Csi-let-7a-5p delivered by extracellular vesicles from a liver fluke activates M1-like macrophages and exacerbates biliary injuries.

Chronic infection with liver flukes (such as Clonorchis sinensis) can induce severe biliary injuries, which can cause cholangitis, biliary fibrosis, and even cholangiocarcinoma. The release of extracellular vesicles by C. sinensis (CsEVs) is of importance in the long-distance communication between the hosts and worms. However, the biological effects of EVs from liver fluke on biliary injuries and the underlying molecular mechanisms remain poorly characterized. In the present study, we found that CsEVs induced M1-like activation. In addition, the mice that were administrated with CsEVs showed severe biliary injuries associated with remarkable activation of M1-like macrophages. We further characterized the signatures of miRNAs packaged in CsEVs and identified a miRNA Csi-let-7a-5p, which was highly enriched. Further study showed that Csi-let-7a-5p facilitated the activation of M1-like macrophages by targeting Socs1 and Clec7a; however, CsEVs with silencing Csi-let-7a-5p showed a decrease in proinflammatory responses and biliary injuries, which involved in the Socs1- and Clec7a-regulated NF-κB signaling pathway. Our study demonstrates that Csi-let-7a-5p delivered by CsEVs plays a critical role in the activation of M1-like macrophages and contributes to the biliary injuries by targeting the Socs1- and Clec7a-mediated NF-κB signaling pathway, which indicates a mechanism contributing to biliary injuries caused by fluke infection. However, molecules other than Csi-let-7a-5p from CsEVs that may also promote M1-like polarization and exacerbate biliary injuries are not excluded.

Evaluation of toxoplasmosis in pregnant women using dot-immunogold-silver staining with recombinant Toxoplasma gondii peroxiredoxin protein.

BACKGROUND: Toxoplasma gondii infection endangers human health and affects animal husbandry. Serological detection is the main method used for epidemiological investigations and diagnosis of toxoplasmosis. The key to effective diagnosis of toxoplasmosis is the use of a standardized antigen and a specific and sensitive detection method. Peroxiredoxin is an antigenic protein and vaccine candidate antigen of T. gondii that has not yet been exploited for diagnostic application. METHODS: In this study, recombinant T. gondii peroxiredoxin protein (rTgPrx) was prepared and used in dot-immunogold-silver staining (Dot-IGSS) to detect IgG antibodies in serum from mice and pregnant women. The rTgPrx-Dot-IGSS method was established and optimized using mouse serum. Furthermore, serum samples from pregnant women were analyzed by rTgPrx-Dot-IGSS. RESULTS: Forty serum samples from mice infected with T. gondii and twenty negative serum samples were analyzed. The sensitivity and specificity of rTgPrx-Dot-IGSS were 97.5 and 100%, respectively, equivalent to those of a commercial ELISA kit for anti-Toxoplasma IgG antibody. Furthermore, 540 serum samples from pregnant women were screened with a commercial ELISA kit. Eighty-three positive and 60 negative serum samples were analyzed by rTgPrx-Dot-IGSS. The positive rate was 95.18%, comparable to that obtained with the commercial ELISA kit. CONCLUSIONS: The Dot-IGSS method with rTgPrx as an antigen might be useful for diagnosing T. gondii infection in individuals.

[Roles and mechanisms of traditional Chinese medicine and its active ingredients in treating epilepsy].

At present,Western medicine is widely used in the treatment of epilepsy.However,about 30%-40% of epileptic patients are resistant to them and are affected by the side effects of these drugs.Traditional Chinese medicine is effective in treating epileptic seizures and relieving complications caused by Western medicine.However,the active ingredients and mechanisms of traditional Chinese medicine remain unclear.This article reviews and summarizes the advances and mechanisms in treating epilepsy,such as Chinese medicine monomer,the extracts of single Chinese medicine and Chinese medicine compound.Chinese medicine monomers,including gastrodin,asarone,rhynchophylline,ligustrazine,tanshinone ⅡA,curcumin,etc.,have antiepileptic effects via regulating excitatory neurotransmitters and receptors,the expression of inflammatory factors,sodium/potassium ion channels and the expression of apoptotic protein,therefore protecting neurons.The extracts of single Chinese herbal including the extracts of Gastrodiae Rhizoma,Acori Tatarinowii Rhizoma,Ginseng Radix et Rhizoma,Ganoderma,Scutellariae Radix and Ginkgo Folium,etc.,have antiepileptic effects related to the inhibition of γ-aminobutyric acid receptor,upregulation of phosphatidylinositol 3-kinase signaling pathway and reduction of glutamate-induced excitotoxicity and oxidative stress response.Furthermore,these extracts can regulate ion channels and reduce oxidative damage of neurons.Chinese medicine compounds including Dianxian Qing Granules,Danxing Ningxian Granules,Huoxue Dingxian formulae,etc.,can improve the therapeutic effect on epilepsy through simultaneously regulating excitatory transmitters,apoptosis factors and cytokines.

