CP-673451, a Selective Platelet-Derived Growth Factor Receptor Tyrosine Kinase Inhibitor, Induces Apoptosis in Opisthorchis viverrini-Associated Cholangiocarcinoma via Nrf2 Suppression and Enhanced ROS.

Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) play essential roles in promoting cholangiocarcinoma (CCA) cell survival by mediating paracrine crosstalk between tumor and cancer-associated fibroblasts (CAFs), indicating the potential of PDGFR as a target for CCA treatment. Clinical trials evaluating PDGFR inhibitors for CCA treatment have shown limited efficacy. Furthermore, little is known about the role of PDGF/PDGFR expression and the mechanism underlying PDGFR inhibitors in CCA related to Opisthorchis viverrini (OV). Therefore, we examined the effect of PDGFR inhibitors in OV-related CCA cells and investigated the molecular mechanism involved. We found that the PDGF and PDGFR mRNAs were overexpressed in CCA tissues compared to resection margins. Notably, PDGFR-α showed high expression in CCA cells, while PDGFR-ß was predominantly expressed in CAFs. The selective inhibitor CP-673451 induced CCA cell death by suppressing the PI3K/Akt/Nrf2 pathway, leading to a decreased expression of Nrf2-targeted antioxidant genes. Consequently, this led to an increase in ROS levels and the promotion of CCA apoptosis. CP-673451 is a promising PDGFR-targeted drug for CCA and supports the further clinical investigation of CP-673451 for CCA treatment, particularly in the context of OV-related cases.

Combined Effects of Fludarabine and Interferon Alpha on Autophagy Regulation Define the Phase of Cell Survival and Promotes Responses in LLC-MK2 and K562 Cells.

Autophagy is a known mechanism of cells under internal stress that regulates cellular function via internal protein recycling and the cleaning up of debris, leading to healthy live cells. However, the stimulation of autophagy by external factors such as chemical compounds or viral infection mostly tends to induce apoptosis/cell death. This study hypothesizes that manipulation of the autophagy mechanism to the pro-cell survival and/or decreased pro-viral niche can be a strategy for effective antiviral and anticancer treatment. Cells susceptible to viral infection, namely LLC-MK2, normal monkey epithelium, and K562, human immune-related lymphocyte, which is also a cancer cell line, were treated with fludarabine nucleoside analog (Fdb), interferon alpha (IFN-α), and a combination of Fdb and IFN-α, and then were evaluated for signs of adaptive autophagy and STAT1 antiviral signaling by Western blotting and immunolabeling assays. The results showed that the low concentration of Fdb was able to activate an autophagy response in both cell types, as demonstrated by the intense immunostaining of LC3B foci in the autophagosomes of living cells. Treatment with IFN-α (10 U/mL) showed no alteration in the initiator of mTOR autophagy but dramatically increased the intracellular STAT1 signaling molecules in both cell types. Although in the combined Fdb and IFN-α treatment, both LLC-MK2 and K562 cells showed only slight changes in the autophagy-responsive proteins p-mTOR and LC3B, an adaptive autophagy event was clearly shown in the autophagosome of the LLC-MK2 cell, suggesting the survival phase of the normal cell. The combined effect of Fdb and IFN-α treatment on the antiviral response was identified by the level of activation of the STAT1 antiviral marker. Significantly, the adaptive autophagy mediated by Fdb was able to suppress the IFN-α-mediated pSTAT1 signaling in both cell types to a level that is appropriate for cellular function. It is concluded that the administration of an appropriate dose of Fdb and IFN-α in combination is beneficial for the treatment of some types of cancer and viral infection.

Distinct cholangiocarcinoma cell migration in 2D monolayer and 3D spheroid culture based on galectin-3 expression and localization.

