ABSTRACT
@#Proteases of nematodes play a crucial role in larval molting and, in addition to their active role in egg hatching, proteases are also considered a crucial factor in tissue invasion and connective tissue remodeling. In Toxocara canis, proteases play important roles throughout the complex life cycle. They can degrade components of a model of extracellular matrix, basement membranes and different physiological substrates. In the present study, measurements of the proteolytic activity of the perivitelline fluid (PF) surrounding Toxocara canis embryos at different stages of development, the hatching fluid (HF) surrounding the infective larvae, as well as the excretory secretory (ES) products of the larvae in the culture media were performed. Measurements were made using casein as substrate following the Sigma non-specific protease activity assay. The results showed that enzyme activity increased as the embryo matured. The infective larvae were found to continuously produce proteases in the surrounding HF and ES products after in vitro cultivation indicating that Toxocara canis proteases might be important for the worm in the egg and the host. Optimal enzymatic activity was found at pH 8. Incubation of the antiserum from infected mice with the HF and ES products decreased their proteolytic activities, suggesting that there may be a link between the proteases present in these fluids and the immune response.
ABSTRACT
BACKGROUND Angiostrongyliasis is caused by the nematode Angiostrongylus cantonensis and can lead to eosinophilic meningitis and meningoencephalitis in humans. The young adult worms play central pathogenic roles in the central nervous system (CNS); however, the underlying mechanism is unclear. Excretory-secretory products (ESPs) are good investigation targets for studying the relationship between a host and its parasite. OBJECTIVES We aimed to profile, identify, and characterise the proteins in the ESPs of A. cantonensis young adults. METHODS The ESPs of young adult worms were collected from culture medium after incubation ranging from 24 to 96 h. Proteomic and bioinformatics analyses were performed to characterise the ESPs. FINDINGS A total of 51 spots were identified, and the highly expressed proteins included two protein disulphide isomerases, one calreticulin, and three uncharacterised proteins. Subsequently, approximately 254 proteins were identified in the ESPs of A. cantonensis young adults via liquid chromatography-mass spectrometry (LC-MS/MS) analysis, and these were further classified according to their characteristics and biological functions. Finally, we identified the immunoreactive proteins from a reference map of ESPs from A. cantonensis young adults. Approximately eight proteins were identified, including a protein disulphide isomerase, a putative aspartic protease, annexin, and five uncharacterised proteins. The study established and identified protein reference maps for the ESPs of A. cantonensis young adults. MAIN CONCLUSIONS The identified proteins may be potential targets for the development of diagnostic or therapeutic agents for human angiostrongyliasis.
Subject(s)
Humans , Adolescent , Adult , Parasite Egg Count , Angiostrongylus cantonensis/parasitology , Feces/parasitologyABSTRACT
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.
Subject(s)
Humans , Carrier Proteins , Cholangiocarcinoma , Cholangitis , Clonorchis sinensis , Cytokines , Fasciola hepatica , Fibrosis , Interleukin-6 , Liver CirrhosisABSTRACT
We investigated the roles of the crude antigen(CA) of Clonorchis sinensis and excretory secretory products (ESPs) in the polarization of Th1 and Th2 cells.Bone marrow-derived cells were generated from BALB/c mice and isolated into immature DCs;immature DCs were then treated with either CA (CA stimulated group),ESPs (ESPs stimulated group),LPS (positive control group) or PBS (negative control group) for 24 hours.Then the CD4+T cells were isolated from mouse spleen by using anti mouse-CD4 Microbeads,and further cocultured with stimulated DCs for another 72 hours.The purities of DCs and CD4+ T cells were evaluated by flow cytometry and the expressing levels of T-bet mRNA and GATA-3 mRNA were detected by real-time PCR.ELISA was used to detect the levels of IFN-γ and IL-4 cytokines in the supernatant.mRNA levels of T-bet and GATA-3 in the ESPs group were higher than those in PBS-stimulated group (P＜0.05).The concentrations of IFN-γ and IL-4 cytokines in the culture were increased in the ESPs group,compared with PBS stimulated group(P＜0.05).IFN-γ but not IL-4 was increased in CA group (P＜0.05).The results implied that CA might play a role in Th1 type immune response,and ESPs likely play roles in both Th1 and Th2 immune responses.