Expression and potential roles of IL-33/ST2 in the immune regulation during Clonorchis sinensis infection.

During clonorchiasis, immune responses of hosts are responsible for the removal of the worms and also are involved in the progress of the pathological damage caused by Clonorchis sinensis. Interleukin-33 (IL-33), a recently described cytokine signaling through the ST2 receptor, has emerged as a potent inducer to bile duct proliferation and fibrosis; however, little is known of this signaling in the pathogen-caused periductal inflammation and fibrosis. In the present study, using immunohistochemistry, real-time PCR, enzyme-linked immunosorbent assay (ELISA), and flow cytometry, we studied the expression of IL-33/ST2 during C. sinensis infection, as well as their potential roles in C. sinensis-induced host immune responses. The results showed that a higher level of IL-33 was detected in the sera of patients of clonorchiasis (n = 45), compared with in those of healthy donors (n = 16). Similarly, in FVB mice experimentally infected with C. sinensis, a higher level of IL-33 was detected at latent stage both in the serum and in the liver, as well as the up-regulated expression of ST2 receptor on the inflammatory cells, especially on CD4(+) T cells in the liver of infected mice. Our results, for the first time, indicated that the increased IL-33/ST2 may be involved in the regulation of immunopathology induced by C. sinensis.

Protective Effect of Salvia Przewalskii Extract on Puromycin-Induced Podocyte Injury.

BACKGROUND: To determine the effect of Salvia przewalskii extract (SPE) from total phenolic acids on puromycin aminonucleoside (PAN)-induced rat podocyte injury. METHODS: The rats were divided into groups that were treated with either PAN only or PAN followed by tacrolimus or SPE. We evaluated the effects of SPE on podocyte injury 5, 10, 15 and 21 days following treatment. RESULTS: (1) Proteinuria was observed starting on day 5 in all groups. The peak levels of proteinuria differed among the groups with tacrolimus and high-dose SPE, which significantly decreased proteinuria relative to the PAN and low- and medium-dose SPE groups. The proteinuria in each group decreased by day 15 and returned to a normal level by day 21. (2) H&E and PAS staining revealed no abnormality in glomerular morphology. With electron microscopy, we observed foot process effacement in the rats of all groups starting on day 5, but rats in the tacrolimus and high-dose SPE groups exhibited a lower degree. (3) IHC staining of nephrin and podocin revealed unaffected expression and better linear distributions in the high-dose SPE and tacrolimus groups. Western blot analysis confirmed that SPE could improve the expression of proteins. (4) The mRNA levels of nephrin and podocin in the tacrolimus and high-dose SPE groups were significantly higher than that in the others. CONCLUSION: In our study, we first demonstrated the ability of SPE to reduce proteinuria, preserve the morphology and structure of podocytes and retain the levels of slit diaphragm proteins on PAN-induced rat podocytes injury.

Anti-inflammatory Effect of Astaxanthin on the Sickness Behavior Induced by Diabetes Mellitus.

