68 Furthermore, patients with low miR-26 expression showed better responses to interferon therapy.83 MiR-122 restoration also sensitized HCC cells to doxorubicin,84 as well as multi-kinase inhibitor Sorafenib,55 indicating miR-122 mimic in combination with anticancer drugs could be a promising therapeutic regimen against HCC. The discovery of miRNA has substantially altered conventional concepts on gene regulation and this class of tiny non-coding RNAs has emerged as novel players in the control of genes expression in cancer. Studies on miRNA profiling have revealed characteristic

miRNA dysregulations in different tumor types and unveiled the importance of miRNA involvement Panobinostat mouse in carcinogenesis. Functional and target association studies on dysregulated miRNAs in HCC have

enabled us to gain a more comprehensive understanding on their roles in the oncogenic signaling pathways. Nevertheless, the mechanistic cause of miRNA dysregulation remains to be fully explored and the characterization of many of the differential expressed miRNAs and their molecular and cell biological targets is still in progress. From a clinical point of view, preliminary studies have highlighted the value of miRNAs in the diagnosis and prognosis of HCC. Differential expressed miRNA patterns may be useful in the stratification of patients to predict disease outcome and recurrence. Recently, there has been considerable interest in the potential use of antagomiRs as anticancer agents, especially for HCC because of their predominant AZD3965 manufacturer uptake by the liver and Sorafenib mouse enhanced hepatic stability.85 Technological advances have also demonstrated the feasibility of utilizing adeno-associated virus to administer miRNAs in a murine HCC model.59 In addition, treatment of chimpanzees with locked nucleic acid (LNA)-modified oligonucleotide suppressed HCV infection.86 The success of miRNA delivery in these animal models may hold promise in the further development of miRNA targeted therapy, which may represent a new avenue for the treatment of HCC. This review was prepared through the support

of a Collaborative Research Fund from the Hong Kong Research Grants Council (Ref. No. CUHK4/CRF/08) “
“Real-time tissue elastography (RTE) is a non-invasive method for the measurement of tissue elasticity using ultrasonography. Liver fibrosis (LF) index is a quantitative method for evaluation of liver fibrosis calculated by RTE image features. This study aimed to investigate the significance of LF index for predicting liver fibrosis in chronic hepatitis C patients. In this prospective study, 115 patients with chronic hepatitis C who underwent liver biopsy were included, and the diagnostic accuracy of LF index and serum fibrosis markers was evaluated. RTE imaging was successfully performed on all patients. Median LF index in patients with F0–1, F2, F3 and F4 were 2.61, 3.07, 3.54 and 4.

[11] The use of BCAA granules was identified as a contributing factor to prolonged survival in a multivariate analysis.[11] The mechanism of the inhibitory effect of BCAA granules against HCC recurrence after RFA needs to be verified in a large-scale prospective

study. BCAA granules may inhibit HCC recurrence in patients who have undergone percutaneous RFA as well as in those who have undergone hepatectomy.[11, 29] Transcatheter arterial chemoembolization is a combination of local chemotherapy through feeding blood vessels and the use of CHIR-99021 research buy embolizing material.[16, 84-87] TACE is most frequently used for the treatment of HCC in Japan, where it was originally developed.[84, 87-90] EASL guidelines recommend TACE for unresectable, Child–Pugh class A or B multiple HCC with no vascular invasion, whereas in Japan the therapy is recommended even for HCC with vascular invasion if it is Vp1 or Vp2.[50, 51] The www.selleckchem.com/products/obeticholic-acid.html factors affecting the survival of HCC patients treated with TACE are: (i) tumor stage; (ii) tumor markers; and (iii) hepatic functional reserve.[84] Preserving hepatic functional reserve is a critical issue in HCC patients who, in general, are treated repeatedly with TACE.[16, 88-92] However, in some patients, hepatic functional reserve decreases after TACE

