High Prevalence of Clonorchis sinensis and Other Zoonotic Trematode Metacercariae in Fish from a Local Market in Yen Bai Province, Northern Vietnam

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.

Bile Ductal Transcriptome Identifies Key Pathways and Hub Genes in Clonorchis sinensis-Infected Sprague-Dawley Rats

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.

Cytokine Production in Cholangiocarcinoma Cells in Response to Clonorchis sinensis Excretory-Secretory Products and Their Putative Protein Components

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.

Characterization of Echinostoma cinetorchis endoribonuclease, RNase H

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.

Expression of MicroRNA in Host Cells Infected with Helicobacter pylori

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.

In Vitro Activity of Diphenyleneiodonium toward Multidrug-Resistant Helicobacter pylori Strains

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.

Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells

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.

Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells

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.

Relationship between Antibody-Positive Rate against Plasmodium vivax Circumsporozoite Protein and Incidence of Malaria

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.

Seroprevalence of Plasmodium vivax in the Republic of Korea (2003-2005) using Indirect Fluorescent Antibody Test

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.

Changes of Cochlear Nerve Terminals after Temporary Noise-Induced Hearing Loss / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Overexposure to intense sound can cause temporary or permanent hearing loss. Post-exposure recovery of thresholds has been assumed to indicate reversal of damage to the inner ear without persistent consequences for auditory function. However, there was a report that acoustic overexposures causing moderate temporary threshold shift caused acute loss of afferent nerve terminals and delayed degeneration of the cochlear ganglion cells while cochlear sensory cells were intact. The purpose of the study was to evaluate the numerical changes of ribbon synapses and efferents to the outer hair cells in ears with temporary noise-induced threshold shifts. MATERIALS AND METHODS: Four-week old CBA mice with normal Preyer's reflexes were used. Mice were exposed to white noise of 110 dB SPL for one hour. Auditory brainstem response (ABR) and distortion-product otoacoustic emission (DPOAE) were recorded before exposure and at four different post-exposure times, 1, 3, 5, and 7 days after noise exposure. Ribbon synapses and efferents near cochlear nerve terminals were stained and calculated in the control group mice at two post-exposure times, 3 and 5 days after the exposure. RESULTS: In the noise-exposed ears, there was no loss of hair cells, in either inner hair cells or outer hair cells. ABR and DPOAE showed maximum threshold shifts after noise-exposure; they returned to the normal pre-exposure values by at day 5. The number of ribbon synapses tended to decrease at 3 days after noise-exposure, but the number of efferent fibers was not statistically different from those of the control mice. CONCLUSION: Our results suggest that the loss of ribbon synapses could be related with the recovery course of temporary threshold shift, even to the point of full hearing recovery.

The relationship between cisplatin resistance and histone deacetylase isoform overexpression in epithelial ovarian cancer cell lines / 부인종양

OBJECTIVE: To investigate the relationship between cisplatin resistance and histone deacetylase (HDAC) isoform overexpression in ovarian cancer cell lines. METHODS: Expression of four HDAC isoforms (HDAC 1, 2, 3, and 4) in two ovarian cancer cell lines, SKOV3 and OVCAR3, exposed to various concentrations of cisplatin was examined by western blot analyses. Cells were transfected with plasmid DNA of each HDAC. The overexpression of protein and mRNA of each HDAC was confirmed by western blot and reverse transcriptase-polymerase chain reaction analyses, respectively. The cell viability of the SKOV3 and OVCAR3 cells transfected with HDAC plasmid DNA was measured using the cell counting kit-8 assay after treatment with cisplatin. RESULTS: The 50% inhibitory concentration of the SKOV3 and OVCAR3 cells can be determined 15-24 hours after treatment with 15 microg/mL cisplatin. The expression level of acetylated histone 3 protein in SKOV3 cells increased after exposure to cisplatin. Compared with control cells at 24 hours after cisplatin exposure, the viability of SKOV3 cells overexpressing HDAC 1 and 3 increased by 15% and 13% (p<0.05), respectively. On the other hand, OVCAR3 cells that overexpressed HDAC 2 and 4 exhibited increased cell viability by 23% and 20% (p<0.05), respectively, compared with control cells 24 hours after exposure to cisplatin. CONCLUSION: In SKOV3 and OVCAR3 epithelial ovarian cancer cell lines, the correlation between HDAC overexpression and cisplatin resistance was confirmed. However, the specific HDAC isoform associated with resistance to cisplatin varied depending on the ovarian cancer cell line. These results may suggest that each HDAC isoform conveys cisplatin resistance via different mechanisms.

