Shunyu Li

Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR

The accuracies of relative gene expressions as determined by quantitative real-time polymerase chain reaction are largely dependent on the variabilities of the reference genes used. Validation of the stabilities of reference genes under experimental conditions is an essential initial step for comparative studies on the expression levels of target genes in experimental groups. Using three total RNA samples extracted independently from Clonorchis sinensis metacercariae and adults, we determined the gene expression stabilities of eight reference gene candidates and the relative transcript levels of three target genes using the geNorm program. The reference genes found to be stably expressed in metacercariae and adults were phosphoglycerate kinase, β-actin, and calcyphosine; reference genes found to be stably expressed under γ-irradiated and non-irradiated conditions were succinate dehydrogenase, small nuclear ribonucleoprotein, and β-actin; and those stably expressed regardless of bile treatment were small nuclear ribonucleoprotein, phosphoglycerate kinase, and succinate dehydrogenase. According to our data, the expression levels of target genes are dependent on normalization factors, such as the CT values of single reference genes and the geometric mean of the CT values of three reference genes. When comparing C. sinensis gene expressions, we propose to employ the geometric mean of the CT values of more than three reference genes validated in the same experimental setting.

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.

Bile components and amino acids affect survival of the newly excysted juvenile Clonorchis sinensis in maintaining media

Clonorchis sinensis thrives on bile juice. The effects of bile and bile acids on newly excysted juvenile C. sinensis (CsNEJ) were studied in terms of survival. Survival of CsNEJs maintained in 1× Locke’s solution, Dulbecco’s modified Eagle’s medium, NCTC 109, Eagle’s, RPMI 1640, and 0.1% glucose was high, but dropped rapidly in 2× Locke’s, 0.85% NaCl, and phosphate-buffered saline. Most amino acids in the media favored CsNEJ survival; however, aspartic and glutamic acids and adenine reduced survival. Survival was also significantly lower in media containing more than 0.1% bile. CsNEJs preconditioned in low bile media survived longer in higher bile media. All bile acids and conjugated bile salts were found to favor CsNEJ survival, except for lithocholic acid (LCA) which was toxic. NCTC 109 medium was found to be optimal for the in vitro maintenance of CsNEJs and 1× Locke’s solution to be suitable for analyzing the biological effects of bioactive compounds and molecules. Based on these results, we propose that bile acids enhance activity of CsNEJs, but LCA deteriorate CsNEJs.

Changing Patterns of Serum and Bile Antibodies in Re-infected Rats with Clonorchis sinensis

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.

In Vitro Maintenance of Clonorchis sinensis Adult Worms

Clonorchis sinensis is a biological carcinogen inducing human cholangiocarcinoma, and clonorchiasis is one of the important endemic infectious diseases in East Asia. The present study investigated survival longevity of C. sinensis adult worms in various in vitro conditions to find the best way of keeping the worms longer. The worms were maintained in 0.85% NaCl, 1×PBS, 1×Lockes solution, RPMI-1640, DMEM, and IMDM media, and in 1×Lockes solution with different supplements. All of the worms died within 3 and 7 days in 0.85% NaCl and 1×PBS, respectively, but survived up to 57 days in 1×Lockes solution. The worms lived for 106 days in DMEM, and 114 days in both RPMI-1640 and IMDM media. The survival rate in RPMI-1640 medium was the highest (50%) compared to that in DMEM (20±10%) and in IMDM (33.3±25.2%) after 3 months. The 1×Lockes solution with 0.005% bovine bile supplement showed increased duration of maximum survival from 42 days to 70 days. Higher concentration of bile supplements than 0.005% or addition of glucose were disadvantageous for the worm survival. The worms died rapidly in solutions containing L-aspartic acid, L-glutamic acid, and adenine compared to L-arginine, L-serine, and L-tryptophan. In conclusion, the 1×Lockes solution best supports the worms alive among inorganic solutions for 57 days, and the RPMI-1640 medium maintains living C. sinensis adults better and longer up to 114 days in vitro than other media.

Factors in the resistance of rats to re-infection and super-infection by Clonorchis sinensis

Rats develop resistance to re-infection by Clonorchis sinensis while humans do not. We investigated factors involved in the development of resistance to re-infection and super-infection in rats. Sprague–Dawley rats were infected by C. sinensis metacercariae and treated with praziquantel, then re-infected after immune modulation. The rats were also subjected to super-imposed infection after primary infection. Resistance to re-infection was observed by lowered rates of worm recovery after various durations from treatment; 1% at 5days and 24.4% at 11months vs 56.2% in the control. Similar significant resistance was observed in the super-infected rats after 3weeks of primary infection. The re-infected or super-infected worms grew very slowly and remained immature. Resistance disappeared in immune-suppressed and nude rats but not in splenectomized rats. Immunization with antigens of C. sinensis produced no resistance. Local tissue reaction and immune response in the infected bile duct may play an important role in the resistance, although the crowding effect may also be implicated in resistance in super-infected rats.