because of complications such as post-TACE syndrome.[93] The usefulness of BCAA granules or BCAA-enriched “snacks” for patients with unresectable HCC treated with TACE has been suggested in several studies.[16, 91, 92] In a randomized controlled trial (RCT) in 56 HCC patients treated with TACE, Takeshita et al. found that the post-TACE decrease

in liver function was suppressed significantly in patients who received an enteral nutritional formula for hepatic failure given as a late-evening snack (LES) compared with the control group.[91, 94] Our retrospective controlled study in 99 HCC patients treated with TACE showed that therapy using BCAA granules significantly inhibited the decrease in hepatic functional reserve at 3 months and 6 months Edoxaban compared with the regular diet group.[16] According to EASL guidelines, if HCC with Child–Pugh class B treated with TACE recurs as Child–Pugh class C, TACE is not indicated for the recurred HCC. The significance of therapy using BCAA granules is considerable in terms of permitting repeated TACE. There had long been a lack of evidence to support systemic chemotherapy for unresectable advanced HCC.[95] However, after the efficacy of a molecular-targeted drug, sorafenib, for unresectable advanced HCC was demonstrated in two RCT (SHARP trial and Asia–Pacific trial), the drug was approved for the treatment of unresectable advanced HCC in Japan in 2009.

Data distribution and gene expression statistical analysis were performed using NCSS statistical and power analysis software 2007. Comparisons of two groups were performed using a Student t test followed by the Mann-Whitney U test where appropriate. P < 0.05 was considered significant. ATPβsynt, ATPβ-synthase; Cpt-1α, carnitine palmitoyl transferase-1α;

COXI, cytochrome c oxidase subunit I; cytC, cytochrome C; DNL, de novo lipogenesis; Dgat1 and 2, diacylglycerol acyltransferase 1 and 2; IL-1β, interleukin β; Idh3α, isocitrate dehydrogenase 3α; Mcad, medium-chain acyl-coenzyme A dehydrogenase; MCD, methionine and choline deficient diet; MCS, methionine and choline supplied diet; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; NEFAs, nonesterified fatty acids; Scd-1, stearoyl Co-A Desaturase 1; pro-col, procollagen; Selleckchem CYC202 Tnfα, tumor necrosis factor α. In order to verify that a constitutive overexpression of PGC-1β in the liver was able to induce its target genes, we first generated a mouse model in which human PGC1-β is selectively overexpressed in the liver (LivPGC-1β mice) by subcloning the hPGC1-β Proteases inhibitor coding sequence

under the control of the apolipoprotein E promoter. The human PGC-1β is expressed only in the liver of transgenic mice (Supporting Fig. 1). In order to characterize the tissue-specific transcriptional scenario activated by PGC-1β, we performed microarray analysis of liver samples from wildtype and LivPGC-1β mice fed a chow diet. The data showed that PGC-1β coactivator overexpression is able to induce a plethora of genes involved in several metabolic pathways (Fig. 1A). The majority of target genes whose expression is enhanced by PGC-1β (1.3-fold or more) encodes for proteins involved in the mitochondrial oxidative phosphorylation. Ceramide glucosyltransferase Other pathways up-regulated

by the hepatic PGC-1β overexpression were ubiquinone and protein biosynthesis, lipid metabolism, TG transport, citrate cycle, gluconeogenesis, and antioxidant systems. These results were confirmed by real-time quantitative (qPCR) analysis of the gene expression levels of cytochrome c (cytC), a component of the respiratory chain, as well as of medium-chain acyl-coenzyme A dehydrogenase (Mcad) and carnitine palmitoyl transferase-1α (Cpt-1α), two key enzymes in fatty acid β-oxidation (Fig. 1B). Moreover, real-time qPCR analysis confirmed that overexpression of PGC1-β was associated with the induction of genes involved in lipid anabolism, including Srebp1c and its target gene, Fas, both involved in fatty acid synthesis. Notably, also the expression of Stearoyl Co-A Desaturase 1 (Scd-1) that catalyzes the biosynthesis of monounsaturated fatty acids, and diacylglycerol acyltransferase 1 and 2 (Dgat1 and 2), fundamental enzymes for TG synthesis, were increased by the overexpression of hepatic PGC1-β (Fig. 1B).

