatation. On ERCP, adult F. hepatica worms were found and were thus removed. Concurrently, clonorchiasis was diagnosed by stool exam and serologic enzyme-linked immunosorbent assay test. Clonorchiasis was treated with praziquantel. Herein, we report a case of intrahepatic bile duct dilatation due to F. hepatica infection with concurrent Clonorchis sinensis infestation.
Adult ; Animals ; Anthelmintics/therapeutic use ; Benzimidazoles/therapeutic use ; Bile Ducts, Intrahepatic ; Cholangiopancreatography, Endoscopic Retrograde ; Clonorchiasis/complications/*diagnosis/drug therapy ; Clonorchis sinensis/immunology/isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Fasciola/isolation & purification ; Fascioliasis/complications/*diagnosis/parasitology ; Humans ; Liver/enzymology ; Male ; Praziquantel/therapeutic use ; Tomography, X-Ray Computed ; Transaminases/metabolism

The prevalence of liver and intestinal fluke infections was determined by surveying inhabitants of Hengxuan, Fusui, and Shanglin villages which were known to be endemic for liver flukes in Guangxi, China in May 2010. A total of 718 people were examined for helminth eggs by the Kato-Katz thick smear technique, ultrasonography, immunoaffinity chromatography, and DNA sequencing. The overall egg positive rate was found to be 59.6% (28.0-70.6%) that included mixed infections with liver and intestinal flukes. Cases showing higher than 20,000 eggs per gram of feces (EPG) were detected between 1.3% and 16.2%. Ultrasonographic findings exhibited overall 28.2% (72 of 255 cases) dilatation rate of the intrahepatic bile duct. Clonorchis sinensis infection was detected serologically in 88.3% (38 of 43 cases) among C. sinensis egg positive subjects by the immunoaffinity chromatography using a specific antigen for C. sinensis. For differential diagnosis of the liver and intestinal flukes, more precise PCR and nucleotide sequencing for copro-DNA were performed for 46 egg positive cases. Mixed infections with C. sinensis and Metagonimus yokogawai were detected in 8 of 46 egg positive cases, whereas 29 specimens were positive for Haplorchis taichui. Ultrasonographic findings and immunoaffinity chromatography results showed usefulness, even in a limited way, in figuring out of the liver fluke endemicity.
Animals ; China/epidemiology ; Chromatography, Affinity ; Clonorchiasis/*epidemiology/parasitology/ultrasonography ; Clonorchis sinensis/genetics/immunology/*isolation & purification ; Coinfection ; DNA, Helminth/chemistry/genetics ; Feces/parasitology ; Female ; Heterophyidae/genetics/*isolation & purification ; Humans ; Intestines/*parasitology ; Liver/*parasitology ; Parasite Egg Count ; Polymerase Chain Reaction ; Prevalence ; Sequence Analysis, DNA ; Trematode Infections/*epidemiology/parasitology/ultrasonography

Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-gamma and TNF-alpha in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-alpha in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10+F4/80+ macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.
Animals ; Antigens, Differentiation/analysis ; Clonorchis sinensis/*enzymology ; Colon/pathology ; Cystatins/*metabolism ; Cytokines/secretion ; Dextran Sulfate/toxicity ; Female ; Helminth Proteins/*metabolism ; Immunologic Factors/*metabolism ; Inflammation/chemically induced/*pathology ; Interleukin-10/analysis ; Intestines/*drug effects/pathology ; Lymph Nodes/immunology ; Macrophages/chemistry/*immunology ; Mice ; Mice, Inbred C57BL ; Severity of Illness Index ; Spleen/immunology