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Objective To observe the effect of excretory/secretory products from Trichinella spiralis adult worms(AES)on cecal ligation and puncture(CLP)?induced sepsis in mice. Methods Forty?eight BALB/c mice were randomly divided into 3 groups:a sham operation group(PBS+sham group,Group A),a CLP?induced sepsis group(PBS+CLP group,Group B)and an AES treatment group(AES+ CLP group,Group C). The mice of each group were intraperitoneally injected with 25 μg of AES or PBS only as a control in a total volume of 200μl. Eight mice from each group were selected randomly for survival analy?sis of 96 hours. The other 8 mice in each group were observed for pathological changes in the lung,liver and kidney tissues by HE staining 12 h after CLP,and then determined for the detection of cytokines including TNF?α,IL?1β,IL?6,IL?10 and TGF? βin the sera by ELISA. Results The difference among the survival rates of mice in the 3 groups was statistically significant (χ2=21.16,P<0.05). Compared to Group A(100%),the survival rate of mice in Group B(0)decreased significantly(P<0.05),and also the pathological damage degrees in the lung,liver and kidney tissues of the mice in Group B increased signifi?cantly after CLP. Compared with the mice in group B,the survival rate of those in Group C(70%)increased significantly(P<0.05),and the pathological damage degrees in the lung,liver and kidney tissues of the mice in Group C decreased significantly after the treatment with AES. The differences among the levels of pro?inflammatory cytokines TNF?α(F=27.11,P<0.05),IL?1β(F=18.75,P<0.05)and IL?6(F=100.93,P<0.05)in the sera of the mice in the three groups were statistically signifi?cant. Compared with the mice in Group A,the levels of the 3 cytokines of those in Group B increased significantly(all P <0.05). However,after the treatment with AES,the levels of the pro?inflammatory cytokines of those in Group C decreased signifi?cantly(all P<0.05). The differences among the levels of immunoregulatory cytokines IL?10(F=10.88,P<0.05)and TGF?β(F=11.37,P<0.05)in the sera of the mice in the three groups were also statistically significant. Compared with the mice in Group B,the levels of IL?10 and TGF?β of those in Group C were higher after treatment with AES(both P<0.05). Conclu?sion T. spiralis AES has a therapeutic potential for alleviating sepsis induced by CLP in mice.
ABSTRACT
Venezuela se encuentra entre los países sudamericanos afectados por la esquistosomiasis y la quimioterapia con praziquantel (PZQ) es la principal estrategia de control. Se determino el efecto cuantitativo del tratamiento con praziquantel sobre la actividad de la Fosfatasa Alcalina (ALP), Fosfatasa Acida (ACP) y Superoxido Dismutasa (SOD), en antígenos solubles (ASG) y productos de excreción-secreción (PESG) de gusanos hembras y machos condición control (ASGHc, ASGMc, PESGHc and PESGMc) o incubados con PZQ in vitro (ASGMpzq, ASGHpzq, PESGMpzq and PESGHpzq). Las proteínas totales se determinaron por colorimetría, la SOD y ACP mediante espectrofotometría y la ALP por fluorometría. Se encontró una mayor concentración de proteínas en las ASG de gusanos no tratados, y en las preparaciones obtenidas luego de la incubación con PZQ in vitro, en los PESG, un incremento en la actividad ACP en los ASG y PESG preparados con gusanos no-tratados, y una disminución de dicha actividad en los ASG y PESG tratados. La SOD, evidenció en los ASG una disminución estadísticamente significativa en los gusanos tratados. La concentración de la ALP disminuyó significativamente en los ASG y PESGH de gusanos tratados en relación a los gusanos no tratados. En conclusión, se observó una disminución en las proteínas totales, actividades enzimáticas ACP y SOD, y concentración de ALP, en ASG y PESG obtenidos con gusanos tratados.
