A small survey was performed to investigate the recent infection status of Clonorchis sinensis and other zoonotic trematode metacercariae in freshwater fish from a local market of Yen Bai city, Yen Bai province, northern Vietnam. A total of 118 fish in 7 species were examined by the artificial digestion method on March 2016. The metacercariae of 4 species of zoonotic trematodes, i.e., C. sinensis, Haplorchis pumilio, Haplorchis taichui, and Centrocestus formosanus, were detected. The metacercariae of C. sinensis were found in 62 (69.7%) out of 89 fish (5 species), and their intensity of infection was very high, 81.2 per fish infected. Prevalences of 3 intestinal flukes, H. pumilio, H. taichui and C. formosanus, were 75.0%, 47.6%, and 31.7% in positive fish species, respectively, with the metacercarial intensities of 15.5, 10.3, and 2.2 per fish infected. From the above results, it has been confirmed that various species of freshwater fish continue to play the role of the infection source of C. sinensis and other zoonotic trematodes in Yen Bai city, Yen Bai province, northern Vietnam. It is of particular note that the prevalence and intensity of C. sinensis metacercariae are much higher than those reported in previous studies in fish in northern Vietnam.

Clonorchis sinensis is a food-borne trematode that infects more than 15 million people. The liver fluke causes clonorchiasis and chronical cholangitis, and promotes cholangiocarcinoma. The underlying molecular pathogenesis occurring in the bile duct by the infection is little known. In this study, transcriptome profile in the bile ducts infected with C. sinensis were analyzed using microarray methods. Differentially expressed genes (DEGs) were 1,563 and 1,457 at 2 and 4 weeks after infection. Majority of the DEGs were temporally dysregulated at 2 weeks, but 519 DEGs showed monotonically changing expression patterns that formed seven distinct expression profiles. Protein-protein interaction (PPI) analysis of the DEG products revealed 5 sub-networks and 10 key hub proteins while weighted co-expression network analysis (WGCNA)-derived gene-gene interaction exhibited 16 co-expression modules and 13 key hub genes. The DEGs were significantly enriched in 16 Kyoto Encyclopedia of Genes and Genomes pathways, which were related to original systems, cellular process, environmental information processing, and human diseases. This study uncovered a global picture of gene expression profiles in the bile ducts infected with C. sinensis, and provided a set of potent predictive biomarkers for early diagnosis of clonorchiasis.

Clonorchis sinensis is a carcinogenic human liver fluke that promotes hepatic inflammatory environments via direct contact or through their excretory-secretory products (ESPs), subsequently leading to cholangitis, periductal fibrosis, liver cirrhosis, and even cholangiocarcinoma (CCA). This study was conducted to examine the host inflammatory responses to C. sinensis ESPs and their putative protein components selected from C. sinensis expressed sequenced tag (EST) pool databases, including TGF-β receptor interacting protein 1(CsTRIP1), legumain (CsLeg), and growth factor binding protein 2 (CsGrb2). Treatment of CCA cells (HuCCT1) with the ESPs or bacterial recombinant C. sinensis proteins differentially promoted the secretion of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) as well as anti-inflammatory cytokines (IL-10, TGF-β1, and TGF-β2) in a time-dependent manner. In particular, recombinant C. sinensis protein treatment resulted in increase (at maximum) of ~7-fold in TGF-β1, ~30-fold in TGF-β2, and ~3-fold in TNF-α compared with the increase produced by ESPs, indicating that CsTrip1, CsLeg, and CsGrb2 function as strong inducers for secretion of these cytokines in host cells. These results suggest that C. sinensis ESPs contribute to the immunopathological response in host cells, leading to clonorchiasis-associated hepatobiliary abnormalities of greater severity.
Carrier Proteins ; Cholangiocarcinoma ; Cholangitis ; Clonorchis sinensis ; Cytokines ; Fasciola hepatica ; Fibrosis ; Humans ; Interleukin-6 ; Liver Cirrhosis