Chronic inflammation appears to play a critical role in sickness behavior caused by diabetes mellitus. Astaxanthin has been used in treating diabetes mellitus and diabetic complications because of its neuroprotective and anti-inflammatory actions. However, whether astaxanthin can improve sickness behavior induced by diabetes and its potential mechanisms are still unknown. The aim of this study was to investigate the effects of astaxanthin on diabetes-elicited abnormal behavior in mice and its corresponding mechanisms. An experimental diabetic model was induced by streptozotocin (150 mg/kg) and astaxanthin (25 mg/kg/day) was provided orally for 10 weeks. Body weight and water consumption were measured, and the sickness behavior was evaluated by the open field test (OFT) and closed field test (CFT). The expression of glial fibrillary acidic protein (GFAP) was measured, and the frontal cortical cleaved caspase-3 positive cells, interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) expression levels were also investigated. Furthermore, cystathionine ß-synthase (CBS) in the frontal cortex was detected to determine whether the protective effect of astaxanthin on sickness behavior in diabetic mice is closely related to CBS. As expected, we observed that astaxanthin improved general symptoms and significantly increase horizontal distance and the number of crossings in the OFT and CFT. Furthermore, data showed that astaxanthin could decrease GFAP-positive cells in the brain and down-regulate the cleaved caspase-3, IL-6, and IL-1ß, and up-regulate CBS in the frontal cortex. These results suggest that astaxanthin provides neuroprotection against diabetes-induced sickness behavior through inhibiting inflammation, and the protective effects may involve CBS expression in the brain.

Recombinant adeno-associated virus 8-mediated inhibition of microRNA let-7a ameliorates sclerosing cholangitis in a clinically relevant mouse model.

BACKGROUND: Primary sclerosing cholangitis (PSC) is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options. Recombinant adeno-associated virus (rAAV) provides a promising platform for gene therapy on such kinds of diseases. A microRNA (miRNA) let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated. AIM: To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8 (rAAV8) on a xenobiotic-induced mouse model of sclerosing cholangitis. METHODS: A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine (DDC) feeding for 2 wk or 6 wk. A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding. Upon sacrifice, the liver and the serum were collected from each mouse. The hepatobiliary injuries, hepatic inflammation and fibrosis were evaluated. The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot. RESULTS: rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk. The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers, and prevent the proliferation of cholangiocytes and biliary fibrosis. Furthermore, inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1, which consequently inhibit of NF-κB-mediated hepatic inflammation. CONCLUSION: Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis, which provides a possible clinical translation of PSC of human.

Inhibition of GLUD1 mediated by LASP1 and SYVN1 contributes to hepatitis B virus X protein-induced hepatocarcinogenesis.

Glutamate dehydrogenase 1 (GLUD1) is implicated in oncogenesis. However, little is known about the relationship between GLUD1 and hepatocellular carcinoma (HCC). In the present study, we demonstrated that the expression levels of GLUD1 significantly decreased in tumors, which was relevant to the poor prognosis of HCC. Functionally, GLUD1 silencing enhanced the growth and migration of HCC cells. Mechanistically, the upregulation of interleukin-32 through AKT activation contributes to GLUD1 silencing-facilitated hepatocarcinogenesis. The interaction between GLUD1 and AKT, as well as α-ketoglutarate regulated by GLUD1, can suppress AKT activation. In addition, LIM and SH3 protein 1 (LASP1) interacts with GLUD1 and induces GLUD1 degradation via the ubiquitin-proteasome pathway, which relies on the E3 ubiquitin ligase synoviolin (SYVN1), whose interaction with GLUD1 is enhanced by LASP1. In hepatitis B virus (HBV)-related HCC, the HBV X protein (HBX) can suppress GLUD1 with the participation of LASP1 and SYVN1. Collectively, our data suggest that GLUD1 silencing is significantly associated with HCC development, and LASP1 and SYVN1 mediate the inhibition of GLUD1 in HCC, especially in HBV-related tumors.

The role of glutamate receptors in the regulation of the tumor microenvironment.

Glutamate, as one of the most important carbon sources in the TCA cycle, is central in metabolic processes that will subsequently influence tumor progression. Several factors can affect the expression of glutamate receptors, playing either a tumor-promoting or tumor-suppressor role in cancer. Thus, the activation of glutamate receptors by the ligand could play a role in tumor development as ample studies have demonstrated the expression of glutamate receptors in a broad range of tumor cells. Glutamate and its receptors are involved in the regulation of different immune cells' development and function, as suggested by the receptor expression in immune cells. The activation of glutamate receptors can enhance the effectiveness of the effector's T cells, or decrease the cytokine production in immunosuppressive myeloid-derived suppressor cells, increasing the antitumor immune response. These receptors are essential for the interaction between tumor and immune cells within the tumor microenvironment (TME) and the regulation of antitumor immune responses. Although the role of glutamate in the TCA cycle has been well studied, few studies have deeply investigated the role of glutamate receptors in the regulation of cancer and immune cells within the TME. Here, by a systematic review of the available data, we will critically assess the physiopathological relevance of glutamate receptors in the regulation of cancer and immune cells in the TME and provide some unifying hypotheses for futures research on the role of glutamate receptors in the immune modulation of the tumor.