An alternative technique is to identify the supraorbital ridge by palpation. The needle is then inserted lateral to the ridge and is advanced medially selleck chemicals into the subcutaneous tissue. After

negative aspiration, the solution is injected. Pressure should be applied to avoid periorbital hematoma. Drugs to use: lidocaine 1%-2% (10-20 mg/mL) and/or bupivacaine 0.25%-0.5% (2.5-5 mg/mL). If a combination of the 2 drugs is used, the recommended volume ratio (lidocaine/bupivacaine) is 1:1-1:3. We do not recommend the use of corticosteroids in this area, or in other trigeminal territories. Volume of injection: 0.2-1.0 mL per nerve. For patients who require repeated injections, the recommended frequency of treatments is once every 2-4 weeks, depending on the response of the individual patient. The SON is the larger of the 2 terminal branches of the frontal nerve. It courses through the supraorbital

notch or foramen and supplies palpebral filaments to the upper eyelid and conjunctiva. It then ascends on the forehead with the supraorbital artery and divides into medial and lateral branches, which supply the skin of the scalp almost as far back as the lambdoid suture. The medial branch pierces the occipitofrontalis muscle to reach the skin, while the lateral branch penetrates the epicranial aponeurosis over the forehead and scalp. Postganglionic sympathetic HCS assay fibers, which innervate the sweat glands of the supraorbital area, are thought to travel in the SON. The supraorbital notch or foramen lies on the superior aspect of an imaginary line coursing caudally and intersecting the pupil, the infraorbital foramen, and the mental foramen. Location of injection: above the supraorbital Idoxuridine notch. Technique of injection: use a 1 mL syringe with a 30-gauge, 0.5-inch needle. Insert

the needle at the corrugator muscle, at the mid-pupillary line (Fig. 2 —). After negative aspiration, the solution may be injected at a depth of 3-4 mm. An alternative technique is to identify the supraorbital notch by palpation, at the superior margin of the orbit, mid-pupillary line. The needle is then advanced medially and inserted at a slight angle to avoid entering the foramen. The solution then may be injected at a depth of 3-4 mm, after negative aspiration. Pressure should be applied to avoid periorbital hematoma. Alternatively, after blocking the STN, redirect the needle laterally and inject. Drugs used and volume of injections are the same as for the STN block. The ATN surfaces onto the face from behind the temporomandibular joint (TMJ) within the superior surface of the parotid gland. It ascends close to the superficial temporal artery, passes over the posterior portion of the zygoma, and divides into superficial temporal branches. The cutaneous branches of the ATN supply the tragus and part of the adjacent auricle of the ear and the posterior part of the temple. The ATN also provides sensory innervation to the majority of the TMJ.

Three days

after pBDL surgery, the serum liver alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and ۷-glutamyl transferase (GGT) increased from baseline by 5. 3, 5. 7, 5. 3 and CP-673451 nmr 12. 1-fold respectively. Serum total bilirubin (TBil) levels increased by ≥100-fold. Seven days after surgery, AST and ALT levels had begun to normalize (3. 0 and 1. 7-fold) while ALP, GGT and total bilirubin values remained high at 4. 9, 22 and 103-fold compared to sham controls. This profile was sustained at 14 days post-surgery with elevations of 6. 8-fold for ALP, 15. 5-fold for GGT and 128-fold for TBil. SBA were also dramatically increased by 28.9-fold (30 uM to 873 uM) 7 days after surgery. SC-435 was administered to the test group