Expression profile of histone deacetylases 1, 2 and 3 in ovarian cancer tissues / 부인종양

OBJECTIVE: To investigate the expression levels of histone deacetylase (HDAC) 1, 2, and 3 in ovarian cancer tissues and normal ovarian tissues. METHODS: Randomly assigned each of six patients with serous, mucinous and endometrioid ovarian cancer were included. Another six patients with normal ovarian tissue were included for comparison. RT-PCR was performed to quantify the levels of HDACs1-3 mRNA in the cancer and normal tissues. Western blot analysis was performed to measure the expression levels of HDACs1-3 protein. The HDACs1-3 expression pattern was also topologically examined by immunohistochemistry. RESULTS: Increased mRNA expressions of HDCA1, HDAC 2 and HDAC 3 were detected in 83%, 67% and 83% of 18 cancer tissue samples, compared to normal tissue samples. The relative densities of HDAC1 mRNA and HDAC3 mRNA in the serous, mucinous and endometrioid cancer tissues, and HDAC2 mRNA in serous cancer tissues were significantly higher than those of the normal tissues, respectively (p<0.05). Overexpression of HDAC1, HDAC2 and HDAC3 proteins were detected in 94%, 72% and 83% of 18 cancer samples, respectively. The relative densities of HDAC1 protein and HDAC3 protein in serous, mucinous and endometrioid cancer, and HDAC2 protein in serous and mucinous cancer tissues were significantly higher than those of normal tissues, respectively (p<0.05). Most cancer tissues expressed moderate to strong staining of HDACs1, 2 and 3 in immunohistochemistry. Staining of HDAC2 was weak in only one endometrioid cancer tissue. CONCLUSION: HDACs1-3 are over expressed in ovarian cancer tissues and probably play a significant role in ovarian carcinogenesis.

The expression of isoforms of peroxiredoxin in normal ovary & epithelial ovarian tumor / 대한산부인과학회지

OBJECTIVE: To assess the expression pattern of all six Prxs in normal ovarian tissue and epithelial ovarian tumor cell using immunohistochemical staining. METHODS: Patients were retrieved from those who had undertaken operation in Obstetrics and Gynecology of our hospital from January 1995 to June 2005. According to the pathologic result, five patients were allocated randomly in each group of malignant serous, malignant mucinous, benign serous and benign mucinous ovarian tumor. And another five with normal ovarian epithelial cell were included for the comparison. Immunohistochemical staining was performed with Prx I to VI antibodies. Using microscopy, we evaluated the immunoreactivities of nucleus and cytoplasm semiquantitatively by dividing into four categories : -; no immunoactivity present, +; weak, ++; moderate, +++; strong staining. RESULTS: The immunopositivity of Prx III in cytoplasm shows weak to moderate and Prx VI moderate to strong in normal ovarian tissue. In mucinous epithelial ovarian tumor cell, cytoplasmic Prx IV shows stronger activity than in normal epithelial cell or serous tumor cell. In malignant epithelial cell, Prx V shows stronger activity in cytoplasm than normal epithelial cell. It shows characteristically granular pattern. Prx VI shows stronger activity in the nucleus of malignant epithelial cell compared to normal epithelial cell or benign tumor epithelial cell. CONCLUSION: Normal ovarian tissue showed higher affinity for Prx III and VI. In epithelial ovarian tumor, cytosolic Prx IV in mucinous tumor, cytosolic Prx V and nuclear Prx VI in malignant tumor were overexpressed.