Hepatitis B virus core protein stabilizes RANGAP1 to upregulate KDM2A and facilitate hepatocarcinogenesis.

PURPOSE: As a vital component of the hepatitis B virus (HBV) nucleocapsid, HBV core protein (HBC) contributes to hepatocarcinogenesis. Here, we aimed to assess the effects of RANGAP1 and KDM2A on tumorigenesis induced by HBC. METHODS: Co-immunoprecipitation (Co-IP) combined with mass spectrometry were utilized to identify the proteins with the capacity to interact with HBC. The gene and protein levels of RANGAP1 and KDM2A in hepatocellular carcinoma (HCC) and HBV-positive HCC tissues were evaluated using different cohorts. The roles of RANGAP1 and KDM2A in HCC cells mediated by HBC were investigated in vitro and in vivo. Co-IP and western blot were used to estimate the interaction of HBC with RANGAP1 and KDM2A and assess RANGAP1 stabilization regulated by HBC. RESULTS: We discovered that HBC could interact with RANGAP1 and KDM2A, the levels of which were markedly elevated in HCC tissues. Relying on RANGAP1 and KDM2A, HBC facilitated HCC cell growth and migration. The increased stabilization of RANGAP1 mediated by HBC was relevant to the disruption of the interaction between RANGAP1 and an E3 ligase SYVN1. RANGAP1 interacted with KDM2A, and it further promoted KDM2A stabilization by disturbing the interaction between KDM2A and SYVN1. HBC enhanced the interaction of KDM2A with RANGAP1 and upregulated the expression of KDM2A via RANGAP1 in HCC cells. CONCLUSIONS: These findings demonstrate a novel mechanism by which HBC facilitates hepatocarcinogenesis. RANGAP1 and KDM2A could act as potential molecular targets for treating HBV-associated malignancy.

CsHscB Derived from a Liver Fluke Clonorchis Sinensis Ameliorates Cholestatic Hepatic Fibrosis in a Mouse Model of Sclerosing Cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by inflammatory fibrosis usually involving the whole biliary tree. However, there are very limited treatment options to treat this disease. Our previous study found a lipid-protein rCsHscB from a liver fluke - Clonorchis sinensis, which had full capacities of immune regulation. Therefore, we investigated the role of rCsHscB in a mouse model of sclerosing cholangitis induced by xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to explore whether this protein had potential therapeutic value for PSC. METHODS: Mice were fed 0.1% DDC for 4 weeks and treated with CsHscB (30 µg/mouse, intraperitoneal injection, once every 3 days); the control group was given an equal amount of PBS or CsHscB under normal diet conditions. All the mice were sacrificed at 4 weeks for the evaluation of biliary proliferation, fibrosis, and inflammation. RESULTS: rCsHscB treatment attenuated DDC-induced liver congestion and enlargement and significantly decreased the upregulation of serum AST and ALT levels. The administration of rCsHscB to DDC-fed mice significantly decreased cholangiocyte proliferation and pro-inflammatory cytokine production compared to mice fed with DDC alone. Also, rCsHscB treatment showed a decreased expression of α-SMA in the liver and other markers of liver fibrosis (Masson staining, Hydroxyproline content, and collagen deposit). More interestingly, DDC-fed mice treated with rCsHscB showed a significant up-regulation of PPAR-γ expression, which was similar to control mice, indicating the involvement of PPAR-γ signaling in the protective action of rCsHscB. CONCLUSION: Overall, our data show that rCsHscB attenuates the progression of cholestatic fibrosis induced by DDC and supports the potential for manipulating the parasite-derived molecule to treat certain immune-mediated disorders.

Clonorchis sinensis infection induces hepatobiliary injury via disturbing sphingolipid metabolism and activating sphingosine 1-phosphate receptor 2.

