Discovery of Serotransferrin Glycoforms: Novel Markers for Diagnosis of Liver Periductal Fibrosis and Prediction of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) caused by chronic liver fluke infection is a major public health problem in Northeast Thailand. Identification of CCA risk groups is urgently needed for the control of CCA in this region. Periductal fibrosis (PDF) induced by chronic inflammation of bile ducts is known as a pre-neoplastic lesion of CCA. We aimed to identify the serum CCA and PDF biomarkers using mass spectrometry (UPLC-ESI-QqQ) with multiple reaction mode (MRM) analysis. Here, serum levels of serotransferrin glycoforms at the glycopeptide level were measured in the sera of CCA (n = 100), PDF (n = 50), and healthy control (n = 100) subjects. The results indicated that serotransferrin peptide levels were generally the same between the control and PDF groups, whereas CCA patients had reduced levels. Moreover, 56 serotransferrin glycoforms were detected, with nine increased in CCA compared to control subjects. Among them, the serum levels of four glycoforms were increased in PDF and CCA patients compared to control subjects. In particular, highly sialylated multi-branched glycans of serotransferrin serum were significantly correlated with poor prognosis and tumor stage in CCA patients. Taken together, these glycoforms could be used as risk biomarkers and prognosis and diagnosis markers of CCA

Long-term persistence of Opisthorchis viverrini antigen in urine: a prospective study in northeast Thailand

Antigen detected in urine for the diagnosis of opisthorchiasis has a low daily variation; however, the longer term variability in antigen concentrations is unknown. In this study, we prospectively monitored Opisthorchis viverrini antigen concentrations for 30 consecutive days and at subsequent monthly intervals in a cohort of opisthorchiasis-positive individuals. On the basis of the monoclonal antibody–based ELISA, the profiles of antigen-positive rate and antigen concentration exhibited no significant change over 30 days with a mean proportion positive of 87.1% (range 73.7%–100%), and the average antigen concentration was 29.7 ± 2.2 ng/mL (mean ± SE). The urine antigen concentration at baseline was similar to the subsequent measurements at 2, 4, 6, and 10 months in the follow-up study (P > 0.05). The consistency and low daily and long-term fluctuation of O. viverrini antigen in urine demonstrates the reliability of urine assay for diagnosis of opisthorchiasis

Production and characterization of antibody against Opisthorchis viverrini via phage display and molecular simulation.

In this study, a key issue to be addressed is the safe disposal of hybridoma instability. Hybridoma technology was used to produce anti-O. viverrini monoclonal antibody. Previous studies have shown that antibody production via antibody phage display can sustain the hybridoma technique. This paper presents the utility of antibody phage display technology for producing the phage displayed KKU505 Fab fragment and using experiments in concomitant with molecular simulation for characterization. The phage displayed KKU505 Fab fragment and characterization were successfully carried out. The KKU505 hybridoma cell line producing anti-O. viverrini antibody predicted to bind to myosin was used to synthesize cDNA so as to amplify the heavy chain and the light chain sequences. The KKU505 displayed phage was constructed and characterized by a molecular modeling in which the KKU505 Fab fragment and -O. viverrini myosin head were docked computationally and it is assumed that the Fab fragment was specific to -O. viverrini on the basis of mass spectrometry and Western blot. This complex interaction was confirmed by molecular simulation. Furthermore, the KKU505 displayed phage was validated using indirect enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry. It is worthy to note that ELISA and immunohistochemistry results confirmed that the Fab fragment was specific to the -O. viverrini antigen. Results indicated that the approach presented herein can generate anti-O. viverrini antibody via the phage display technology. This study integrates the use of phage display technology together with molecular simulation for further development of monoclonal antibody production. Furthermore, the presented work has profound implications for antibody production, particularly by solving the problem of hybridoma stability issues

General acts passed by the General Court of Massachusetts

Imprint varies.Vols. for 1915-19 published in 2 v.: General acts; Special acts.Vols. for some years issued in parts.Separate vols. issued for extra session, 1916, and for extra session, 1933.Vol. 12 (May 1831-Mar. 1833) in Jan. session, 1833; Jan. 1834-Apr. 1836 in vol. for extra session 1835/Jan. session 1836; May 1824-Mar. 1828; June 1828-June 1831, Jan. 1832-Apr. 1834, Jan. 1835-Apr. 1838, each bound with corresponding vol.Resolves issued separately, 1780-1838

