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

Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.
Animal Structures/chemistry ; Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/chemistry/*genetics/immunology/*isolation & purification ; Cloning, Molecular ; Dirofilaria immitis/chemistry/*genetics/immunology ; Disease Models, Animal ; Dogs ; Escherichia coli/genetics ; Gene Expression ; Molecular Sequence Data ; Molecular Weight ; Recombinant Fusion Proteins/chemistry/genetics/immunology/isolation & purification ; Sequence Analysis, DNA ; Tumor Markers, Biological/chemistry/*genetics/immunology/*isolation & purification

Toxocariasis is a worldwide zoonosis caused by larvae of ascarid nematodes of dogs or cats, Toxocara canis or T. cati. Diagnosis of human toxocariasis currently relies on serology that uses T. canis excretory-secretory antigen to detect specific IgG antibodies by ELISA. We investigated the serodiagnostic efficacy of ELISA using crude antigen of T. canis larvae (TCLA). Serum specimens of 64 clinically confirmed toxocariasis, 115 healthy controls, and 119 other tissue-invading helminthiases were screened by ELISA using TCLA. The ELISA using TCLA showed 92.2% (59/64 patient samples) sensitivity and 86.6% (103/119) specificity. Its positive diagnostic predictivity was 78.7% and negative predictivity was 97.8%. No serum of healthy controls reacted but that of anisakiasis (45.5%), gnathostomiasis (19.2%), clonorchiasis (15.8%), sparganosis (11.1%), and cysticercosis (6.3%) cross-reacted. Immunoblot analysis on TCLA recognized antigenic proteins of 28- and 30-kDa bands in their dominant protein quantity and strong blotting reactivity. The present results indicate that the ELISA using our TCLA antigen is acceptable by the sensitivity and specificity for serodiagnosis of human toxocariasis. ELISA with TCLA is recommended to make differential diagnosis for patients with any sign of organ infiltration and eosinophilia.
Adolescent ; Adult ; Aged ; Animals ; Antigens, Helminth/chemistry/*diagnostic use/immunology/isolation & purification ; Cats ; Dogs ; Enzyme-Linked Immunosorbent Assay/*methods ; Female ; Humans ; Larva/chemistry/immunology ; Male ; Middle Aged ; Serologic Tests ; Toxocara canis/chemistry/*immunology ; Toxocariasis/*diagnosis/immunology/parasitology ; Young Adult

The author reported previously on separation of the outer tegument of the spargana (plerocercoids of Spirometra mansoni) using high concentration of urea solution. To determine which layer of the tegument is separated by this method, an electron microscopic analysis has been processed in this study. It was confirmed that the basement layer of the tegument is separated from the parenchyme of the sparganum. In addition, the antigenicity of the separated outer tegument against the human sparganosis patient sera was evaluated. Numerous antigenic proteins, including 16 and 55 kDa proteins, were noticed in the separated tegument; however, there were no diagnostic 31/36 kDa molecules in this tegument. The molecules reactive with the patient sera in the tegument are to be characterized in future studies.
Animal Structures/immunology/ultrastructure ; Animals ; Antigens, Helminth/chemistry/*immunology/isolation & purification ; Helminth Proteins/chemistry/*immunology/isolation & purification ; Humans ; Immunoblotting ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron ; Molecular Weight ; Sparganum/*immunology/*ultrastructure