Clonorchis sinensis (C. sinensis) infection induces severe hepatobiliary injuries, which can cause inflammation, periductal fibrosis, and even cholangiocarcinoma. Sphingolipid metabolic pathways responsible for the generation of sphingosine-1-phosphate (S1P) and its receptor S1P receptors (S1PRs) have been implicated in many liver-related diseases. However, the role of S1PRs in C. sinensis-mediated biliary epithelial cells (BECs) proliferation and hepatobiliary injury has not been elucidated. In the present study, we found that C. sinensis infection resulted in alteration of bioactive lipids and sphingolipid metabolic pathways in mice liver. Furthermore, S1PR2 was predominantly activated among these S1PRs in BECs both in vivo and in vitro. Using JTE-013, a specific antagonist of S1PR2, we found that the hepatobiliary pathological injuries, inflammation, bile duct hyperplasia, and periductal fibrosis can be significantly inhibited in C. sinensis-infected mice. In addition, both C. sinensis excretory-secretory products (CsESPs)- and S1P-induced activation of AKT and ERK1/2 were inhibited by JTE-013 in BECs. Therefore, the sphingolipid metabolism pathway and S1PR2 play an important role, and may serve as potential therapeutic targets in hepatobiliary injury caused by C. sinensis-infection.

ß2-Adrenergic Receptor Enhances the Alternatively Activated Macrophages and Promotes Biliary Injuries Caused by Helminth Infection.

The autonomic nervous system has been studied for its involvement in the control of macrophages; however, the mechanisms underlying the interaction between the adrenergic receptors and alternatively activated macrophages (M2) remain obscure. Using FVB wild-type and beta 2 adrenergic receptors knockout, we found that ß2-AR deficiency alleviates hepatobiliary damage in mice infected with C. sinensis. Moreover, ß2-AR-deficient mice decrease the activation and infiltration of M2 macrophages and decrease the production of type 2 cytokines, which are associated with a significant decrease in liver fibrosis in infected mice. Our in vitro results on bone marrow-derived macrophages revealed that macrophages from Adrb2-/- mice significantly decrease M2 markers and the phosphorylation of ERK/mTORC1 induced by IL-4 compared to that observed in M2 macrophages from Adrb2+/+ . This study provides a better understanding of the mechanisms by which the ß2-AR enhances type 2 immune response through the ERK/mTORC1 signaling pathway in macrophages and their role in liver fibrosis.

[Effect of hepatitis B virus X gene on the apoptosis of HepG2 cells].

OBJECTIVE: To investigate the effect of hepatitis B virus X gene on the apoptosis in X gene-transfected HepG2 cells. METHODS: HBX gene eukaryon expression vector pcDNA3.1-X was transfected into HepG2 cells by lipid-mediated transfection to establish HepG2/HBX cell model for HBX expression. HepG2 transfected with pcDNA3.1 was used as controls. At 24 h, 48 h, 72 h and 96 h after transfection, cell apoptotic rates were detected by flow cytometry. RT-PCR and Western blot were applied to evaluate the expression levels of HBX, Fas, FasL, and the levels of p-JNK and p-c-Jun protein. RESULTS: HBX mRNA was detected in HepG2/ HBX cells 24 h after transfection, and the expression of HBX mRNA level was gradually up-regulated after transfection (P < 0.05); whereas no expression of HBX gene in the control cells. At 24 h, 48 h, 72 h and 96 h after transfection, all of the apoptotic rate in HepG2/HBX group were much higher than those in the controls (P < 0.05). The expression levels of Fas and FasL protein as well as the levels of p-JNK and p-c-Jun protein were significantly higher than those in the controls (P < 0.05). A positive correlationship was observed between the expression of HBX mRNA level and the hepatocyte apoptotic rate (P < 0.05), the same correlation of HBX mRNA level with the levels of Fas, FasL, p-JNK and p-c-Jun were also observed. (P < 0.05). CONCLUSION: HBX can induce hepatocyte apoptosis by up-regulating the levels of p-JNK and p-c-Jun to promote the expression of FasL.

TLR4 Deficiency Exacerbates Biliary Injuries and Peribiliary Fibrosis Caused by Clonorchis sinensis in a Resistant Mouse Strain.