Introduction: Cholangiocarcinoma (CCA) is difficult to cure due to its ineffective treatment and advanced stage diagnosis. Thoroughly mechanistic understandings of CCA pathogenesis crucially help improving the treatment success rates. Using three-dimensional (3D) cell culture platform offers several advantages over a traditional two-dimensional (2D) culture as it resembles more closely to in vivo tumor. Methods: Here, we aimed to establish the 3D CCA spheroids with lowly (KKU-100) and highly (KKU-213A) metastatic potentials to investigate the CCA migratory process and its EMT-associated galectin-3 in the 3D setting. Results and discussion: Firstly, the growth of lowly metastatic KKU-100 cells was slower than highly metastatic KKU-213A cells in both 2D and 3D systems. Hollow formation was observed exclusively inside the KKU-213A spheroids, not in KKU-100. Additionally, the migration activity of KKU-213A cells was higher than that of KKU-100 cells in both 2D and 3D systems. Besides, altered expression of galectin-3 were observed across all CCA culture conditions with substantial relocalization from inside the 2D cells to the border of spheroids in the 3D system. Notably, the CCA migration was inversely proportional to the galectin-3 expression in the 3D culture, but not in the 2D setting. This suggests the contribution of culture platforms to the alternation of the CCA cell migration process. Conclusions: Thus, our data revealed that 3D culture of CCA cells was phenotypically distinct from 2D culture and pointed to the superiority of using the 3D culture model for examining the CCA cellular mechanisms, providing knowledges that are better correlated with CCA phenotypes in vivo.

Biochemical and proteomic analyses of venom from a new pit viper, Protobothrops kelomohy.

Background: A new pit viper, Protobothrops kelomohy, has been recently discovered in northern and northwestern Thailand. Envenoming by the other Protobothrops species across several Asian countries has been a serious health problem since their venom is highly hematotoxic. However, the management of P. kelomohy bites is required as no specific antivenom is available. This study aimed to investigate the biochemical properties and proteomes of P. kelomohy venom (PKV), including the cross-neutralization to its lethality with antivenoms available in Thailand. Methods: PKV was evaluated for its neutralizing capacity (ER50), lethality (LD50), procoagulant and hemorrhagic effects with three monovalent antivenoms (TAAV, DSAV, and CRAV) and one polyvalent (HPAV) hematotoxic antivenom. The enzymatic activities were examined in comparison with venoms of Trimeresurus albolabris (TAV), Daboia siamensis (DSV), Calloselasma rhodostoma (CRV). Molecular mass was separated on SDS-PAGE, then the specific proteins were determined by western blotting. The venom protein classification was analyzed using mass spectrometry-based proteomics. Results: Intravenous LD50 of PKV was 0.67 µg/g. ER50 of HPAV, DSAV and TAAV neutralize PKV at 1.02, 0.36 and 0.12 mg/mL, respectively. PKV exhibited procoagulant effect with a minimal coagulation dose of 12.5 ± 0.016 µg/mL and hemorrhagic effect with a minimal hemorrhagic dose of 1.20 ± 0.71 µg/mouse. HPAV was significantly effective in neutralizing procoagulant and hemorrhagic effects of PKV than those of TAAV, DSAV and CRAV. All enzymatic activities among four venoms exhibited significant differences. PKV proteome revealed eleven classes of putative snake venom proteins, predominantly metalloproteinase (40.85%), serine protease (29.93%), and phospholipase A2 (15.49%). Conclusions: Enzymatic activities of PKV are similarly related to other viperid venoms in this study by quantitatively hematotoxic properties. Three major venom toxins were responsible for coagulopathy in PKV envenomation. The antivenom HPAV was considered effective in neutralizing the lethality, procoagulant and hemorrhagic effects of PKV.

Baicalein Inhibits Metastatic Phenotypes in Nasopharyngeal Carcinoma Cells via a Focal Adhesion Protein Integrin ß8.

Baicalein, a prominent flavonoid from the indigenous herbal plant Scutellaria baicalensis Georgi, possesses broad-spectrum anticancer activities. However, the biological effects of baicalein on nasopharyngeal carcinoma (NPC) and its underlying mechanisms remain unclarified. Thus, in this study, we examined the effects of baicalein on NPC cell lines and investigated the corresponding molecular mechanism through transcriptome profiling. In the study, four NPC cell lines were treated with various concentrations of baicalein at different time points. Cellular toxicity and proliferative inhibition of baicalein were examined by MTT assay. Metastatic phenotypes of NPC cells were investigated by wound healing, transwell, and adhesion assays. Additionally, microarray experiments were performed to determine the cellular pathways affected by baicalein. The expression and localization of the integrin ß8 were validated by western immunoblotting and immunofluorescence. Our results revealed that baicalein exhibited its cytotoxicity and antiproliferative activity on all tested NPC cell lines. It also significantly inhibited metastatic phenotypes at sub-lethal concentrations. Transcriptomic analysis showed that baicalein significantly affected the focal adhesion pathway in NPC, where integrin ß8 was greatly diminished. Thus, the present study results suggested that baicalein inhibits the metastatic phenotypes of NPC cells by modulating integrin ß8, one of the major molecules in a focal adhesion pathway.