by once daily oral gavage at 10 mg/kg starting one day prior to pBDL surgery. Seven days after surgery ASBTi treatment had significantly reduced ALP by 58%, GGT by 48%, TBil by 49% and SBA by 52% compared to the untreated control group (p < 0.03 for all parameters). By 14 days post-surgery, ALP was reduced 75%, GGT by 65% and TBil by 67% compared to the untreated control group (p < 0.05 for all parameters). CONCLUSIONS: The pBDL model in HSD rats results in significant increases in SBA and serum liver enzymes from 3 to 14 days TGF-beta inhibitor after surgery that are characteristic of cholestasis and liver injury. Blocking bile acid recycling with SC-435 prevents dramatic increases in total SBA and liver biomarkers Thiamine-diphosphate kinase suggesting that an ASBTi may provide a new therapeutic option for the treatment of cholestatic liver disease by decreasing the accumulation of toxic bile acids and reducing the severity of cholestatic liver injury. Disclosures: Bradley T. Keller – Employment: Lumena Pharmaceuticals, Rivervest Venture Partners Bronislava

Gedulin – Employment: Lumena Pharmaceuticals The following people have nothing to disclose: Svetlana Nikoulina, Nicolaus Nazarenkov Background: Nucleotide oligomerization domain 2 (Nod2), a member of the Nod-like receptor (NLR) family of intracellular immune receptors, plays an important role in the defense against bacterial infection through binding to its ligand muramyl dipeptide (MDP). The aim of our study was to test whether Nod2 plays a role in experimental liver fibrosis. Methods and Results: In wild type and Nod2 mice cholestatic liver disease was induced by bile duct ligation for 3 weeks, and toxic liver disease was induced by 12 intraperitoneal injections of carbon tetrachloride. Nod2 deficiency protected mice from cholestatic but not toxin-induced liver injury and fibrosis. Bile duct ligated Nod2-/- showed significantly less liver injury (plasma ALT levels), and less fibrosis (Sirius red staining and collagen α1(I) QPCR); liver inflammation was not changed.

[31] In these mice, hepatic expression of OPN protein was markedly increased at 1 day after the beginning of MCD diet and persisted

check details up to 8 weeks, whereas OPN mRNA expression was increased at 4 weeks.[31] OPN protein expression was predominantly localized to hepatocytes, not inflammatory cells, assessed by immunohistochemistry at 3 days and at 8 weeks. Moreover, hepatic inflammation induced by MCD diet was markedly reduced in OPN–/– mice compared with OPN+/+ mice, while histological steatosis and liver triglyceride levels were similar among these mice.[31] This may be because mice fed MCD diet lose weight and do not show insulin resistance, unlike human or other diet-induced rodent models of NAFLD. Osteopontin mRNA was increased during culture-related activation of rat quiescent hepatic stellate cells to myofibroblastic stellate cells.[32] Furthermore, incubation of hepatic stellate cells with OPN induced their collagen production, transforming growth factor-β receptor upregulation, proliferation and migration.[32] These results suggested a potential role for OPN in the progression of hepatic fibrosis. Hepatic fibrosis induced by MCD diet was HKI-272 mw attenuated in OPN–/–

mice compared with OPN+/+ mice.[31, 33] Moreover, hepatic OPN mRNA and protein levels were not affected by feeding an MCD diet in genetically leptin-deficient, obese and diabetic ob/ob mice, which developed steatohepatitis but not liver fibrosis after the feeding.[34] In contrast, the diet had a stimulatory effect on OPN mRNA and protein levels in hyperleptinemic, obese and diabetic db/db mice, which exhibited a lesser degree of steatosis, but greater histological inflammation and marked pericellular fibrosis by the diet.[34]

