Although liver fibrosis reflects disease severity in chronic hepatitis patients, there has been no simple and accurate system to evaluate the therapeutic effect based on fibrosis. We developed a glycan-based immunoassay, FastLec-Hepa, to fill this unmet need. FastLec-Hepa automatically detects unique fibrosis-related glyco-alteration in serum hyperglycosylated Mac-2 binding protein within 20 min. The serum FastLec-Hepa counts increased with advancing fibrosis and illustrated significant differences in medians between all fibrosis stages. FastLec-Hepa is sufficiently sensitive and quantitative to evaluate the effects of PEG-interferon-α/ribavirin therapy in a short post-therapeutic interval. The obtained fibrosis progression is equivalent to -0.30 stages/year in patients with sustained virological response, and 0.01 stages/year in relapse/nonresponders. Furthermore, long-term follow-up of the severely affected patients found hepatocellular carcinoma developed in patients after therapy whose FastLec-Hepa counts remained above a designated cutoff value. FastLec-Hepa is the only assay currently available for clinically beneficial therapy evaluation through quantitation of disease severity.

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Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and highthroughput manner. Thus, the established system should provide a powerful pipeline in support of ongoing efforts in glyco-biomarker discovery. Molecular & Cellular Proteomics 8:99 -108, 2009.

A moderately water-swollen hydrogel with a molecularly ordered structure was prepared by copolymerizing an acrylic monomer with a hydrophobic long alkyl side group-ra-stearyl acrylate (SA)-with acrylic acid (AA). The long alkyl ester side chains of the gel form lamellar layers with a thickness of ca. 5 nm, and the lamellar distance of the swollen gels is nearly 0.8 nm larger compared to that of their dry state. This indicates that water molecules in the polymer gel are preferentially adsorbed between two aggregates of stearyl groups perpendicularly aligned to the main chain. We have also found that the poly(SA-co-AA) gel undergoes a reversible order-disorder transition with change in temperature and a dramatic change in its Young's modulus at a certain temperature.

Cholangiocarcinoma (CC) is an aggressive malignant tumor for which useful markers are not presently available for early and precise diagnosis. The aim of this study was therefore to identify a high-performance diagnostic marker with a special focus on glyco-alteration of glycoproteins. In the course of study, we found that Wisteria floribunda agglutinin (WFA) is the best probe to differentiate intrahepatic cholangiocarcinoma (ICC) lesions from normal bile duct epithelia (BDE) (P < 0.0001). The subsequent histochemical study confirmed ICC-specific WFA staining on 165 tissue specimens. On the other hand, the WFA staining was shown to be closely associated with that of MY.1E12 established previously against sialylated mucin 1 (MUC1) by double-staining experiments. Moreover, glyco-alteration of MUC1 could be verified by western blotting of WFA-captured bile samples from patients with CC patients. Thus, we attempted to construct an enzyme-linked immunosorbent assay system for more convenient CC diagnosis, where WFA-coated plates, the specific monoclonal antibody MY.1E12, and the bile specimens from CC including ICC (n 5 30) and benign diseases (n 5 38) were combined. As a result, CC was clearly distinguished from benign diseases with statistical scores (sensitivity 5 90.0%, specificity 5 76.3%, and area under the curve 5 0.85). As a particular note, the obtained sensitivity is the highest score among those having been so far reported. Conclusion: Our approach focusing significant glyco-alteration of a particular glycoprotein yielded a novel diagnostic system for CC with satisfactory clinical scores. (HEPATOLOGY 2010;52:174-182) C holangiocarcinoma (CC) is an aggressive malignant tumor arising from the epithelial lining of the intrahepatic biliary tract. Although it contributes to only 15% of the total incidence of primary liver cancer, 1 recent epidemiological reports show that the CC incidence has increased significantly in Abbreviations: AFP, alpha-fetoprotein; AUC, area under the curve; BDE, bile duct epithelia; BSA, bovine serum albumin; CA19-9, carbohydrate antigen 19-9;