Venezuela is among South American countries affected by schistosomiasis and chemotherapy with praziquantel (PZQ) is the main control strategy. We determined the quantitative effect of treatment with PZQ on alkaline phosphatase activity (ALP), acid phosphatase (ACP) and superoxide dismutase (SOD) in soluble antigens of worms (SWAP) and excretion-secretion products (EEP) of male and female worms (SMWAPc, SFWAPc, ESPWMc and ESPWHc) or incubated with PZQ in vitro (SMWAP PZQ, SFWAP PZQ, ESPWM PZQ and ESPWH PZQ). Total proteins were determined by colorimetry, SOD and ACP by spectrophotometry and fluorometry ALP. There was higher protein concentration in the untreated worms EG, and the preparations obtained after incubation with PZQ in vitro, in the EG, an increase in ACP activity in the EG and PG prepared with non-treated worms and a decrease of such activity on the EG and treated PG. On the other hand, SOD activity, the EG showed statistical significance in the treated worms. In the PG showed the same behavior, but those differences were not statistically significant. Similarly, there was a decrease in the concentration of ALP noticeable in the EG and worm PGh treated worms relative to untreated statistically significant. In conclusion, we observed a decrease in total protein, ACP and SOD enzyme activities and concentration of ALP, and EG in PG treated worms.
ABSTRACT
Clonorchis sinensis is one of the most prevalent parasitic helminths in Korea. Although cholangiocarcinoma can be induced by C. sinensis infection, the underlying mechanism is not clearly understood. To assess the role of C. sinensis infection in carcinogenesis, an in vitro system was established using the human epithelial cell line HEK293T. In cells exposed to the excretory/secretory products (ESP) of C. sinensis and the carcinogen dimethylnitrosamine (DMN), cellular proliferation and the proportion of cells in the G2/M phase increased. Moreover, the expression of the cell cycle proteins E2F1, p-pRb, and cyclin B was dramatically increased when ESP and DMN were added together. Similarly, the transcription factor E2F1 showed its highest level of activity when ESP and DMN were added simultaneously. These findings indicate that DMN and ESP synergistically affect the regulation of cell cycle-related proteins. Our results suggest that exposure to C. sinensis and a small amount of a carcinogen such as DMN can promote carcinogenesis in the bile duct epithelium via uncontrolled cellular proliferation and the upregulation of cell cycle-related proteins.
Subject(s)
Animals , Humans , Carcinogens/metabolism , Cell Cycle/drug effects , Cell Line , Cell Proliferation , Clonorchis sinensis/metabolism , Dimethylnitrosamine/toxicity , Epithelial Cells/drug effectsABSTRACT
Eosinophil degranulation is considered to be a key effector function for the killing of helminthic worms and tissue inflammation at worm-infected lesion sites. However, relatively little data are available with regard to eosinophil response after stimulation with worm-secreted products which contain a large quantity of cysteine proteases. In this study, we attempted to determine whether the degranulation of human eosinophils could be induced by the direct stimulation of the excretory-secretory products (ESP) of Paragonimus westermani, which causes pulmonary paragonimiasis in human beings. Incubation of eosinophils for 3 hr with Paragonimus-secreted products resulted in marked degranulation, as evidenced by the release of eosinophil-derived neurotoxin (EDN) in the culture supernatants. Moreover, superoxide anion was produced by eosinophils after stimulation of the ESP. The ESP-induced EDN release was found to be significantly inhibited when the ESP was pretreated with protease inhibitor cocktail or the cysteine protease inhibitor, E-64. These findings suggest that human eosinophils become degranulated in response to P. westermani-secreted proteases, which may contribute to in vivo tissue inflammation around the worms.
Subject(s)
Animals , Humans , Cell Degranulation , Cysteine Endopeptidases/metabolism , Eosinophil-Derived Neurotoxin/metabolism , Eosinophils/physiology , Paragonimus westermani/enzymology , Superoxides/metabolism , Time FactorsABSTRACT
The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.