Recently, it was reported that OPN was induced by Hedgehog signaling and directly promoted profibrogenic responses in steatohepatitis (Fig. 2). Hedgehog pathway can promote activation of quiescent hepatic stellate cells to myofibroblastic stellate cells.[35] In patients with NAFLD, accumulation of Hedgehog ligands and expression of Hedgehog-target genes were significantly correlated with Amylase hepatic fibrosis stage.[36] Furthermore, Hedgehog-mediated accumulation of natural killer T (NKT) cells contributed to fibrosis progression of NASH in mice and humans.[37] As shown in Figure 3, after the binding of the Hedgehog ligand to the Patched (Ptc) receptor, glioma-associated oncogene (Gli) is activated by release from a large protein complex and translocated to the nucleus to function as a transcriptional activator. The consensus DNA-binding sequences of Gli-1 were indentified in the 5′ regions of OPN, and gel shift analysis confirmed Gli-1 protein could bind to the oligonucleotides of OPN promoter region.[38] Syn et al. analyzed hepatic OPN expression and fibrosis grade, using Ptc-deficient (Ptc+/−) mice with haploinsufficiency of Ptc, which exhibit overly active Hedgehog signaling.

An unusual set of circumstances allowed us to record the vocalizations of photo-identified individuals within a single social unit over a 41 d period. Using click interpulse intervals, we were able to assign codas to individuals and investigate coda production at the individual level within a social unit for the first time. Adult females in the unit vocalized at approximately equal rates. A calf and juvenile, both male, vocalized less often than the adult females. Repertoires were selleck kinase inhibitor indistinguishable for all unit members apart from a

mother and her calf, which possessed significantly different repertoires—even from one another. We suggest that similarity among the coda repertoires of most unit members indicates a function in advertising unit identity. In contrast, the distinctive repertoires of the calf and its mother may facilitate reunions between these whales. We hypothesize that sperm BI 6727 whales may be able to vary their vocal repertoires as

their reproductive status alters the trade-off between the benefits of individual and group identification. “
“Wide-ranging marine central place foragers often exhibit foraging site fidelity to oceanographic features over differing spatial scales (i.e., localized coastal upwellings and oceanic fronts). Few studies have tested how the degree of site fidelity to foraging areas varies in relation to the type of ocean features used. In order to determine how foraging site fidelity varied between continental shelf and oceanic foraging habitats, 31 lactating New Zealand fur seals (Arctocephalus australis forsteri1) were satellite tracked over consecutive foraging trips (14–108 d). Thirty-seven foraging trips were recorded from 11 females that foraged on the continental shelf, in a region associated with a coastal upwelling, while 65 foraging trips were recorded from 20 females that foraged in oceanic

waters. There were no significant differences in the mean bearings (to maximum distance) of individual’s consecutive foraging trips, suggesting individual fidelity to foraging areas. However, overlap in area and time spent in area varied considerably between continental shelf and oceanic foragers. Females that foraged on the continental Galactosylceramidase shelf had significantly greater overlap in consecutive foraging trips when compared to females that foraged in oceanic waters (overlap in 5 × 5 km grid cells visited on consecutive trips 55.9%± 20.4% and 13.4%± 7.6%, respectively). Females that foraged on the continental shelf also spent significantly more time within the same grid cell than females that foraged in oceanic waters (maximum time spent in 5 × 5 km grid cells: 14%± 5% and 4%± 2%, respectively). This comparatively high foraging site fidelity may reflect the concentration of productivity associated with a coastal upwelling system, the Bonney Upwelling.

5), containing 1 mM of EDTA, 2 mM of MgCl2, 50 mM of KCl, 1 mM of dithiothreitol, and protease inhibitors by incubating at 4°C for 30 minutes and centrifuging at 14,000 rpm for 5 minutes. A detailed protocol and antibodies used are described in Supporting Materials and Methods. Liver sections were stained for

