Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Animals ; Antigens, Helminth ; isolation & purification ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; chemistry ; pharmacology ; Gene Expression Regulation ; Hedgehog Proteins ; agonists ; antagonists & inhibitors ; genetics ; immunology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; parasitology ; prevention & control ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; drug effects ; immunology ; Models, Biological ; Monocytes ; cytology ; drug effects ; metabolism ; Pentoxifylline ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; RNA, Messenger ; genetics ; immunology ; Schistosoma japonicum ; chemistry ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology ; Zinc Finger Protein GLI1 ; genetics ; immunology ; Zygote ; chemistry

Strongyloides stercoralis can cause systemic infection, termed strongyloidiasis, and gastrointestinal ulcer disease in immunocompromised patients. However, to our knowledge, there are no reported cases of comorbid gastric adenocarcinoma and S. stercoralis infection. Here, we report a case of an 81-year-old Korean man who presented with S. stercoralis infection coexisting with early gastric adenocarcinoma (T1aN0M0). S. stercoralis eggs, rhabditiform larvae, and adult females were observed in normal gastric and duodenal crypts. They were also observed in atypical glands representative of adenocarcinoma and adenoma. Preliminary laboratory tests revealed mild neutrophilic and eosinophilic leukocytosis. A routine stool test failed to detect rhabditiform larvae in the patient's fecal sample; however, S. stercoralis was identified by PCR amplification and 18S rRNA sequencing using genomic DNA extracted from formalin-fixed paraffin-embedded tissues. Postoperatively, the patient had a persistent fever and was treated with albendazole for 7 days, which alleviated the fever. The patient was followed-up by monitoring and laboratory testing for 4 months postoperatively, and no abnormalities were observed thus far. The fact that S. stercoralis infection may be fatal in immunocompromised patients should be kept in mind when assessing high-risk patients.
Adenocarcinoma/*complications/*diagnosis/pathology ; Aged, 80 and over ; Albendazole/therapeutic use ; Animals ; Anthelmintics/therapeutic use ; DNA, Helminth/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Endoscopy, Digestive System ; Female ; Histocytochemistry ; Humans ; Korea ; Male ; RNA, Ribosomal, 18S/genetics ; Sequence Analysis, DNA ; Stomach Neoplasms/*complications/*diagnosis/pathology ; Strongyloides stercoralis/*isolation & purification ; Strongyloidiasis/*complications/*diagnosis/drug therapy/pathology ; Treatment Outcome

Strongyloides stercoralis can cause systemic infection, termed strongyloidiasis, and gastrointestinal ulcer disease in immunocompromised patients. However, to our knowledge, there are no reported cases of comorbid gastric adenocarcinoma and S. stercoralis infection. Here, we report a case of an 81-year-old Korean man who presented with S. stercoralis infection coexisting with early gastric adenocarcinoma (T1aN0M0). S. stercoralis eggs, rhabditiform larvae, and adult females were observed in normal gastric and duodenal crypts. They were also observed in atypical glands representative of adenocarcinoma and adenoma. Preliminary laboratory tests revealed mild neutrophilic and eosinophilic leukocytosis. A routine stool test failed to detect rhabditiform larvae in the patient's fecal sample; however, S. stercoralis was identified by PCR amplification and 18S rRNA sequencing using genomic DNA extracted from formalin-fixed paraffin-embedded tissues. Postoperatively, the patient had a persistent fever and was treated with albendazole for 7 days, which alleviated the fever. The patient was followed-up by monitoring and laboratory testing for 4 months postoperatively, and no abnormalities were observed thus far. The fact that S. stercoralis infection may be fatal in immunocompromised patients should be kept in mind when assessing high-risk patients.
Adenocarcinoma/*complications/*diagnosis/pathology ; Aged, 80 and over ; Albendazole/therapeutic use ; Animals ; Anthelmintics/therapeutic use ; DNA, Helminth/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Endoscopy, Digestive System ; Female ; Histocytochemistry ; Humans ; Korea ; Male ; RNA, Ribosomal, 18S/genetics ; Sequence Analysis, DNA ; Stomach Neoplasms/*complications/*diagnosis/pathology ; Strongyloides stercoralis/*isolation & purification ; Strongyloidiasis/*complications/*diagnosis/drug therapy/pathology ; Treatment Outcome

OBJECTIVETo examine the expression profile and immunofluorescence localization of the major egg antigen p40 of Schistosoma japonicum (Sjp40) during granuloma formation in the liver of infected New Zealand white rabbits.