Roles of Zinc Finger Protein 423 in Proliferation and Invasion of Cholangiocarcinoma through Oxidative Stress

Zinc finger protein 423 (ZNF423) is a transcriptional factor involved in the development and progression of cancers but has not yet been examined in cholangiocarcinoma (CCA), an oxidative stress-driven cancer of biliary epithelium. In this study, we hypothesized that oxidative stress mediated ZNF423 expression regulates its downstream genes resulting in CCA genesis. ZNF423 protein expression patterns and 8-oxodG (an oxidative stress marker) formation in CCA tissues were investigated using immunohistochemical analysis. The results showed that ZNF423 was overexpressed in CCA cells compared to normal bile duct cells adjacent of the tumor. Notably, ZNF423 expression was positively correlated with 8-oxodG formation. Moreover, ZNF423 expression in an immortalized cholangiocyte cell line (MMNK1) was increased by hydrogen peroxide-treatment, suggesting that oxidative stress induces ZNF423 expression. To investigate the roles of ZNF423 in CCA progression, ZNF423 mRNA was silenced using specific siRNA in CCA cell lines, KKU-100 and KKU-213. Silencing of ZNF423 significantly inhibits cell proliferation and invasion of both CCA cell lines. Taking all these results together, the present study denoted that ZNF423 is an oxidative stress-responsive gene with an oncogenic property contributing to the regulation of CCA genesis

Antifibrotic effect of xanthohumol in combination with praziquantel is associated with altered redox status and reduced iron accumulation during liver fluke-associated cholangiocarcinogenesis

Cholangiocarcinoma (CCA) caused by infection of the liver fluke Opisthorchis viverrini, (Ov) is the major public health problem in northeast Thailand. Following Ov infection the subsequent molecular changes can be associated by reactive oxygen species (ROS) induced chronic inflammation, advanced periductal fibrosis, and cholangiocarcinogenesis. Notably, resistance to an activation of cell death in prolonged oxidative stress conditions can occur but some damaged/mutated cells could survive and enable clonal expansion. Our study used a natural product, xanthohumol (XN), which is an anti-oxidant and anti-inflammatory compound, to examine whether it could prevent Ov-associated CCA carcinogenesis. We measured the effect of XN with or without praziquantel (PZ), an anti-helminthic treatment, on DNA damage, redox status change including iron accumulation and periductal fibrosis during CCA genesis induced by administration of Ov and N-dinitrosomethylamine (NDMA) in hamsters. Animals were randomly divided into four groups: group I, Ov infection and NDMA administration (ON); group II, Ov infection and NDMA administration and PZ treatment (ONP); the latter 2 groups were similar to group I and II, but group III received additional XN (XON) and group IV received XN plus PZ (XONP). The results showed that high 8-oxodG (a marker of DNA damage) was observed throughout cholangiocarcinogenesis. Moreover, increased expression of CD44v8-10 (a cell surface in regulation of the ROS defense system), whereas decreased expression of phospho-p38MAPK (a major ROS target), was found during the progression of the bile duct cell transformation. In addition, high accumulation of iron and expression of transferrin receptor-1 (TfR-1) in both malignant bile ducts and inflammatory cells were detected. Furthermore, fibrosis also increased with the highest level being on day 180. On the other hand, the groups of XN with or without PZ supplementations showed an effective reduction in all the markers examined, including fibrosis when compared with the ON group. In particular, the XONP group, in which a significant reduction DNA damage occurred, was also found to have iron accumulation and fibrosis compared to the other groups. Our results show that XN administered in combination with PZ could efficiently prevent CCA development and hence provide potential chemopreventive benefits in Ov-induced cholangiocarcinogenesis

Prolonged oxidative stress down-regulates Early B cell factor 1 with inhibition of its tumor suppressive function against cholangiocarcinoma genesis

