AIM：To characterize the IFN-response and its modulation by the antiviral compound lamivudine in HBV- transfected HepG2.2.15 cells. METHODS： HepG2.2.15 and HepG2 cells were stimulated with various ...concentrations of IFN-α 2a in the presence or absence of lamivudine. Then, total RNA was extracted and analysed by customised cDNA arrays and northern blot for interferon-inducible genes （ISGs）. In addition, cellular proteins were extracted for EMSA and western blot. HBV replication was assessed by southern blot or ELISAs for HBsAg and HBeAg. RESULTS： Two genes （MxA, CigS） with completely abolished and 4 genes （IFITM1, -2, -3, and 6-16） with partially reduced IFN-responses were identified in HepG2.2.15 cells. In 2 genes （IFITM1, 6-16）, the response to IFN-α could be restored by treatment with lamivudine. This effect could not be explained by a direct modulation of the Jak/Stat signalling pathway since EMSA and western blot experiments revealed no suppression of Statl activation and ISGF3 formation after stimulation with IFN-α in HepG2.2.15 compared to HepG2 cells. CONCLUSION： These results are consistent with the assumption that chronic hepatitis B may specifically modulate the cellular response to IFN by a selective blockage of some ISGs. Antiviral treatment with lamivudine may partially restore ISG expressionby reducing HBV gene expression and replication.
The proteome analysis of bile fluid represents a promising strategy to identify biomarker candidates for various diseases of the hepatobiliary system. However, to obtain substantive results in ...biomarker discovery studies large patient cohorts necessarily need to be analyzed. Consequently, this would lead to an unmanageable number of samples to be analyzed if sample preparation protocols with extensive fractionation methods are applied. Hence, the performance of simple workflows allowing for "one sample, one shot" experiments have been evaluated in this study. In detail, sixteen different protocols implying modifications at the stages of desalting, delipidation, deglycosylation and tryptic digestion have been examined. Each method has been individually evaluated regarding various performance criteria and comparative analyses have been conducted to uncover possible complementarities. Here, the best performance in terms of proteome coverage has been assessed for a combination of acetone precipitation with in-gel digestion. Finally, a mapping of all obtained protein identifications with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) revealed several proteins easily detectable in bile fluid. These results can build the basis for future studies with large and well-defined patient cohorts in a more disease-related context.
Human bile fluid is a proximal body fluid and supposed to be a potential source of disease markers. However, due to its biochemical composition, the proteome analysis of bile fluid still represents a challenging task and is therefore mostly conducted using extensive fractionation procedures. This in turn leads to a high number of mass spectrometric measurements for one biological sample. Considering the fact that in order to overcome the biological variability a high number of biological samples needs to be analyzed in biomarker discovery studies, this leads to the dilemma of an unmanageable number of necessary MS-based analyses. Hence, easy sample preparation protocols are demanded representing a compromise between proteome coverage and simplicity.
In the presented study, such protocols have been evaluated regarding various technical criteria (e.g. identification rates, missed cleavages, chromatographic separation) uncovering the strengths and weaknesses of various methods. Furthermore, a cumulative bile proteome list has been generated that extends the current bile proteome catalog by 248 proteins. Finally, a mapping with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) derived from tissue-based studies, revealed several of these proteins being easily and reproducibly detectable in human bile. Therefore, the presented technical work represents a solid base for future disease-related studies.
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•Protocols for "one sample, one shot" proteome analyses of bile fluid were evaluated•In-gel digestion was found to be superior to in-solution digestion•The current bile proteome catalog was extended by 248 proteins•Several putative HCC and CCC markers were easily detectable in bile fluid
The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and ...bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver.
Multi-OMICS approaches aim on the integration of quantitative data obtained for different biological molecules in order to understand their interrelation and the functioning of larger systems. This ...paper deals with several data integration and data processing issues that frequently occur within this context. To this end, the data processing workflow within the PROFILE project is presented, a multi-OMICS project that aims on identification of novel biomarkers and the development of new therapeutic targets for seven important liver diseases. Furthermore, a software called CrossPlatformCommander is sketched, which facilitates several steps of the proposed workflow in a semi-automatic manner.
Application of the software is presented for the detection of novel biomarkers, their ranking and annotation with existing knowledge using the example of corresponding Transcriptomics and Proteomics data sets obtained from patients suffering from hepatocellular carcinoma. Additionally, a linear regression analysis of Transcriptomics vs. Proteomics data is presented and its performance assessed.
It was shown, that for capturing profound relations between Transcriptomics and Proteomics data, a simple linear regression analysis is not sufficient and implementation and evaluation of alternative statistical approaches are needed. Additionally, the integration of multivariate variable selection and classification approaches is intended for further development of the software.
Although this paper focuses only on the combination of data obtained from quantitative Proteomics and Transcriptomics experiments, several approaches and data integration steps are also applicable for other OMICS technologies. Keeping specific restrictions in mind the suggested workflow (or at least parts of it) may be used as a template for similar projects that make use of different high throughput techniques. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.
•A workflow for integrating multi-OMICS data is presented.•The suggested workflow may serve as a template for similar projects.•The suggested CrossPlatformCommander software facilitates several workflow steps.•Capturing multi-OMICS correlations requires sophisticated statistical approaches.
Hepatocellular carcinoma (HCC) is a major lethal cancer worldwide. Despite sophisticated diagnostic algorithms, the differential diagnosis of small liver nodules still is difficult. While imaging ...techniques have advanced, adjuvant protein-biomarkers as glypican3 (GPC3), glutamine-synthetase (GS) and heat-shock protein 70 (HSP70) have enhanced diagnostic accuracy. The aim was to further detect useful protein-biomarkers of HCC with a structured systematic approach using differential proteome techniques, bring the results to practical application and compare the diagnostic accuracy of the candidates with the established biomarkers.