METHODSNew Zealand white rabbits were infected with S. japonicum cercariae, and the livers were harvested at 29 and 45 days post-infection (dpi). The total RNA of the liver tissues was extracted for expression profiling of Sjp40 by quantitative reverse transcription-PCR (qRT-PCR) with GAPDH of S. japonicum as the endogenous reference gene. The expression of Sjp40 in the liver were detected by Western blotting using anti-Sjp40 monoclonal antibody (mAb) 9G7 or anti-Toxoplasma gondii tSAG1 mAb Y3A8 (control) as the primary antibody. Paraffin sections of the liver were prepared for observing egg granuloma formation using HE staining and for indirect immunofluorescence assay of Sjp40 location in the trapped eggs and egg granulomas.

RESULTSThe level of Sjp40 mRNA in the eggs trapped in rabbit livers was significantly higher at 45 dpi than that at 29 dpi (P<0.05), and Western blotting confirmed the presence of Sjp40 protein in the rabbit livers at both 29 and 45 dpi. Immunofluorescence assay demonstrated localized expression of Sjp40 in the immature eggs in the rabbit liver at 29 dpi, but at 45 dpi fluorescence was detected in clusters of mature eggs containing miracidium and in the surrounding egg granulomas.

CONCLUSIONSThe transcriptional levels of Sjp40 significantly increased with the maturation of eggs trapped in the rabbit livers. Sjp40 protein spread from the eggs to the surrounding egg granuloma at 45 dpi when acute liver granulomatous lesions occur, suggesting that Sjp40 plays a key role in egg granulomas formation in the livers of infected New Zealand white rabbits.

Animals ; Antibodies, Monoclonal ; Antigens, Helminth ; metabolism ; Fluorescent Antibody Technique ; Gene Expression Profiling ; Granuloma ; parasitology ; Helminth Proteins ; metabolism ; Liver ; parasitology ; RNA, Messenger ; Rabbits ; Schistosoma japonicum ; Schistosomiasis japonica

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples.
Adult ; Animals ; Ascariasis/diagnosis/history/*parasitology ; Ascaris/classification/genetics/*isolation & purification ; Base Sequence ; Cytochromes b/genetics ; DNA Primers/genetics ; DNA, Helminth/*genetics ; DNA, Mitochondrial/*genetics/history ; Female ; Fossils/history/parasitology ; History, Ancient ; Humans ; Male ; Molecular Sequence Data ; Mummies/history/*parasitology ; Ovum/chemistry/classification ; Phylogeny ; RNA, Ribosomal, 18S/genetics

The rumen parasite, Gastrothylax crumenifer (Platyhelminthes: Gastrothylacidae), is a highly pathogenic trematode parasite of goat (Capra hircus). It sucks blood that causes acute disease like anemia, and severe economic losses occur due to morbidity and mortality of the ruminant infected by these worms. The study of these rumen paramphistomes, their infection, and public health importance remains unclear in India especially in the western part of state Uttar Pradesh (U.P.), Meerut, India, where the goat meat consumption is very high. This paper provides the molecular characterization of G. crumenifer recovered from the rumen of Capra hircus from Meerut, U.P., India by the partial sequence of 28S rDNA. Nucleotide sequence similarity searching on BLAST of 28S rDNA from parasites showed the highest identity with those of G. crumenifer from the same host Capra hircus. This is the first report of molecular identification of G. crumenifer from this part of India.
Animals ; Cluster Analysis ; DNA, Helminth/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Goat Diseases/*parasitology ; Goats ; India ; Microscopy, Electron, Scanning ; Molecular Sequence Data ; Phylogeny ; Platyhelminths/*classification/genetics/*isolation & purification/ultrastructure ; RNA, Ribosomal, 28S/genetics ; Rumen/parasitology ; Sequence Analysis, DNA ; Trematode Infections/parasitology/*veterinary

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5+/-0.2degrees C, 79.0+/-0.3degrees C, 76.8+/-0.1degrees C, and 79.9+/-0.1degrees C, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.
Animals ; Blood/*parasitology ; Brugia/classification/genetics/*isolation & purification ; Cats ; Culicidae/*parasitology ; Dirofilaria immitis/classification/genetics/*isolation & purification ; Dogs ; Humans ; Male ; Parasitology/*methods ; RNA, Helminth/genetics ; RNA, Ribosomal, 5S/genetics ; Real-Time Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; Transition Temperature ; Wuchereria bancrofti/classification/genetics/*isolation & purification