Bile-induced genes in Clonorchis sinensis metacercariae

Bile stimulates many intestinal parasites, and newly excysted juvenile Clonorchis sinensis (CsNEJ) responds chemotactically to bile and matures in the bile duct. In this study, using annealing control primer-based polymerase chain reaction (PCR), 16 differentially expressed genes (DEGs) were found to be upregulated in C. sinensis metacercariae incubated in bile. Using contigs retrieved from a C. sinensis-expressed sequence tag pool, DEG sequences were extended further by DNA-walking. Of these, five DEGs were annotated to functional genes and confirmed to have been upregulated by more than twofold by quantitative real-time PCR. The gene products of these DEGs were cytochrome c oxidase subunit 2, phosphoenolpyruvate carboxykinase 2, and mitochondrial phosphate carrier protein, which are involved in energy generation, and HLA-B-associated transcript 3 and zinc finger protein, which are regulatory proteins associated with apoptosis and/or proliferation signaling pathways. Based on these results, it is suggested that bile stimulates the expressions of genes that produce the energy required by CsNEJs to migrate to the bile duct and to modulate the regulatory signals of cell proliferation associated with adult development.

Molecular cloning and characterization of WD40-repeat protein from Clonorchis sinensis

WD40-repeat proteins have four to eight repeating units flanked by Gly-His (GH) and Trp-Asp (WD) at both termini and folds into a β-propeller. A polypeptide deduced from a Clonorchis sinensis cDNA clone analyzed to have seven WD40-repeats and predicted to form a β-propeller (CsWD1). The CsWD1 protein was expressed stage-specifically in the metacercariae and localized in the tegumental syncytium. The CsWD1 protein is suggested to serve as a platform for interacting partner proteins in the tegumental syncytium of C. sinensis metacercariae.

Partner proteins that interact with Clonorchis sinensis WD40-repeat protein.

WD40-repeat proteins have four to eight repeat units, which have Gly–His (GH) and Trp–Asp (WD) at both termini and fold into a beta-propeller. In particular, the WD40-repeat protein of Clonorchis sinensis (CsWD1) has seven WD-repeat units and is expressed stage-specifically in metacercariae. By yeast two-hybrid screening, putative interacting protein cDNAs were cloned from a C. sinensis metacercaria cDNA library and purified further by higher stringency screening and lacZ colony-lift assay. After assessing their nucleotide and polypeptide sequences, 21 putative partner protein cDNAs were selected and assembled into 14 clones. Using YRG2 strain yeast, 12 putative partner protein clones were confirmed to interact with CsWD1 protein. These 12 proteins were grouped into functional categories, i.e., signal proteins, transporters, proteases, and muscle proteins. These results suggest that CsWD1 protein is associated with intracellular protein translocation and cell cycle control in C. sinensis metacercaria.

Influencing Factors for Cure of Clonorchiasis by Praziquantel Therapy: Infection Burden and CYP3A5 Gene Polymorphism

Chemotherapy of clonorchiasis with praziquantel (PZQ) is effective but about 15% of treated cases have been reported uncured. The present study investigated correlation of single nucleotide polymorphisms (SNPs) of the cytochrome P450 gene, CYP3A5 and cure of clonorchiasis. A total of 346 egg passing residents were subjected and treated by 3 doses of 25 mg/kg PZQ. Reexamination recognized 33 (9.5%) uncured and 313 cured. Numbers of eggs per gram of feces (EPGs) before treatment were significantly lower in the cured group than in the uncured group (2,011.2±3,600.0 vs 4,998.5±7,012.0, P<0.001). DNAs of the subjects were screened for SNPs at 7 locations of CYP3A5 using PCR. In the uncured group, the SNP frequencies at g.-20555G>A and g.27526C>T of CYP3A5 were 15.2% and 9.1% while those were 3.8% and 1.0%, respectively, in the cured group. The cure rate was significantly lower in the cases with SNP at g.27526C>T and EPGs≥1,000. In conclusion, EPGs and SNPs of CYP3A5 are factors which influence cure of clonorchiasis by PZQ therapy. It is strongly suggested to recommend 2-day medication for individuals with high EPGs≥1,000.