Early B cell factor 1 (EBF1) is a transcription factor involved in the differentiation of several stem cell lineages and it is a negative regulator of estrogen receptors. EBF1 is down-regulated in many tumors, and is believed to play suppressive roles in cancer promotion and progression. However, the functional roles of EBF1 in carcinogenesis are unclear. Liver fluke-infection-associated cholangiocarcinoma (CCA) is an oxidative stress-driven cancer of bile duct epithelium. In this study, we investigated EBF1 expression in tissues from CCA patients, CCA cell lines (KKU-213, KKU-214 and KKU-156), cholangiocyte (MMNK1) and its oxidative stress-resistant (ox-MMNK1-L) cell lines. The formation of 8-oxo-7,8-dihydro-2â²-deoxyguanosine (8-oxodG) was used as an oxidative stress marker. Our results revealed that EBF1 expression was suppressed in cancer cells compared with the individual normal bile duct cells at tumor adjacent areas of CCA tissues. CCA patients with low EBF1 expression and high formation of 8-oxodG were shown to correlate with poor survival. Moreover, EBF1 was suppressed in the oxidative stress-resistant cell line and all of CCA cell lines compared to the cholangiocyte cell line. This suggests that prolonged oxidative stress suppressed EBF1 expression and the reduced EBF1 level may facilitate CCA genesis. To elucidate the significance of EBF1 suppression in CCA genesis, EBF1 expression of the MMNK1 cell line was down-regulated by siRNA technique, and its effects on stem cell properties (CD133 and Oct3/4 expressions), tumorigenic properties (cell proliferation, wound healing and cell migration), estrogen responsive gene (TFF1), estrogen-stimulated wound healing, and cell migration were examined. The results showed that CD133, Oct3/4 and TFF1 expression levels, wound healing, and cell migration of EBF1 knockdown-MMNK1 cells were significantly increased. Also, cell migration of EBF1-knockdown cells was significantly enhanced after 17β-estradiol treatment. Our findings suggest that EBF1 down-regulation via oxidative stress induces stem cell properties, tumorigenic properties and estrogen responses of cholangiocytes leading to CCA genesis with aggressive clinical outcomes. Keywords: Cholangiocarcinom, EBF1, Stem cells, Estrogen, Oxidative stres

In vitro and molecular chemosensitivity in human cholangiocarcinoma tissues.

Adjuvant chemotherapy is required for cholangiocarcinoma (CCA) patients after surgical treatment. Gemcitabine and gemcitabine plus cisplatin are considered the appropriate regimen; however, the response spectrum to chemotherapy differs between patients. Thus, the present study aims to evaluate the response pattern of individual CCA patients by using an in vitro method, histoculture drug response assay (HDRA), to predict the chemosensitivity of individual patients in a prospective study. Moreover, we also investigate the expression of gemcitabine and cisplatin sensitivity factors in CCA tissues in the same cases. Based on the dose response curve, 1000 and 1500 μg/ml of gemcitabine were used as the testing concentrations. For cisplatin, concentrations of 20 and 25 μg/ml were selected for testing and for the combination regimen, 1000 μg/ml of gemcitabine and 20 μg/ml of cisplatin were chosen. The median %IR of each drug was measured as the cut-off to categorize the response pattern into response and non-response groups. In addition, we compared the effectiveness of the chemotherapy regimens between gemcitabine alone and gemcitabine plus cisplatin. The %IR of the combination of gemcitabine and cisplatin was significantly higher than gemcitabine alone. The relationship between the expression level of gemcitabine and cisplatin sensitive factors and the individual response pattern as well as clinicopathological data of CCA patients were analyzed. The results indicated that a low expression of the gemcitabine sensitive factor hENT-1 was significantly associated with the non-response group in vitro (p = 0.002). Moreover, the low expression of hENT-1 was also significantly associated with advanced stages CCA in the patients (p = 0.025). A low expression of MT and ERCC1 was significantly correlated with the response group in the in vitro experiments (p = 0.015 and p = 0.037 for MT and ERCC1, respectively). Therefore, HDRA may serve as an aid to selecting chemotherapy, and the expression of hNET-1, MT and ERCC1 may serve as biomarkers for predicting chemotherapy success