Co-Clinical Trials: An Innovative Drug Development Platform for Cholangiocarcinoma.

Cholangiocarcinoma (CCA), a group of malignancies that originate from the biliary tract, is associated with a high mortality rate and a concerning increase in worldwide incidence. In Thailand, where the incidence of CCA is the highest, the socioeconomic burden is severe. Yet, treatment options are limited, with surgical resection being the only form of treatment with curative intent. The current standard-of-care remains adjuvant and palliative chemotherapy which is ineffective in most patients. The overall survival rate is dismal, even after surgical resection and the tumor heterogeneity further complicates treatment. Together, this makes CCA a significant burden in Southeast Asia. For effective management of CCA, treatment must be tailored to each patient, individually, for which an assortment of targeted therapies must be available. Despite the increasing numbers of clinical studies in CCA, targeted therapy drugs rarely get approved for clinical use. In this review, we discuss the shortcomings of the conventional clinical trial process and propose the implementation of a novel concept, co-clinical trials to expedite drug development for CCA patients. In co-clinical trials, the preclinical studies and clinical trials are conducted simultaneously, thus enabling real-time data integration to accurately stratify and customize treatment for patients, individually. Hence, co-clinical trials are expected to improve the outcomes of clinical trials and consequently, encourage the approval of targeted therapy drugs. The increased availability of targeted therapy drugs for treatment is expected to facilitate the application of precision medicine in CCA.

Proteomics and immunocharacterization of Asian mountain pit viper (Ovophis monticola) venom.

The venomic profile of Asian mountain pit viper Ovophis monticola is clarified in the present study. Using mass spectrometry-based proteomics, 247 different proteins were identified in crude venom of O. monticola found in Thailand. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant proteins included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%), C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). The immunoreactivity of this viper's venom to a monovalent antivenom against green pit viper Trimeresurus albolabris, or to a polyvalent antivenom against hemotoxic venom was investigated by indirect ELISA and two-dimensional (2D) immunoblotting. Polyvalent antivenom showed substantially greater reactivity levels than monovalent antivenom. A titer for the monovalent antivenom was over 1:1.28x107 dilution while that of polyvalent antivenom was 1:5.12x107. Of a total of 89 spots comprising 173 proteins, 40 spots of predominantly SVMP, SVSP and PLA2 were specific antigens for antivenoms. The 49 unrecognized spots containing 72 proteins were characterized as non-reactive proteins, and included certain types of CTLs and CRISPs. These neglected venom constituents could limit the effectiveness of antivenom-based therapy currently available for victims of pit viper envenomation.

Effect of Combining EGFR Tyrosine Kinase Inhibitors and Cytotoxic Agents on Cholangiocarcinoma Cells.

PURPOSE: The potential of members of the epidermal growth factor receptor (ErbB) family as drug targets in cholangiocarcinoma (CCA) has not been extensively addressed. Although phase III clinical trials showed no survival benefits of erlotinib in patients with advanced CCA, the outcome of the standard-of-care chemotherapy treatment for CCA, gemcitabine/cisplatin, is discouraging so we determined the effect of other ErbB receptor inhibitors alone or in conjunction with chemotherapy in CCA cells. MATERIALS AND METHODS: ErbB receptor expression was determined in CCA patient tissues by immunohistochemistry and digital-droplet polymerase chain reaction, and in primary cells and cell lines by immunoblot. Effects on cell viability and cell cycle distribution of combination therapy using ErbB inhibitors with chemotherapeutic drugs was carried out in CCA cell lines. 3D culture of primary CCA cells was then adopted to evaluate the drug effect in a setting that more closely resembles in vivo cell environments. RESULTS: CCA tumors showed higher expression of all ErbB receptors compared with resection margins. Primary and CCA cell lines had variable expression of erbB receptors. CCA cell lines showed decreased cell viability when treated with chemotherapeutic drugs (gemcitabine and 5-fluorouracil) but also with ErbB inhibitors, particularly afatinib, and with a combination. Sequential treatment of gemcitabine with afatinib was particularly effective. Co-culture of CCA primary cells with cancer-associated fibroblasts decreased sensitivity to chemotherapies, but sensitized to afatinib. CONCLUSION: Afatinib is a potential epidermal growth factor receptor targeted drug for CCA treatment and sequential treatment schedule of gemcitabine and afatinib could be explored in CCA patients.

