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Klemmer I.,RWTH Aachen | Yagi S.,Kyoto University | Yagi S.,Institute for Laboratory Animal Science and Experimental Surgery | Gressner O.A.,Wisplinghoff Medical Laboratories | Gressner O.A.,RWTH Aachen
Hepatology Research | Year: 2011

Aim: Several epidemiological studies suggest a beneficial effect of coffee consumption on the formation and progression of fibrogenic diseases, particularly of the liver. Recent data now point to a modulation of transforming growth factor-β (TGF-β) signaling by paraxanthine (1,7-dimethylxanthine [1,7-DMX]), the demethylated primary metabolite of caffeine Methods: Twenty adult Sprague-Dawley rats were bile duct ligated (BDL) or sham operated with or without concomitant oral 1,7-DMX (1 mM) application. Serum was investigated by standard biochemical analysis, in-house connective tissue growth factor (CTGF), enzyme linked immunosorbent assay (ELISA) or liquid chromatography-mass spectrometry analysis. Liver tissue was stained using hematoxylin-eosin (HE) and Sirius-red staining. Whole liver lysates, primary rat hepatocytes (PC) and hepatic stellate cells (HSC) were investigated by CTGF, and total Smad2/3 Western blot analysis, CTGF reporter gene assay or an in-house malondialdehyde ELISA. Results: The in vitro 50% inhibitory dose (ID50) of 1,7-DMX was 0.95 mM by for CTGF promoter activity and protein expression in PC and 1.25 mM for protein expression in HSC. Oral 1,7-DMX application (1 mM) attenuated cholestatic hepatocellular injury in vivo as determined by biochemical serum analysis and reduced intercellular collagen deposition in the cholestatic rat liver (HE-and Sirius-red staining). Western Blot analysis of whole liver lysates revealed a reduction of intrahepatic concentrations of Smad2/3 and CTGF following oral 1,7-DMX intake. However, serum CTGF concentrations were not reduced in 1,7-DMX treated BDL rats. Oral 1,7-DMX furthermore led to a reduction of intrahepatic lipid peroxidation (malondialdehyde concentrations) as markers of oxidative stress in BDL rats. Conclusion: Our pilot study warrants further studies of 1,7-DMX as a potential new drug to fight fibrotic processes, not just of the liver. © 2011 The Japan Society of Hepatology.

Gressner O.A.,Wisplinghoff Medical Laboratories | Gao C.,Shanghai University
Clinica Chimica Acta | Year: 2014

The clinical course of chronic liver diseases is significantly dependent on the progression rate of fibrosis which is the unstructured replacement of injured parenchyma by extracellular matrix. Despite intensive studies, the clinical opportunities for patients with fibrosing liver diseases have not improved. This will be changed by increasing knowledge of new pathogenetic mechanisms, which complement the "canonical principle" of fibrogenesis. The latter is based on the activation of hepatic stellate cells and their transdifferentiation to myofibroblasts induced by hepatocellular injury and consecutive inflammatory mediators such as TGF-β. Stellate cells express a broad spectrum of matrix components. New mechanisms indicate that the heterogeneous pool of (myo-)fibroblasts can be supplemented by epithelial-mesenchymal transition (EMT) from cholangiocytes and potentially also from hepatocytes to fibroblasts, by influx of bone marrow-derived fibrocytes in the damaged liver tissue and by differentiation of a subgroup of monocytes to fibroblasts after homing in the damaged tissue. These processes are regulated by the cytokines TGF-β and BMP-7, chemokines, colony-stimulating factors, metalloproteinases and numerous trapping proteins. They offer innovative diagnostic and therapeutic options. As an example, modulation of TGF-β/BMP-7 ratio changes the rate of EMT, and so the simultaneous determination of these parameters and of the connective tissue growth factor (CTGF) in serum might provide information on fibrogenic activity. Also, proteomic and glycomic approaches of serum are under investigation to set up specific protein profiles in patients with liver fibrosis. The aim of this article is to present the current pathogenetic concepts of liver fibrosis and to discuss established and novel diagnostic approaches to reflect the process of hepatic fibrogenesis in the medical laboratory. © 2014 Elsevier B.V.

Wang H.,Shanghai University | Wang A.-H.,Shanghai University | Gressner O.A.,Wisplinghoff Medical Laboratories | Fang M.,Shanghai University | And 5 more authors.
Clinical and Experimental Medicine | Year: 2015

This study aimed to investigate the association between HBV mutations and intrahepatic HBV status and to determine the clinical features and the contribution of HBV mutations to postoperative prognosis for hepatocellular carcinoma (HCC) patients with HBsAg (+) in China. Using TaqMan fluorescent real-time PCR, HBV covalently closed circular DNA (cccDNA) and total DNA (tDNA) were both quantified in 106 pairs of tumor tissues (TT) and adjacent non-tumor tissues (ANTT) obtained from HCC patients who received no antiviral treatment before surgical treatment. The prevalence of 19 hot spot mutations was evaluated by Sanger sequencing. HBV cccDNA and tDNA were lower in TT than in ANTT. The loads of cccDNA and tDNA in ANTT were associated with serum HBV DNA and HBeAg. Three Pre-S mutants (A2962G and C2964A in Pre-S1 and C105T in Pre-S2) were associated with higher tumor cccDNA levels (P < 0.05), and A2962G/C2964A mutants were associated with higher AFP levels. This would assist to disclose the virological features, to implement a more clinically personalized therapy and to improve the prognosis of HBV-related HCC patients. © 2014, Springer-Verlag Italia.