After label-free and gel-based proteomics (n=18 HCC/corresponding non-tumorous liver tissue (NTLT)) biomarker candidates were tested for diagnostic accuracy in immunohistochemical analyses (n=14 HCC/NTLT). Suitable candidates were further tested for consistency in comparison to known protein-biomarkers in HCC (n=78), hepatocellular adenoma (n=25; HCA), focal nodular hyperplasia (n=28; FNH) and cirrhosis (n=28).
Of all protein-biomarkers, 14-3-3Sigma (14-3-3S) exhibited the most pronounced up-regulation (58.8×) in proteomics and superior diagnostic accuracy (73.0%) in the differentiation of HCC from non-tumorous hepatocytes also compared to established biomarkers as GPC3 (64.7%) and GS (45.4%). 14-3-3S was part of the best diagnostic three-biomarker panel (GPC3, HSP70, 14-3-3S) for the differentiation of HCC and HCA which is of most important significance. Exclusion of GS and inclusion of 14-3-3S in the panel (>1 marker positive) resulted in a profound increase in specificity (+44.0%) and accuracy (+11.0%) while sensitivity remained stable (96.0%).
14-3-3S is an interesting protein biomarker with the potential to further improve the accuracy of differential diagnostic process of hepatocellular tumors. This article is part of a Special Issue entitled: Medical Proteomics.
•Tissue protein biomarker candidates were identified and validated in liver tumors.•13 candidates were selected after differential proteomics for further validation.•Since clinically most important we focused on hepatocellular carcinoma and adenoma.•14-3-3Sigma is a superior biomarker candidate also in relation to known biomarkers.•A new 3-biomarker panel including 14-3-3Sigma enhances the diagnostic process.
Background and Objectives
Up to now the diagnosis of early stage cholangiocarcinoma (CC) has remained difficult, with low sensitivities reported for current diagnostic methods. Based on recent ...promising findings about circulating U2 small nuclear RNA fragments (RNU2-1f) as novel blood-based biomarkers for pancreatic and colorectal adenocarcinoma, we studied the utility of RNU2-1f as a diagnostic marker of CC in bile fluid.
Methods
Bile fluid was collected from patients with CC (
n
= 12), controls (patients with choledocholithiasis) (
n
= 11) and with primary sclerosing cholangitis (PSC;
n
= 11). RNU2-1f levels were measured by real-time polymerase chain reaction normalized to cel-54.
Results
Measurement of RNU2-1f levels in bile fluids enabled the differentiation of patients with CC from controls in all cases. Furthermore, RNU2-1f levels in bile fluids of patients with CC were significantly higher than in patients with PSC, resulting in a receiver-operating characteristic curve area of 0.856, with sensitivity of 67 % and specificity of 91 %.
Conclusions
Our data suggest that the measurement of RNU2-1 fragments detected in the bile fluid can be used as a diagnostic marker for CC and should be included in future prospective diagnostic studies for this disease entity.
Background The aim of the study was to determine the role of Vascular Endothelial Growth Factor (VEGF) on the microvasculature and on angiogenetic gene expression after partial hepatectomy (PH) in ...the rat model. Methods To determine the effect of exogenous and endogenous VEGF after PH, rats were subjected to 70% PH and treated either with VEGF, anti-VEGF or NaCl. Postoperatively (3–168 h), vessel density (VD), vessel diameter (VDi), and intersinusoidal space, liver body weight ratio (LBR), hepatic proliferation and biochemical markers were assessed. To further elucidate the underlying molecular mechanisms hepatic gene expression was determined by customized cDNA arrays and quantitative RT-PCR. Results In the VEGF group, VD, VDi, and LBR were significantly increased compared with anti-VEGF or controls. Blockage of endogenous VEGF led to a marked increase of biochemical markers. Anti-VEGF almost completely suppressed and VEGF markedly enhanced hepatic proliferation in the first 24 h after surgery. This was associated with a modulation of cell cycle control genes (PC4, Gadd45a, Tis21/BTG2), v-jun, and CD14 by VEGF. Conclusions VEGF plays an important role in liver regeneration and this may be due in part through its effects on neovascularization. Whether it may, when given therapeutically, represent a strategy to optimize liver regeneration in problematic patients needs to be clarified.
Sorafenib, a protein kinase inhibitor, is a systemic drug that has been licensed for the treatment of hepatocellular carcinoma (HCC). This retrospective study assessed whether the administration of ...sorafenib can result in a reduction of the hepatopulmonary shunt (HPS) before selective internal radiation therapy (SIRT). After exclusion from SIRT because of high HPS, computed tomography scan indicated a shunt reduction in seven patients with HCC receiving sorafenib. Repeated measurements revealed HPS reduction (from 26.5% to 7.5% on average), and subsequent SIRT became possible. In conclusion, sorafenib may reduce HPS in patients with advanced HCC in some cases.
Context and objective: Human hepatocellular carcinoma (HCC) is a severe malignant disease, and accurate and reliable diagnostic markers are still needed. This study was aimed for the discovery of ...novel marker candidates by quantitative proteomics.
Methods and results: Proteomic differences between HCC and nontumorous liver tissue were studied by mass spectrometry. Among several significantly upregulated proteins, translocator protein 18 (TSPO) and Ras-related protein Rab-1A (RAB1A) were selected for verification by immunohistochemistry in an independent cohort. For RAB1A, a high accuracy for the discrimination of HCC and nontumorous liver tissue was observed.
Conclusion: RAB1A was verified to be a potent biomarker candidate for HCC.
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