5′-bromo-2′-deoxyuridine (BrdU)-positive nuclei with the BrdU labeling and detection kit (Roche, Indianapolis, IN), according to manufacturer’s instructions. Ten randomly selected high-power fields (40×) of liver Torin 1 sections from 4-6 mice per group were analyzed. The number of BrdU-positive cells were counted and expressed as a percentage of the total number of cells, as visualized by hematoxylin-eosin staining. For evaluation of eNOS gene expression, RNA was isolated from liver tissues harvested at 0.5-72 hours post-PH using the Qiagen RNeasy minikit, according to the manufacturer’s

instructions (Qiagen Sciences, Germantown, MD). Reverse transcription (RT) was performed using 2 μg of total RNA in a first-strand complementary DNA (cDNA) synthesis reaction with the high-capacity cDNA RT kit (Applied Biosystems, Foster city, CA), as recommended by the manufacturer. The cDNA product was amplified by quantitative RT polymerase chain reaction (qRT-PCR) in an ABI prism 7700 sequence-detection system buy GDC-0199 (Applied Biosystems) with primers specific for mouse eNOS and cyclophilin, as described previously.15 Hepatocytes were isolated from 6-8-week-old WT and eNOS−/− male mice by the two-step collagenase perfusion protocol, as described previously, Celecoxib with modifications optimized for

mice.16 For hepatocyte proliferation assays, cell preparations with viability over 95%, as screened by Trypan Blue exclusion assay, were seeded at a low density of 200,000 cells/35 mm of Primaria tissue-culture wells (BD Labware, Franklin Lakes, NJ) in Williams E complete media, with additives for 3 hours to ensure hepatocyte adherence to plates. Subsequently, hepatocytes were maintained in Williams E minimal media free from serum and growth factors for 20 hours before treatment with growth factors. A detailed protocol of doses and duration of EGF treatment of hepatocytes in vitro and pretreatment of cell-signaling-pathway–specific inhibitors, to assess the role of eNOS in EGF-mediated mitogenic signaling and proliferation, is described in Supporting Materials and Methods. Data are represented as the mean ± standard deviation (SD) of at least three independent experiments. The statistical significance of difference between groups was analyzed by the unpaired Student’s t-test. Values of P < 0.05 were considered statistically significant. Activation of MAPKs and immediate early genes are hallmarks of early events activated within minutes of PH.

4 %and smaller

p-values for gender (p=0.0008), IFNL4 rs12979860 genotype (ptrend=0.02) and race (p=0.03). Conclusion: The very high SVR rates observed among certain subgroups of patients treated for 8 weeks with ledipasvir/sofosbuvir support conducting shorter trials of this regimen in selected patients. A briefer course of therapy could yield substantial healthcare savings; assuming treatment costs of $1000/day, every 100,000 patients treated with ledipas-vir/sofosbuvir for two fewer weeks (i.e., 6 weeks rather than 8 weeks) would reduce healthcare costs by $1.4 billion dollars. Disclosures: Thomas R. O’Brien – Patent Held/Filed: National Cancer Institute The following people have nothing to disclose: Krystle A. Kuhs, Ruth M. Pfeiffer Background: The new oral single tablet regimen of LDV/SOF has been shown to have excellent efficacy and tolerability Palbociclib nmr in treatment-naive (TN) and treatment-experienced (TE) patients with HCV GT1. A decision-analytic model evaluated the health outcomes of LDV/SOF compared with

current recommended options, including SOF with pegylated interferon alfa and ribavirin (PR) at 12 weeks, simeprevir (SMV) with PR at 24-48 weeks, and no treatment. NVP-AUY922 supplier Methods: The analysis modeled cohorts of 10,000 HCV TN or TE GT1 patients with an average age of 52 and varying level of fibrosis from a US third party payer perspective for a lifetime horizon. LDV/SOF for 8 weeks was compared to LDV/SOF for 12 weeks, SOF+PR for 12 weeks, SMV+PR for 12 weeks (+PR for 12-36 weeks per prescribing Selleck Alectinib information), and no treatment.