Dysregulation of microRNA in cholangiocarcinoma identified through a meta-analysis of microRNA profiling.

BACKGROUND: In the past decades, the potential of microRNA (miRNA) in cancer diagnostics and prognostics has gained a lot of interests. In this study, a meta-analysis was conducted upon the pooled miRNA microarray data of cholangiocarcinoma (CCA). AIM: To identify differentially expressed (DE) miRNAs and perform functional analyses in order to gain insights to understanding miRNA-target interactions involved in tumorigenesis pathways of CCA. METHODS: Raw data from 8 CCA miRNA microarray datasets, consisting of 443 samples in total, were integrated and statistically analyzed to identify DE miRNAs via comparison of levels of miRNA expression between CCA and normal bile duct samples using t-tests (P < 0.001). The 10-fold cross validation was performed in order to increase the robustness of the t-test results. RESULTS: Our data showed 70 up-regulated and 48 down-regulated miRNAs in CCA. Gene Ontology and pathway enrichment analyses revealed that mRNA targets of DE miRNAs were significantly involved in several biological processes. The most prominent dysregulated pathways included phosphatidylinositol-3 kinases/Akt, mitogen-activated protein kinase and Ras signaling pathways. CONCLUSION: DE miRNAs found in our meta-analysis revealed dysregulation in major cancer pathways involved in the development of CCA. These results indicated the necessity of understanding the miRNA-target interactions and the significance of dysregulated miRNAs in terms of diagnostics and prognostics of cancers.

Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus macrops and T. hageni).

The two venomous pit vipers, Trimeresurus macrops and T. hageni, are distributed throughout Thailand, although their abundance varies among different areas. No species-specific antivenom is available for their bite victims, and the only recorded treatment method is a horse antivenom raised against T. albolabris crude venom. To facilitate assessment of the cross-reactivity of heterologous antivenoms, protein profiles of T. macrops and T. hageni venoms were explored using mass-spectrometry-based proteomics. The results show that 185 and 216 proteins were identified from T. macrops and T. hageni venoms, respectively. Two major protein components in T. macrops and T. hageni venoms were snake venom serine protease and metalloproteinase. The toxicity of the venoms on human monocytes and skin fibroblasts was analyzed, and both showed a greater cytotoxic effect on fibroblasts than monocytic cells, with toxicity occurring in a dose-dependent rather than a time-dependent manner. Exploring the protein composition of snake venom leads to a better understanding of the envenoming of prey. Moreover, knowledge of pit viper venomics facilitates the selection of the optimum heterologous antivenoms for treating bite victims.

Downregulation of ABCA1 and ABCG1 transporters by simvastatin in cholangiocarcinoma cells.

Disturbances in cholesterol homeostasis of the bile duct epithelium, including transport interruption and the hyperaccumulation of intracellular cholesterol can lead to the initiation and progression of cholangiocarcinoma (CCA). Statins, which are lipid-lowering drugs, have been previously documented to exhibit anti-cancer properties. The role of statins in CCA cell cholesterol transport through the expression and function of ATP-binding cassette (ABC) A1 and ABCG1 was investigated in the current study. In four CCA cell lines, ABCA1 and ABCG1 expression was identified. However, neither ABCG5 nor ABCG8 expression was observed. Immunocytochemistry revealed that the expression of ABCA1 was localized in the proximity of the nucleus, while ABCG1 was more dispersed throughout the cytoplasm of KKU-100 cells. A cholesterol efflux assay was performed using bodipy cholesterol, and the translocation of cholesterol via ABCA1 and ABCG1 to Apo-A1 and high density lipoprotein was confirmed, respectively. Simvastatin and atorvastatin demonstrated the inhibitory effects on CCA cell viability. A reduction in intracellular lipid level and a lower expression of ABCA1 and ABCG1 were observed in KKU-100 cells under simvastatin treatment. The pre-exposure of KKU-100 cells to cholesterol diminished the statin effect. Furthermore, when KKU-100 cells were pre-loaded with cholesterol, ABCA1 and ABCG1-mediated exports were unaffected even though they were treated with simvastatin. The results of the current study indicated the limitations of the use of statin in CCA therapy, particularly under hypercholesterolemia conditions.