Gressner O.A.,RWTH Aachen | Gressner O.A.,Wisplinghoff Medical Laboratories | Peredniene I.,RWTH Aachen | Peredniene I.,Synlab Laboratory Services | Gressner A.M.,RWTH Aachen
World Journal of Gastroenterology | Year: 2011

AIM: To investigate the mechanisms involved in a possible modulator role of interleukin (IL)-6 signalling on CYR61-CTGF-NOV (CCN) 2/connective tissue growth factor (CTGF) expression in hepatocytes (PC) and to look for a relation between serum concentrations of these two parameters in patients with acute infammation. METHODS: Expression of CCN2/CTGF, p-STAT3, p-Smad 3/1 and p-Smad2 was examined in primary freshly isolated rat or cryo-preserved human PC exposed to various stimuli by Western blotting, electrophoretic mobility shift assay (EMSA), reporter-gene-assays and reverse-transcriptase polymerase chain reaction. RESULTS: IL-6 strongly down-regulated CCN2/CTGF protein and mRNA expression in PC, enhanceable by extracellular presence of the soluble IL-6 receptor gp80, and supported by an inverse relation between IL-6 and CCN2/CTGF concentrations in patients' sera. The inhibition of TGFβ1 driven CCN2/CTGF expression by IL-6 did not involve a modulation of Smad2 (and Smad1/3) signalling. However, the STAT3 SH2 domain binding peptide, a selective inhibitor of STAT3 DNA binding activity, counteracted the inhibitory effect of IL-6 on CCN2/CTGF expression much more pronounced than pyrrolidine-dithiocarbamate, an inhibitor primarily of STAT3 phosphorylation. An EMSA confirmed STAT3 binding to the proposed proximal STAT binding site in the CCN2/CTGF promoter. CONCLUSION: CCN2/CTGF is identifed as a hepato-cellular negative acute phase protein which is down-regulated by IL-6 via the STAT3 pathway through interaction on the DNA binding level © 2011 Baishideng. All rights reserved.

Gressner O.A.,Wisplinghoff Medical Laboratories | Fang M.,Shanghai University | Li H.,Shanghai University | Lu L.G.,Shanghai JiaoTong University | And 2 more authors.
Clinica Chimica Acta | Year: 2013

Still a challenging medical problem is the non-invasive monitoring of patients with a variety of chronic liver diseases being on risk to develop fibrosis, cirrhosis, and, finally, primary liver cell carcinoma. Previously, we have shown that CTGF/CCN2, a down-stream mediator of TGF-β, in serum might be a promising non-invasive biomarker of fibrosis, which is extended in the following study to cirrhosis and liver cell carcinoma.Healthy individuals (n. = 56), as well as fibrotic (n. = 77), cirrhotic (n. = 17), and HCC-patients (n. = 72) with chronic hepatitis B (HBV) infection, clinically, biochemically and histopathologically well characterized and classified, were included for the measurements of CTGF-concentrations in serum using a newly developed CTGF-enzyme immunoassay.A statistical significant increase of the mean serum CTGF-concentrations was associated with different stages of fibrosis, ranging from 15.9. μg/L (S0), 20.3. μg/L (S1/2) to 36.9. μg/L (S3/4). The highest CTGF-concentrations were measured in cirrhotic patients (43.6. μg/L), compared to healthy subjects (17.7. μg/L), followed by a decrease in cirrhotic HCC-patients (38.5. μg/L; p= 0.001). Of note, HCC patients without underlying cirrhosis (n. = 8) had CTGF levels (13.5 ± 13.2. μg/L) comparable to those in healthy controls. No statistical relation between CTGF levels and parameters of liver injury (e.g. AST, ALT) was noticed, but CTGF levels are correlated negatively with serum albumin levels (p= 0.007) and platelet counts (p= 0.0032), respectively. The latter was negatively correlated with the stage of fibrosis (p= 0.025). In HCC patients, CTGF concentrations decreased with tumor progression and size, with lower levels in TNM stage II (30.5. μg/L) and stage III (33.6. μg/L) compared to TNM stage I (41.6. μg/L).Our data suggest a valuable diagnostic impact of CTGF in serum for the follow-up of patients suffering from chronic liver diseases developing fibrosis, cirrhosis and finally HCC. CTGF serum levels in HCC are most likely due to underlying fibrosis/cirrhosis but not due to malignancy per se. © 2013 Elsevier B.V.

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