Sustained viro-logic response (SVR) (listed in Table 1) and adverse rates were based on phase III clinical trials Transition probabilities and utility were based on literature review, public sources, and consensus by a panel of 4 hepatologists. Results: LDV/SOF regimen for 8-or-12 weeks resulted in better health outcomes compared with SOF+PR, SMV+PR, and no treatment, with the lowest number of liver-related complications. Patients on LDV/ SOF regimens also demonstrated the highest life year gains and quality adjusted life years (QALYs) compared to other comparator regimens, among TN and TE patients. Conclusions: Compared to current recommended options, LDV/SOF demonstrated better overall health outcomes. Large discrepancies in efficacy, side effects, and adherence rates have been reported for currently available regimens between real-world and clinical trial settings. Additional real-world analyses are necessary to determine the potential impact of the greater expected real-world differences between LDV/SOF regimen and other available therapies. Disclosures: Sammy Saab – Advisory Committees or Review Panels: BMS, Gilead, Merck, Genentech; Grant/Research Support: Merck, Gilead; Speaking and Teaching: BMS, Gilead, Merck, Genentech, Salix, Onyx, Bayer, Janssen; Stock Shareholder: Salix, Johnson and Johnson, BMS, Gilead Aijaz Ahmed – Consulting: BMS, Gilead, Vertex, Genentech, Onyxx Stuart C.

The study protocol conformed to the ethical guidelines of the Declaration of Helsinki (1975). All patients provided written

informed consent for the analysis of the biopsy specimens or drainage bile. The protocol for this study was approved by the ethical committee of Kanazania University, Tokyo Women’s Medical University and University of Tsukuba. Differential glycan profiling of tissue sections was performed essentially as described.21 Briefly, formalin-fixed, paraffin-embedded ICC tissue sections were deparaffinized, and the relevant tissue fragments including cancerous (n = 45) and normal bile duct epithelia (BDE) (n = 38) lesions (corresponding to 1.0 mm square and 5 μm thickness, respectively) were then scratched www.selleckchem.com/products/erastin.html from the glass slide using a needle (gauge size: 21 G) under a microscope. Total protein extracts from the scratched tissue fragments thus obtained were fluorescence-labeled with 10 μg of Cy3-succimidyl ester (SE; Amersham selleck chemical Biosciences, Tokyo, Japan). After blocking free Cy3-SE with 0.5 M glycine in Tris-buffered saline containing 1% Triton X-100 (TBSTx), an aliquot (¼) was applied to a lectin microarray slide. Fluorescence

signals were measured on a GlycoStation scanner (Moritex Co., Tokyo, Japan). The obtained lectin microarray data were analyzed on the basis of normalized signal intensities as described,23 where the lectin showing the strongest signal intensity (max intensity) was assigned a value of 1.0. The values are presented as the median ± standard error of the mean (SEM). A two-sided Welch or Student t test was used to compare the clinicopathological data between groups. All calculations were performed using Origin version 7.5 software

for Windows (OriginLab Co., Northampton, MA). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the differences between ICC and benign disease on the bases of sensitivity and specificity at various cutoff levels. An area under the ROC curve (AUC) of 1.0 indicates perfect discrimination, whereas an area of GNA12 0.5 indicates that the test discriminates no better than chance.24 WFA staining was performed using biotinylated WFA (Vector Co., Burlingame, UK). Detection was made with Histofine Simple Stain MAX-PO (Nichirei Co., Tokyo, Japan). The tissue sections were deparaffinized and then autoclaved to enhance the WFA reactivity. After cooling to room temperature, endogenous peroxidase was blocked by incubating the sections in methanol containing 0.3% hydrogen peroxide. The tissue sections were blocked with phosphate-buffered saline (PBS) containing 1% (wt/vol) bovine serum albumin (BSA), and the sections were incubated with 2 μg/mL of biotinylated WFA in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid for 1 hour at room temperature. The sections were incubated with streptavidin–peroxidase reagents, reacted with 3,3′-diaminobenzidine tetrahydrochloride for visualization, and counterstained with hematoxylin.