Andrographolide Inhibits Cholangiocarcinoma Cell Migration by Down-Regulation of Claudin-1 via the p-38 Signaling Pathway.

Andrographolide, a bioactive phytochemical from Andrographis paniculata, is emerging as a promising anticancer agent against various cancers. This study aims to investigate anticancer activities of andrographolide against cholangiocarcinoma (CCA) and to understand the underlying mechanism. The anti-proliferative activity of andrographolide was evaluated in a range of cholangiocarcinoma (CCA) cell lines including HuCCA-1, KKU-100, KKU-M213, and RMCCA-1. The anti-migration activity and the corresponding mechanism were studied in highly metastatic KKU-M213 cells. The results indicated that andrographolide significantly inhibited the proliferation of CCA cells with the 50% inhibitory growth concentration (IC50) of ∼120 µM. Andrographolide also inhibited CCA cell migration and invasion. Our further explorations demonstrated that andrographolide decreased the expression of claudin-1, a major tight junction protein, while it up-regulated the expression of Snail, a transcriptional repressor of claudin-1. Moreover, andrographolide induced the phosphorylation of Jun N-terminus kinase (JNK) and p-38 Mitogen-activated protein kinase (MAPK). Treatment with the p-38-specific inhibitor recovered the claudin-1 expression and migration ability of CCA cells. This work demonstrated the potential anticancer effects of andrographolide, indicating that andrographolide could inhibit CCA cell migration via suppression of claudin-1 through the activation of p-38 MAPK signaling pathway. This compound would be useful for development of alternative therapeutic agent for CCA.

CD207+ Langerhans cells constitute a minor population of skin-derived antigen-presenting cells in the draining lymph node following exposure to Schistosoma mansoni.

Infectious cercariae of Schistosoma mansoni gain entry to the mammalian host through the skin where they induce a transient inflammatory influx of mononuclear cells. Some of these cells have antigen-presenting cell function (MHCII+) and have been reported to migrate to the skin-draining lymph nodes (sdLN) where they have the potential to prime CD4+ cells of the acquired immune response. Here, in mice exposed to vaccinating radiation-attenuated schistosome larvae, which induce high levels of protective immunity to challenge infection, we describe the parasite-induced migration of Langerhans cells (LCs) from the epidermal site of immunisation to the sdLN using a specific monoclonal antibody that recognises langerin (CD207). CD207+ cells with dendritic morphology were abundant in the epidermis at all times and their migration into the dermis was detected soon after vaccination. All CD207+ LCs were MHCII+ but not all MHCII+ cells in the skin were CD207+. LCs migrated from the dermis in enhanced numbers after vaccination, as detected in dermal exudate populations recovered after in vitro culture of skin biopsies. Elevated numbers of CD207+ LCs were also detected in the sdLN from 24h to 4 days after vaccination. However, compared with other dermal-derived antigen-presenting cells that were CD207-MHCII+ or CD207-CD11c+, the relative numbers of CD207+ cells in the dermal exudate population and in the sdLN were very small. Furthermore, the migration of CD207+ cells after exposure to 'protective' radiation-attenuated, compared with 'non-protective' normal cercariae, was similar in terms of numbers and kinetics. Together, these studies suggest that CD207+ LCs are only a minor component of the antigen-presenting cell population that migrates from the epidermis and they are unlikely to be important in the priming of protective CD4+ cells in the sdLN.

An Omics Perspective on Molecular Biomarkers for Diagnosis, Prognosis, and Therapeutics of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy arising from the epithelial bile duct. The lack of early diagnostic biomarkers as well as therapeutic measures results in severe outcomes and poor prognosis. Thus, effective early diagnostic, prognostic, and therapeutic biomarkers are required to improve the prognosis and prolong survival rates in CCA patients. Recent advancement in omics technologies combined with the integrative experimental and clinical validations has provided an insight into the underlying mechanism of CCA initiation and progression as well as clues towards novel biomarkers. This work highlights the discovery and validation of molecular markers in CCA identified through omics approaches. The possible roles of these molecules in various cellular pathways, which render CCA carcinogenesis and progression, will also be discussed. This paper can serve as a reference point for further investigations to yield deeper understanding in the complex feature of this disease, potentially leading to better approaches for diagnosis, prognosis, and therapeutics.