Seroprevalence of the IgG antibodies for Clonorchis sinensis, Paragonimus westermani, Taenia solium metacestode (cysticercus), and Spirometra erinacei plerocercoid (sparganum) was measured using enzyme-linked immunosorbent assay (ELISA) in sera of patients in Korea from 1993 to 2006. A total of 74,448 specimens referred nationwide from 121 hospitals revealed an IgG positive rate of 7.6% for the 4 parasites. The IgG positive rate (18.7%) for the 4 parasites in 1993 decreased gradually to 6.6% in 2006. Individual positive rate decreased from 5.2% (1993) to 1.6% (2006) for C. sinensis, from 2.8% (1993) to 1.1% (2006) for P. westermani, from 8.3% (1993) to 2.2% (2006) for cysticercus, and from 2.6% (1993) to 1.6% (2006) for sparganum. The positive rate was highest (21.2%) in the group of patients who ranged in age from 50-59 yr old, and in the group that was referred from the Seoul area (55.9%). In conclusion, our results suggest that tissue invading parasitic infections should always be included in differential diagnosis for patients with eosinophilia associated lesions of the central nervous system, liver, and lungs in Korea.
Adolescent ; Adult ; Age Factors ; Aged ; Aged, 80 and over ; Animals ; Antibodies, Helminth/*blood ; Child ; Child, Preschool ; Clonorchiasis/diagnosis/*epidemiology ; Clonorchis sinensis/immunology/isolation & purification ; Cysticercosis/diagnosis/*epidemiology ; Cysticercus/immunology/isolation & purification ; Diagnosis, Differential ; Enzyme-Linked Immunosorbent Assay ; Eosinophilia/immunology ; Female ; Humans ; Immunoglobulin G/blood ; Infant ; Male ; Middle Aged ; Paragonimiasis/diagnosis/*epidemiology ; Paragonimus westermani/immunology/isolation & purification ; Republic of Korea/epidemiology ; Seroepidemiologic Studies ; Sparganosis/diagnosis/*epidemiology ; Sparganum/immunology/isolation & purification

Paramyosin is a myofibrillar protein present in helminth parasites and plays multifunctional roles in host-parasite interactions. In this study, we identified the gene encoding paramyosin of Clonorchis sinensis (CsPmy) and characterized biochemical and immunological properties of its recombinant protein. CsPmy showed a high level of sequence identity with paramyosin from other helminth parasites. Recombinant CsPmy (rCsPmy) expressed in bacteria had an approximate molecular weight of 100 kDa and bound both human collagen and complement 9. The protein was constitutively expressed in various developmental stages of the parasite. Imunofluorescence analysis revealed that CsPmy was mainly localized in the tegument, subtegumental muscles, and the muscle layer surrounding the intestine of the parasite. The rCsPmy showed high levels of positive reactions (74.6%, 56/75) against sera from patients with clonorchiasis. Immunization of experimental rats with rCsPmy evoked high levels of IgG production. These results collectively suggest that CsPmy is a multifunctional protein that not only contributes to the muscle layer structure but also to non-muscular functions in host-parasite interactions. Successful induction of host IgG production also suggests that CsPmy can be applied as a diagnostic antigen and/or vaccine candidate for clonorchiasis.
Amino Acid Sequence ; Animal Structures/chemistry ; Animals ; Antibodies, Helminth/blood ; Cloning, Molecular ; Clonorchis sinensis/chemistry/*genetics ; Collagen/metabolism ; Complement C9/metabolism ; Helminth Proteins/chemistry/*genetics/immunology/metabolism ; Immunoglobulin G/blood ; Molecular Sequence Data ; Molecular Weight ; Protein Binding ; Rats ; Rats, Sprague-Dawley ; Sequence Alignment ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Tropomyosin/chemistry/*genetics/immunology/metabolism

OBJECTIVETo 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.

METHODSCsESAs 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.

RESULTSAfter 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.

CONCLUSIONIntraperitoneal 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.

Actins ; metabolism ; Animals ; Antigens, Helminth ; immunology ; Clonorchiasis ; parasitology ; Clonorchis sinensis ; immunology ; pathogenicity ; Hepatic Stellate Cells ; pathology ; Liver Cirrhosis ; immunology ; parasitology ; Male ; Rats ; Rats, Wistar

Rats develop strong resistance to re-infection and super-infection by Clonorchis sinensis. The present study investigated the antibodies present in the sera and bile juice of rats that were primary infected and re-infected with C. sinensis. The serum level of specific IgG antibodies, which were elevated 2 wk of the primary infection, peaked at 4 wk and subsequently remained unchanged even during re-infection. The total IgE level in serum increased slowly from 388 ng / ml to 3,426 ng / ml beginning 2 wk after the primary infection, and remained high up to 8 wk but dropped to a normal level (259 ng / ml) after treatment. In resistant re-infected rats, the serum IgE level increased rapidly and peaked within 1 wk, whereas no increase was observed in immunosuppressed rats. The serum level of specific IgA antibodies was elevated beginning 1 wk after infection, and decreased 4 wk after treatment. The total bile IgA level unchanged during the primary infection but increased in treated and re-infected rats. The elevated levels of serum IgE and bile IgA indicate that these immunoglobulins may be correlated with the development of resistance to re-infection by C. sinensis in rats.
Animals ; Antibodies, Helminth/analysis/*blood/*immunology/metabolism ; Bile/*immunology ; Clonorchiasis/blood/*immunology ; Clonorchis sinensis/*immunology/*physiology ; Immunoglobulin A/analysis/blood ; Male ; Rats ; Rats, Sprague-Dawley ; Time Factors