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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.

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.

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.

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.

A new pit viper,PKV was evaluated for its neutralizing capacity (ERIntravenous LDEnzymatic 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.

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.

Material surface properties are considered as critical factor for the study of in Vitro cell growth and activities. It provides advantages not only for study of cellular activity but also for biomedical technology such as tissue engineering. Neuron is a typical cell type widely used to examine cell communications, alignments and differentiations. In the present study, a simple pattern of indium tin oxide (ITO) for neuron culture is introduced. Circular inter-digitated design of ITO platform was fabricated by using simple photolithography techniques. In order to enhance cell attachment, the fabricated electrode surface was modified by poly-L-lysine, a positive charge molecule that allows binding site for cell adhesion. SH-SY5Y cell, a human neuroblastoma cell line, was cultured on the modified surface. Results show that cells preferably adhere on the ITO surface while some of them align itself according to the ITO pattern. In addition, the possibility of fabricated nanostructure platform can indicate the potential control of cell precisely guidance and pattern on surface.

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, 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.

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.

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.

[6]-Gingerol (6 % w/v)-loaded cellulose acetate (12 % w/v CA; MW ~ 3 × 104 g/mol) fibers (375 ± 107 nm) were prepared by electrospinning at 7.5 kV. ATR-FTIR spectra indicated that the mixture was miscible at this composition. Differential scanning calorimetry revealed that [6]-gingerol was uniformly dispersed in the CA matrix and interrupted the hydrogen bond formation among the CA chains. Controlled release study showed that ~97 % of the loaded [6]-gingerol could be released from the loaded fibers to the acetate buffer solution at 37 °C, whereas only ~74 % of it could be done from the corresponding films. About 92 % of [6]-gingerol in the fibers was dramatically released within 4 h. Release was mainly governed by a diffusion-controlled mechanism. The radical scavenging assay showed antioxidant activity of the loaded fibers. The in vitro cytotoxicity test revealed that the viability of L-929 mouse fibroblast cells to the loaded fibers was ~65 %.

The antiviral activities of Houttuynia cordata (H. cordata) aqueous extract against dengue virus serotype 2 (DEN-2), strain 16681, were evaluated in this study. The results showed that pre- and post-incubation of H. cordata extract (10–100 µg/mL) with HepG2 cells significantly reduced intracellular DEN-2 RNA production correlating with the decrease in dengue protein expression. In the direct blocking mode, the extract bound with DEN-2 and strongly inhibited the intracellular viral RNA replication with an effective dose (EC50) of 0.8 µg/mL. Concentrations as low as 10–40 µg/mL of H. cordata extract also exhibited protective effect on virion release from infected LLC-MK2 cells. High-performance liquid chromatography analysis of H. cordata extract indicated that hyperoside was the predominant bioactive compound, and was likely to play a role in this inhibition. The extract was also shown to have no genotoxic effect on human blood cells. PRACTICAL APPLICATIONS Houttuynia cordata is recognized as medicinal vegetable consumed by people in East and Southeast Asia. It has multilateral activities in health promotion and regulation of inflammatory reactions induced by several pathogens. Dengue viral infection is a global health problem since licensed vaccines or specific antiviral drug treatments are not yet available. The current study provides scientific data to support the phytomedicinal properties of H. cordata aqueous extract on anti-dengue virus in three modes of action (prevention, treatment and virucidal). A major constituent in the extract, hyperoside, seems to be the bioactive compound effectively acting against dengue infection. H. cordata aqueous extract was shown to be nontoxic and hyperoside may be considered a lead compound possessing drug potential for further development.

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.

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.

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.

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 1α and interleukin-1β (IL-1β) 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, CD8α, 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.

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.

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 antigenpresenting 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 24 h 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 antigenpresenting cell population that migrates from the epidermis and they are unlikely to be important in the priming of protective CD4 + cells in the sdLN.