Different Responses in MMP/TIMP Expression of U937 and HepG2 Cells to Dengue Virus Infection.

Disease severities following dengue virus (DV) infection are the result of increased vascular permeability leading to hypovolemic shock. Matrix metalloproteinases (MMPs) are believed to play a key role in promoting such severities. A previous study reported that supernatants of DV-infected dendritic cells (DCs), which contained high levels of MMP-2 and MMP-9, induced vascular leakage in a mouse model. In the present study, we investigated whether hepatocytes (HepG2) and monocytes (U937) could be additional sources of MMPs during DV infection. HepG2 and U937 cells were exposed to DV serotype 2 strain 16681. The secretion of MMP-2 and MMP-9 was detected using gelatin zymography. We found that DV infection in the HepG2 cells promoted MMP-2 production while that in the U937 cells promoted MMP-9 production. Semi-quantitative RT-PCR results also confirmed that DV infection in the HepG2 cells up-regulated the expression of MMP-2 mRNA, whereas that in the U937 cells enhanced the expression of MMP-9 mRNA. We monitored the expression of endogenous TIMP-1 and TIMP-2. DV infection induced TIMP-1 expression in the U937 cells. However, lower expression of TIMP-2 was observed in the infected HepG2 cells. We believed that following DV infection, monocytes and hepatocytes can act as MMP-9 and MMP-2 producers, respectively. Their responses could be attributed to the disturbance of TIMP expression by DV in different cell types.

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication.

Insertion of green fluorescent protein (GFP) encoding-gene into virus genes has provided a valuable tool for flavivirus research. This study aimed to develop dengue virus (DENV) replicons expressing GFP reporter that would provide a fast in vitro system to analyze functional roles of specific DENV sequences in viral replication. Two classes of recombinant replicon constructs were generated; one was a RNA-launched replicon with a GFP gene directly inserted into a full-length DENV genome (FL-DENV/GFP), and the other consisted of 4 types of DNA-launched DENV subgenomic replicons with GFP replacement at various structural genes (Δ-DENV/GFP). The FL-DENV/GFP resulted in GFP expression in transfected cells with no viable DENV being recovered from the transfection. The Δ-DENV/GFP constructs with partial structural gene deletion (ΔC-, ΔCprM/M-, ΔprM/M-, or ΔE-) expressed bright and long lasting GFP. The GFP expression intensity in living cells correlated well with the level of RNA replication. Various mutations in the 5'noncoding region of DENV-2 previously shown to be important genetic determinants for virus replication and mouse virulence were incorporated into the 5 different replicon constructs. Characterizations of 29 mutants demonstrated that these replicons can provide a useful platform for a quick and powerful in vitro system to analyze genetic determinants of DENV replication. These constructs can also be useful for development of vectors expressing foreign genes for various researches.

Estrogen-related fecundity reduction of Lymnaea ollula following Fasciola gigantica infection.

Infection by trematode parasites generally affects life history traits of their intermediate hosts. Reduction in life expectancy and reproductive capacity have previously been documented in Fasciola gigantica-infected Lymnaea ollula, but the influence of the endocrine system on this specific host­parasite interaction has not been previously examined. In the present study, we observed survival, growth pattern, and reproductive output of L. ollula following exposure to F. gigantica. Both the survival and the growth pattern of infected snails were similar to those of the non-infected control group. However, a significant difference was apparent in fecundity, as infected snails consistently showed lower levels of egg and embryo production throughout a 7-wk observation period. Semi-quantitative RT-PCR also revealed down-regulation of estrogen receptor expression in infected snails during the first 4 wk of infection. Nonetheless, the inhibition of estrogen signaling was transient, as they regained expression in the later phase of infection. It is, therefore, suggested that other hormones of the complex endocrine system may be involved in the reduced fecundity of L. ollula following F. gigantica infection.

Expression of ATP-binding cassette multidrug transporters in the giant liver fluke Fasciola gigantica and their possible involvement in the transport of bile salts and anthelmintics.