Mice with different genetic backgrounds have various susceptibilities to infection with Clonorchis sinensis, although the mechanisms underlying are largely unknown. Toll-like receptor 4 (TLR4) as one of the most important pattern recognition receptors (PPRs) is essential for the invasion, survival, pathogenesis, and elimination of worms. The roles played by TLR4 in C. sinensis infection may vary due to the different genetic backgrounds of mice. In the present study, a relatively resistant mouse strain-C57BL/10 to C. sinensis was used for investigation on the possible roles of TLR4 in the biliary injuries and peribiliary fibrosis. TLR4 wild type (TLR4 wild ) and TLR4 defective (TLR4 def ) mice were orally infected with 45 metacercariae of C. sinensis, and all C. sinensis-infected mice and non-infected groups were anesthetized on day 28 post-infection. The liver and serum from each mouse were collected for assessment of the biliary injuries and biliary fibrosis. Meanwhile, hepatic leukocytes were isolated and detected for the activation of M1 or M2 macrophage using flow cytometry. The hepatic type 1 immune response and type 2 immune responses -relative molecules were also evaluated using ELISA and quantitative PCR. The data showed that TLR4 def aggravated liver inflammatory cell infiltrations, bile duct proliferation, biliary and hepatocellular injuries, and ECM deposition in C. sinensis-infected mice, compared with TLR4 wild mice when they were intragastrically administered with the same amounts of C. sinensis metacercaria. Furthermore, the M2-like macrophages and type 2 immune responses were significantly predominant induced in TLR4 def mice, compared with that of TLR4 wild mice following C. sinensis infection. But the type 1 immune response were significantly decreased in TLR4 def mice, compared with TLR4 wild mice after C. sinensis infection. These data demonstrate that TLR4 deficiency exacerbates biliary injuries and peribiliary fibrosis caused by C. sinensis in C57BL/10 strain mice, which is contributed by augments of type 2 immune responses and decrease pro-inflammatory responses.

Soluble egg antigen of Schistosoma japonicum induces pyroptosis in hepatic stellate cells by modulating ROS production.

BACKGROUND: Inflammation-induced dysfunction of hepatic stellate cells (HSCs) is involved in schistosomiasis-associated liver fibrosis, and soluble egg antigen (SEA) is a crucial pathogen-associated molecular pattern associated with liver injury in schistosomiasis. In addition, numerous studies have shown that caspase-1-mediated pyroptosis participates in the development of multiple inflammation-related diseases. However, whether pyroptotic cell death of HSCs is involved in SEA-mediated liver damage is not well understood. METHODS: Primary cultured HSCs and Schistosoma japonicum-infected mouse liver tissue were analysed for histological changes and caspase-1 activation, and the role of pyroptosis in the mechanisms underlying SEA-induced HSC death was investigated. Accumulation of reactive oxygen species (ROS) in infected livers and SEA-stimulated HSCs was measured by flow cytometry and immunofluorescence. RESULTS: Caspase-1 activity was elevated in both liver tissues and HSCs of S. japonicum-infected mice. Furthermore, SEA stimulation increased the proportion of pyroptotic HSCs, as shown by lactate dehydrogenase (LDH) release assays and by flow cytometric analysis of propidium iodide (PI) and caspase-1 double staining in cells. In addition, ROS generation was elevated in infected liver tissues and SEA-stimulated HSCs, and ROS inhibition downregulated SEA-induced caspase-1 activation and pyroptosis in HSCs. CONCLUSIONS: Our present study demonstrates that pyroptotic cell death in HSCs induced by SEA via ROS-mediated caspase-1 activation may serve as a significant mechanism to initiate the inflammatory response and thereby exacerbate liver injury during S. japonicum infection.

Membrane-bound IgE on B cells is increased during Clonorchis sinensis infection.