The complete coding sequence of Haemonchus (H.) contortus HC29 cDNA was generated by rapid amplification of cDNA ends in combination with PCR using primers targeting the 5'- and 3'-ends of the partial mRNA sequence. The cloned HC29 cDNA was shown to be 1,113 bp in size with an open reading frame of 507 bp, encoding a protein of 168 amino acid with a calculated molecular mass of 18.9 kDa. Amino acid sequence analysis revealed that the cloned HC29 cDNA contained the conserved catalytic triad and dimer interface of selenium-independent glutathione peroxidase (GPX). Alignment of the predicted amino acid sequences demonstrated that the protein shared 44.7~80.4% similarity with GPX homologues in the thioredoxin-like family. Phylogenetic analysis revealed close evolutionary proximity of the GPX sequence to the counterpart sequences. These results suggest that HC29 cDNA is a GPX, a member of the thioredoxin-like family. Alignment of the nucleic acid and amino acid sequences of HC29 with those of the reported selenium-independent GPX of H. contortus showed that HC29 contained different types of spliced leader sequences as well as dimer interface sites, although the active sites of both were identical. Enzymatic analysis of recombinant prokaryotic HC29 protein showed activity for the hydrolysis of H2O2. These findings indicate that HC29 is a selenium-independent GPX of H. contortus.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary/genetics/isolation & purification ; Glutathione Peroxidase/*genetics/*metabolism ; Goat Diseases/parasitology ; Goats ; Haemonchiasis/parasitology/prevention & control/*veterinary ; Haemonchus/*enzymology/*genetics ; Hydrogen Peroxide/metabolism ; Molecular Sequence Data ; Phylogeny ; RNA, Helminth/chemistry/genetics ; Random Amplified Polymorphic DNA Technique ; Rats ; Rats, Sprague-Dawley ; Sequence Alignment ; Sequence Analysis, DNA

The present study was performed to describe 2 human cases infected by the horsehair worm, Parachordodes sp., in Japan. Two gordiid worms were collected in the vomit and excreta of an 80-year-old woman in November 2009 in Kyoto city, and in the mouth of 1-year-old boy in December 2009 in Nara city, Japan, respectively. Both worms were males having bifurcated posterior ends and male gonads in cross sectional specimens. They were identified as Parachordodes sp. (Nematomorpha: Chordodidae) based on the characteristic morphologies of cross sections and areoles in the cuticle. DNA analysis on 18S rRNA partial sequence arrangements was also carried out and both worms were assumed to be close to the genus Paragordionus based on tree analysis, and far from Gordius sp. which has already been reported in humans in Japan. DNA sequencing of the Parachordodes worm does not appear on the database; therefore, more information on the gene sequences of the genus Parachordodes from humans, animals, or intermediates is required.
Aged, 80 and over ; Animals ; Cluster Analysis ; DNA, Helminth/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Female ; Helminthiasis/*diagnosis/*parasitology/pathology ; Helminths/anatomy & histology/classification/genetics/*isolation & purification ; Humans ; Infant ; Japan ; Male ; Microscopy ; Phylogeny ; RNA, Ribosomal, 18S/genetics ; Sequence Analysis, DNA

OBJECTIVETo study the effect of RNA interference (RNAi) on small heat shock protein (sHSP) Sjp40 of Schistosoma japonicum and its synergistic effect on the expression of SjHSP60, SjHSP70, and SjHSP90 mRNA, and observe the mRNA expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 in different stages of S.japonicum.

METHODSDouble-stranded RNA (dsRNA) of Sjp40 (dsSjp40) and a control dsRNA of green fluorescent protein (dsGFP) were generated by in vitro transcription and transfected into adult worm by immersing the worm in dsRNA solution. The total RNA and proteins were isolated simultaneously from the adult worms using TRIzol reagent 7 days after transfection. The expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 mRNA and the expression level of Sjp40 protein were determined by quantitative real-time PCR (qPCR) and Western blotting, respectively. The mRNA expression of HSPs of S. japonicum in different stages was evaluated by qPCR.

RESULTSCompared with those in the control worms transfected with dsGFP, Sjp40 mRNA level was decreased by 80% in the worms transfected with dsSjp40, and the level of Sjp40 protein showed also a significant decrease. The mRNA expression levels of SjHSP60, SjHSP70, and SjHSP90 did not show an obvious synergism after Sjp40 RNAi. The expression profiles of Sjp40, SjHSP60, SjHSP70, and SjHSP90 showed significant differences in different stages of S. japonicum, and the expression level of Sjp40 mRNA in the egg stage was much higher than that of other HSP genes.

CONCLUSIONdsSjp40-RNAi can induce effective suppression of Sjp40 gene expression at both mRNA and protein levels, but no obvious synergism occurs in the mRNA expressions of SjHSP60, SjHSP70, and SjHSP90.

Animals ; Gene Expression Profiling ; Heat-Shock Proteins, Small ; genetics ; Helminth Proteins ; genetics ; RNA Interference ; RNA, Double-Stranded ; RNA, Messenger ; genetics ; Schistosoma japonicum ; genetics