ATP-binding cassette (ABC) transporters belong to one of the largest protein families that either import or export a wide spectrum of different substrates. Certain members of this superfamily have been implicated in multidrug resistance in various types of cancer as well as in pathogenic microorganisms. The role of ABC proteins in parasitic multidrug resistance becomes increasingly evident. However, studies on ABC transporters in helminths have been limited to MDR1 and MRP orthologues. In the present study, we reported, for the first time, the expression and localization of ABC proteins including orthologues of MDR1, MRP1, BCRP, and BSEP in the giant liver fluke Fasciola gigantica. Furthermore, the functional activities of these ABC transporters were characterized in isolated fluke cells using a fluorescent substrate, rhodamine. The results revealed the inhibition of rhodamine efflux by cyclosporin A, a potent inhibitor of ABC transporters. Interestingly, our data suggested that these proteins might play a role in the export of bile salts, in particular, taurocholate. Although, we did not observe any substantial changes in rhodamine transport in the presence of anthelmintics under experimental conditions, however, our findings altogether shed light on the possible involvement of several members of ABC proteins in the mechanism of drug resistance as well as detoxification process in helminths to survive inside their hosts.

IL-10 regulates early IL-12-mediated immune responses induced by the radiation-attenuated schistosome vaccine.

Radiation-attenuated (RA) schistosomes penetrate the host via the skin where they stimulate intense inflammatory reactions and the release of pro-inflammatory IL-12, important for T(h)1-type immune responses which are partially host protective. However, RA larvae also induce the secretion of regulatory IL-10. We now show that following vaccination of IL-12p40(-/-) mice, abundant IL-10 was produced by in vitro cultured skin biopsies between days 4 and 14, corresponding to the down-regulation of MHC II expression by cells in the dermis and cells that emigrate from the skin. In IL-10(-/-) mice, inflammation of the vaccination site was increased with larger numbers of IL-12p40(+), MHC II(+) and CD86(+) cells in the dermal exudate, and was associated with elevated levels of skin-derived IL-12p40 and IL-1beta. These changes in IL-10(-/-) mice were also reflected by an increased number of cells in the skin-draining lymph nodes (sdLN) and greater levels of lymphocyte proliferation. Moreover, such mice had increased numbers of CD4(+) sdLN cells that were CD25(+), CD28(+) or CD152(+) and accessory cells that were CD40(+) or MHC II(+). Finally, the secretion of IFN-gamma (and IL-12p40) by in vitro cultured sdLN cells was substantially raised in IL-10(-/-) mice, but much reduced in IL-12p40(-/-) mice, resulting in the development of highly polarized T(h)1 and T(h)2 cytokine profiles in the two groups of mice respectively. We conclude that IL-10 has an important role early in the regulation of IL-12-mediated priming of acquired immune responses, and effectively contains excessive dermal inflammation and prevents the development of highly polarized T(h)1-type responses.

Interleukin-12 p40 secretion by cutaneous CD11c+ and F4/80+ cells is a major feature of the innate immune response in mice that develop Th1-mediated protective immunity to Schistosoma mansoni.

Radiation-attenuated (RA) schistosome larvae are potent stimulators of innate immune responses at the skin site of exposure (pinna) that are likely to be important factors in the development of Th1-mediated protective immunity. In addition to causing an influx of neutrophils, macrophages, and dendritic cells (DCs) into the dermis, RA larvae induced a cascade of chemokine and cytokine secretion following in vitro culture of pinna biopsy samples. While macrophage inflammatory protein 1alpha and interleukin-1beta (IL-1beta) were produced transiently within the first few days, the Th1-promoting cytokines IL-12 and IL-18 were secreted at high levels until at least day 14. Assay of C3H/HeJ mice confirmed that IL-12 secretion was not due to lipopolysaccharide contaminants binding Toll-like receptor 4. Significantly, IL-12 p40 secretion was sustained in pinnae from vaccinated mice but not in those from nonprotected infected mice. In contrast, IL-10 was produced from both vaccinated and infected mice. This cytokine regulates IL-12-associated dermal inflammation, since in vaccinated IL-10(-/-) mice, pinna thickness was greatly increased concurrent with elevated levels of IL-12 p40. A significant number of IL-12 p40(+) cells were detected as emigrants from in vitro-cultured pinnae, and most were within a population of rare large granular cells that were Ia(+), consistent with their being antigen-presenting cells. Labeling of IL-12(+) cells for CD11c, CD205, CD8alpha, CD11b, and F4/80 indicated that the majority were myeloid DCs, although a proportion were CD11c(-) F4/80(+), suggesting that macrophages were an additional source of IL-12 in the skin.