The present study was performed to estimate the seroprevalence of larval Anisakis simplex infection among the residents health-examined in 3 hospitals in southern parts of Korea. A total of 498 serum samples (1 serum per person) were collected in 3 hospitals in Busan Metropolitan city, Masan city, and Geoje city in Gyeongsangnam-do (Province) and were examined by IgE-ELISA and IgE-western blotting with larval A. simplex crude extract and excretory-secretory products (ESP). The prevalence of antibody positivity was 5.0% and 6.6% with ELISA against crude extracts and ESP, respectively. It was also revealed that infection occurred throughout all age groups and higher in females than in males. A specific protein band of 130 kDa was detected from 10 patients with western blot analysis against crude extract and ESP among those who showed positive results by ELISA. Our study showed for the first time the seroprevalence of anisakiasis in Korea. The allergen of 130 kDa can be a candidate for serologic diagnosis of anisakiasis.
Adolescent ; Adult ; Age Distribution ; Aged ; Aged, 80 and over ; Animals ; Anisakiasis/*epidemiology ; Anisakis/*immunology ; Antibodies, Helminth/*blood ; Antigens, Helminth/chemistry/diagnostic use ; Blotting, Western ; Child ; Enzyme-Linked Immunosorbent Assay ; Female ; Hospitals ; Humans ; Immunoglobulin E/blood ; Korea/epidemiology ; Larva/immunology ; Male ; Middle Aged ; Molecular Weight ; Seroepidemiologic Studies ; Sex Distribution ; Young Adult

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

Differential diagnosis of Taenia asiatica infection from other human taeniases by serology has been tested. An enzyme-linked immunoelectrotransfer blot (EITB) was applied to subjected human sera and tapeworm materials. Thirty-eight proteins reactive to serum IgG were observed between 121 and 10 kDa in adult worms, and more than 22 serum-reactive components between 97 kDa and 21.5 kDa were observed in eggs of T. asiatica. Antigens of adult T. asiatica revealed immunoblot bands between 120 and 21.5 kDa against T. asiatica infected sera. Antigens of adult Taenia saginata revealed 110-100, 66, 58-56, and 46 kDa immunoblot bands against T. asiatica infected sera. Antigens of adult Taenia solium also revealed 99-97, 68-66, and 46 kDa bands against T. asiatica infected sera. The immunoblot band of 21.5 kDa exhibited specificity to T. asiatica.
Animals ; Antibodies, Helminth/immunology ; Antigens, Helminth/chemistry/immunology ; Humans ; Immunoblotting ; Molecular Weight ; Taenia/chemistry/*immunology ; Taeniasis/*immunology/parasitology

After collecting calcareous corpuscles from plerocercoid of Spirometra mansoni (sparganum), we evaluated the antigenic values of calcareous corpuscles binding proteins obtained from the cyst fluid of Taenia solium metacestodes. Immunoblot analysis revealed that cysticercosis patient sera strongly recognized 10 and 95 kDa calcareous corpuscles binding proteins. This result demonstrated that calcareous corpuscles are bound with major secretory antigenic proteins, which is possibly involved in the secretory pathways of the 10 and 95 kDa proteins presenting in the cyst fluid of T. solium metacestodes.
Animals ; Antigens, Helminth/*analysis ; Carrier Proteins/*immunology ; Cysticercosis/diagnosis/immunology ; Helminth Proteins/*immunology ; Humans ; Immunoblotting/methods ; Molecular Weight ; Serologic Tests ; Sparganum ; Taenia solium/*chemistry/immunology

We purified specific 31/36 kDa antigenic molecules from sparganum in different intermediate hosts (snakes and mice) and analyzed their monosaccharide compositions. Compositional analysis showed that glucose and mannose concentrations were 2-3 fold higher in the 31/36 kDa molecule purified from snakes than those from mice. This result implies that antigenic glycoproteins of sparganum from snakes might be modified in mammalian sparganosis with respect to their carbohydrate composition.
Animals ; Antigens, Helminth/*chemistry ; Human ; Mice ; Monosaccharides/*analysis ; Snakes/parasitology ; Sparganum/immunology ; Spirometra/*immunology ; Support, Non-U.S. Gov't

A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.
Animals ; Antigens, Helminth/*analysis/chemistry/immunology/metabolism ; Carbohydrates/analysis/immunology ; Chromatography, Affinity ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Epitopes/analysis/immunology ; Glucosaminidase/metabolism ; Human ; Peptide-N4- (N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism ; Periodic Acid/chemistry ; Sparganosis/*parasitology ; Sparganum/immunology/metabolism ; Spirometra/immunology/*metabolism ; Support, Non-U.S. Gov't