BACKGROUND/AIMS: MicroRNAs (miRNAs) regulate gene expression. We assess miRNA regulation by Helicobacter pylori infection and elucidate their role in H. pylori-infected gastric epithelial cells. METHODS: The relationship between miRNA expression and DNA methylation was examined. Cells were treated with the nuclear factor-kappaB (NF-κB) inhibitor Bay 11-7082 to determine the relationship between miRNA expression and NF-κB signal transduction. RESULTS: In the negative control cells infected with H. pylori 26695, the expression of six miRNAs was increased, whereas the expression of five miRNAs was decreased. The expression of upregulated miRNAs was increased when the host cells were treated with H. pylori and an NF-κB inhibitor. miR-127-5p, -155, and -181 were associated with increased interleukin 6 (IL-6) secretion in H. pylori infected cells treated with anti-miRNA. The expression of miR-155, -127-5p, -195, -216, -206, and -488 increased by approximately 3-fold following treatment with the methylation inhibitor Aza. CONCLUSIONS: We found novel miR-NAs in H. pylori-infected negative control cells using miRNA microarrays. Upregulated miRNA expression was inversely related to the transcription of NF-κB. miR-195 and miR-488 appear to play a pivotal role in controlling IL-6 activity in H. pylori infection. miRNA expression in H. pylori infection was affected by methylation.
Bays ; Cytokines ; DNA Methylation ; Epithelial Cells ; Gene Expression ; Helicobacter pylori* ; Helicobacter* ; Interleukin-6 ; Methylation ; MicroRNAs* ; NF-kappa B ; Signal Transduction

Echinostoma cinetorchis is an oriental intestinal fluke causing significant pathological damage to the small intestine. The aim of this study was to determine a full-length cDNA sequence of E. cinetorchis endoribonuclease (RNase H; EcRNH) and to elucidate its molecular biological characters. EcRNH consisted of 308 amino acids and showed low similarity to endoribonucleases of other parasites (<40%). EcRNH had an active site centered on a putative DDEED motif instead of DEDD conserved in other species. A recombinant EcRNH produced as a soluble form in Escherichia coli showed enzymatic activity to cleave the 3′-O-P bond of RNA in a DNA-RNA duplex, producing 3′-hydroxyl and 5′-phosphate. These findings may contribute to develop antisense oligonucleotides which could damage echinostomes and other flukes.
Amino Acids ; Catalytic Domain ; DNA, Complementary ; Echinostoma* ; Endoribonucleases ; Escherichia coli ; Intestine, Small ; Oligonucleotides, Antisense ; Parasites ; Ribonuclease H* ; Ribonucleases* ; RNA ; Trematoda

BACKGROUND/AIMS: The increased resistance of Helicobacter pylori to antibiotics has increased the need to develop new treatments for this bacterium. The aim of our study was to identify new drugs with anti-H. pylori activity. METHODS: We screened a small molecule library—the library of pharmacologically active compounds (LOPAC), which includes 1,280 pharmacologically active compounds—to identify inhibitors of H. pylori growth. The minimal inhibitory concentrations (MICs) of antibiotics against multidrug-resistant H. pylori strains were determined using the agar dilution method. RESULTS: We identified diphenyleneiodonium (DPI) as a novel anti-H. pylori agent. The MIC values for DPI were <0.03 μg/mL against all tested H. pylori strains. DPI also exhibited strong antibacterial activity against common gram-negative and gram-positive pathogenic bacteria. CONCLUSIONS: DPI may be a candidate anti-H. pylori drug for future development.
Agar ; Anti-Bacterial Agents ; Bacteria ; Drug Resistance, Multiple ; Helicobacter pylori* ; Helicobacter* ; In Vitro Techniques* ; Methods

Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.
Antibodies ; Bile Ducts ; Cholangiocarcinoma* ; Clonorchiasis ; Clonorchis sinensis* ; Free Radicals ; Humans ; Immunity, Innate ; Inflammation ; RNA, Messenger ; Signal Transduction ; Toll-Like Receptors

Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.
Antibodies ; Bile Ducts ; Cholangiocarcinoma* ; Clonorchiasis ; Clonorchis sinensis* ; Free Radicals ; Humans ; Immunity, Innate ; Inflammation ; RNA, Messenger ; Signal Transduction ; Toll-Like Receptors

The relationship between anti-Plasmodium vivax circumsporozoite protein (CSP) antibody levels and the prevalence of malaria in epidemic areas of South Korea was evaluated. Blood samples were collected from inhabitants of Gimpo-si (city), Paju-si, and Yeoncheon-gun (county) in Gyeonggi-do (province), as well as Cheorwon-gun in Gangwon-do from November to December 2004. Microscopic examinations were used to identify malaria parasites. ELISA was used to quantitate anti-circumsporozoite protein (CSP) antibodies against P. vivax. A total of 1,774 blood samples were collected. The overall CSP-ELISA-positive rate was 7.7% (n=139). The annual parasite incidences (APIs) in these areas gradually decreased from 2004 to 2005 (1.09 and 0.80, respectively). The positive rate in Gimpo (10.4%, 44/425) was the highest identified by CSP-ELISA. The highest API was found in Yeoncheon, followed by Cheorwon, Paju, and Gimpo in both years. The positive rates of CSP-ELISA were closely related to the APIs in the study areas. These results suggest that seroepidemiological studies based on CSP may be helpful in estimating the malaria prevalence in certain areas. In addition, this assay can be used to establish and evaluate malaria control and eradication programs in affected areas.
Adolescent ; Adult ; Antibodies, Protozoan/*blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Incidence ; Malaria, Vivax/*blood/*epidemiology/parasitology ; Male ; Middle Aged ; Plasmodium vivax/*immunology/physiology ; Prevalence ; Protozoan Proteins/immunology ; Republic of Korea/epidemiology ; Seroepidemiologic Studies ; Young Adult

Plasmodium vivax reemerged in the Republic of Korea (ROK) in 1993, and is likely to continue to affect public health. The purpose of this study was to measure levels of anti-P. vivax antibodies using indirect fluorescent antibody test (IFAT) in border areas of ROK, to determine the seroprevalence of malaria (2003-2005) and to plan effective control strategies. Blood samples of the inhabitants in Gimpo-si, Paju-si, and Yeoncheon-gun (Gyeonggi-do), and Cheorwon-gun (Gangwon-do) were collected and kept in Korea Centers for Disease Control and Prevention (KCDC). Out of a total of 1,774 serum samples tested, the overall seropositivity was 0.94% (n=17). The seropositivity was the highest in Paju-si (1.9%, 7/372), followed by Gimpo-si (1.4%, 6/425), Yeoncheon-gun (0.67%, 3/451), and Cheorwon-gun (0.19%, 1/526). The annual parasite incidence (API) in these areas gradually decreased from 2003 to 2005 (1.69, 1.09, and 0.80 in 2003, 2004, and 2005, respectively). The highest API was found in Yeoncheon-gun, followed by Cheorwon-gun, Paju-si, and Gimpo-si. The API ranking in these areas did not change over the 3 years. The seropositivity of Gimpo-si showed a strong linear relationship with the API of 2005 (r=0.9983, P=0.036). Seropositivity data obtained using IFAT may be useful for understanding malaria prevalence of relevant years, predicting future transmission of malaria, and for establishing and evaluating malaria control programs in affected areas.
Antibodies, Protozoan/*blood ; Fluorescent Antibody Technique, Indirect ; Humans ; Incidence ; Malaria, Vivax/*epidemiology ; Plasmodium vivax/*immunology ; Republic of Korea/epidemiology ; Seroepidemiologic Studies