A high level of serum IgE is a hallmark of helminthic disease. Secretory IgE can bind FcεRI or FcεRII/CD23. The combination of IgE and FcεRI, a high-affinity interaction, has long received attention and is believed to facilitate helminth control, while the properties of CD23-bound IgE have long been unexplored. Here, we established a Clonorchis sinensis (C. sinensis) infection model with different mouse strains and investigated membrane-bound IgE on B cells during infection. We show that after infection, the increase in CD23 expression on B cells was obvious, even in relatively resistant C57BL/6 mice, as well as in susceptible BALB/c and FVB mice. Although the serum IgE amount was lower in C57BL/6 mice than in BALB/c and FVB mice, the level of IgE binding to peripheral B cells was also elevated. Additionally, the IgE on B cells was soon undetectable in vitro due to dissociable binding. The results of the present study demonstrate the dramatic increase in CD23-bound IgE on B cells after C. sinensis infection. The significance of CD23-bound IgE in Ag transport and presentation has gained consideration in allergy development for its potential ability to promote the Th2 response. Therefore, even though the association of IgE and CD23 is not as substantial as that of IgE and FcεRI, membrane-bound IgE on B cells may be worth further study regarding clonorchiasis and other parasitic infections.

Diversity of Helicobacter pylori isolates in expression of antigens and induction of antibodies.

AIM: To obtain evidence for selection of antigens used in genetically engineered vaccine against Helicobacter pylori (H pylori). METHODS: Enzyme linked immunoabsorbent assay (ELISA) was established on the basis of recombinant protein antigens rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB of H pylori to detect expression rates of the antigens in bacterial isolates as well as positive rates of the antibodies in sera from H pylori-infected patients. PCR was applied to the detection of carrying rates of the genes encoding antigens in the isolates. RESULTS: The outputs of rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB were approximately 35%, 32%, 15%, 23%, 56%, 25% and 20% of the total bacterial proteins, respectively. One hundred and fifty-one strains of H pylori were isolated from 347 biopsy specimens of chronic gastritis, peptic ulcer or gastric adenocarcinoma, with a positive rate of 43.5%. All of the isolates expressed UreB, HpaA, FlaA and FlaB while 52.3%, 92.1% and 93.4% of the isolates expressed VacA, CagA and NapA, respectively. In the sera of 151 H pylori-infected patients, the positive rates of IgG antibodies against UreB, HpaA, VacA, CagA, NapA, FlaA and FlaB were 100%, 87.4%, 43%, 71.5%, 89.4%, 84.8% and 79.5%, respectively. Furthermore, the expression frequencies of VacA and NapA were found to be relative to the severity of gastric diseases (P=0.016 and P<0.0001, respectively). CONCLUSION: UreB antigen is the top option of developing genetically engineered vaccine against H pylori followed by NapA or HpaA.

The Regulatory Roles of Toll-Like Receptor 4 in Secretions of Type 1/Type 2 Relative Cytokines by Splenocytes and Dendritic Cells Exposed to Clonorchis sinensis Excretory/Secretory Products.

The roles of TLR4 in mediation of innate immune response and in regulation of adaptive immune responses triggered by Clonorchis sinensis remain unknown. In the present study, splenocytes derived from C3H/HeN (TLR4 wild ) and C3H/Hej mice (TLR4 mut ) that were infected with 45 metacercariae of C. sinensis were harvested, then stimulated by C. sinensis excretory/secretory products (ESP) or medium (control) for 48 h, respectively. Meanwhile, bone marrow-derived dendritic cells (BMDCs) from normal C3H/HeN and C3H/Hej mice were prepared and stimulated with medium, ESP, LPS, or ESP+LPS for 24 h, respectively. The supernatants were collected, and the concentrations of type 1 and type 2 relative cytokines were determined by ELISA. The maturation of BMDCs indicated by surface markers of CD80, CD86, and MHC II was evaluated by flow cytometry. The results showed that the levels of IFN-γ, IL-6, TNF-α, and IL-10 in the splenocytes from C. sinensis-infected TLR4 mut mice were significantly lower than those from TLR4 wild mice when they were further exposed to ESP. For BMDCs, the productions of the cytokines IL-12p70 and IL-10, but not IL-4, in the BMDCs from TLR4 mutation mice were predominantly decreased compared with those from TLR4 wild mice when the BMDCs were co-stimulated by ESP combined with LPS. Flow cytometry analysis showed that ESP could significantly decrease the high levels of CD80, CD86, and MHC II which were elevated by LPS. In conclusion, these data suggest that TLR4 may play a regulatory role in type 1 immune responses during C. sinensis infection.