Repolarization Precedes Oval Cell-mediated Hepatocytic Regeneration in the CDE Diet Mouse Model.

The liver has a unique ability to recover from injury unlike any other organ. A poorly understood aspect of liver regeneration is the role of hepatocellular polarization. Neighbor of Punc E11 (Nope) is an oncofetal stem/progenitor cell marker, which is expressed by depolarized adult hepatocytes after cholestatic liver injury and in hepatocellular carcinoma. Liver injury induced by a choline-deficient and ethionine-supplemented diet is reversible if followed by an additional dietary stop interval and enabled us to study the expression of Nope during the induction of chronic liver injury and during subsequent liver regeneration. We could show by quantitative RT-PCR, Western blotting, and immunohistochemistry that the expression of Nope is induced in depolarized adult hepatocytes during injury. However, after another 2 weeks of a normal diet, the polarization of hepatocytes was almost completely restored and the expression of Nope remained limited to bile ducts and oval cells. Using an inducible CK19-lineage tracing model, we could demonstrate that oval cell-mediated hepatocyte regeneration is rare and was preceded by repolarization of hepatocytes. In conclusion, polarization of hepatocytes is an important part of liver regeneration and precedes oval cell-mediated regeneration of the liver. This process can be visualized by a characteristic expression pattern of Nope.

First evaluation of Neighbor of Punc E11 (NOPE) as a novel marker in human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second most common cause of cancer death worldwide and the search for clinically useful biomarkers is ongoing. Neighbor of Punc E11 (NOPE) is an established biomarker of murine HCC that remains undetectable in normal liver and at preneoplastic stages. OBJECTIVE: The aim of our study was to evaluate the presence of NOPE in human HCC. METHODS: Histologically confirmed HCC and corresponding non-tumor liver samples from 20 patients were analyzed for expression of NOPE using qRT-PCR and mRNA-in-situ technology in a conserved tissue context. RESULTS: In our cohort, 30% of HCC samples were expressing NOPE which proved particularly useful in non-cirrhotic HCC samples with up to 155-fold higher expression than in adult liver. Using mRNA-in-situ technology, NOPE was clearly identified within epithelial tumor cells of NOPE positive human HCCs. In our analyzed cohort, the combination of AFP with NOPE did not reach more than 40% sensitivity while GPC-3 and NOPE were complementary to each other reaching a combined sensitivity of 85.7%. CONCLUSIONS: This is the first characterization of NOPE as a potential biomarker for human HCC. Our results underline the value of NOPE as a complementing biomarker for human HCC.

Case report of patient with a Cronkhite-Canada syndrome: sustained remission after treatment with corticosteroids and mesalazine.

BACKGROUND: Cronkhite-Canada syndrome is a rare disease of unknown etiology and the optimal treatment for this syndrome is unknown. CASE PRESENTATION: We present the case of a man who at the age of 66.0 years was diagnosed with Cronkhite-Canada syndrome (CCS). In addition to watery diarrhea, alopecia, and a complete loss of toenails and fingernails, the patient had been suffering from dysgeusia and rapid weight loss of more than 10.0 kg within a few months. The patient had recently incurred a distal radius fracture. During the initial endoscopy an extensive polyposis of the stomach and jejunum was found. The diagnosis of CCS was made and after initiation of a steroid therapy his diarrhea improved immediately. A discontinuation of the steroid therapy was not possible and mesalazine (1000 mg t.i.d.) was added to prednisolone (10.0 mg/d). This therapy led to a remission within 6.0 months with weight gain and normalization of serum albumin levels. The prednisolone dose was reduced to 7.5 mg/d. During the following year, the steroids could be further reduced and nails had regrown again. Within three years, all polyps had disappeared and the steroid therapy was finished while the dosage of mesalazine was reduced in a stepwise fashion. Four years later, the mesalazine was stopped and more than 14.0 years after the initial diagnosis the patient is still in complete remission without any treatment. CONCLUSION: The optimal treatment for CCS is unknown. In our case, the initial combination therapy of corticosteroids plus mesalazine followed by a mesalazine monotherapy has led to a remarkable long-lasting remission with complete resolution of all intestinal polyps.

Depolarized Hepatocytes Express the Stem/Progenitor Cell Marker Neighbor of Punc E11 After Bile Duct Ligation in Mice.

There is a medical need of biomarkers for disease stratification in cholestatic liver diseases that come along with changes in hepatocyte polarity. Neighbor of Punc E11 (Nope) is an oncofetal marker that is lost after final differentiation and polarization of hepatocytes. We analyzed the expression pattern of Nope and connexin (Cx) 26 as markers of hepatocyte polarization during murine liver development as well as in adult liver with or without bile duct ligation (BDL) by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), western blotting (WB), and immunohistochemistry. Nope is highly expressed in fetal and postnatal liver but barely detectable thereafter. Cx26, however, is much higher expressed in adult than in fetal liver. Postnatally, Nope is directed to the sinusoidal membrane of early hepatocytes while Cx26 remains distributed over the whole membrane indicating limited polarization. In the adult liver, only Cx26 is detectable and restricted to the bile canalicular domain indicating fully polarized hepatocytes. After BDL, Nope is again >300-fold upregulated while Cx26 is reduced rapidly. By immunohistochemistry, Nope identifies a subset of hepatocytes with randomly distributed Cx26. In summary, Nope identifies depolarized adult hepatocytes after cholestatic liver injury resembling early postnatal hepatocytes. Therefore, Nope might be a valuable histochemical biomarker allowing stage-specific stratifications in cholestatic liver diseases.

A Novel Easy-to-Use Prediction Scheme for Upper Gastrointestinal Bleeding: Cologne-WATCH (C-WATCH) Risk Score.

Acute upper gastrointestinal bleeding (UGIB) is the leading indication for emergency endoscopy. Scoring schemes have been developed for immediate risk stratification. However, most of these scores include endoscopic findings and are based on data from patients with nonvariceal bleeding. The aim of our study was to design a pre-endoscopic score for acute UGIB--including variceal bleeding--in order to identify high-risk patients requiring urgent clinical management. The scoring system was developed using a data set consisting of 586 patients with acute UGIB. These patients were identified from the emergency department as well as all inpatient services at the University Hospital of Cologne within a 2-year period (01/2007-12/2008). Further data from a cohort of 322 patients who presented to our endoscopy unit with acute UGIB in 2009 served for external/temporal validation.Clinical, laboratory, and endoscopic parameters, as well as further data on medical history and medication were retrospectively collected from the electronic clinical documentation system. A multivariable logistic regression was fitted to the development set to obtain a risk score using recurrent bleeding, need for intervention (angiography, surgery), or death within 30 days as a composite endpoint. Finally, the obtained risk score was evaluated on the validation set. Only C-reactive protein, white blood cells, alanine-aminotransferase, thrombocytes, creatinine, and hemoglobin were identified as significant predictors for the composite endpoint. Based on the regression coefficients of these variables, an easy-to-use point scoring scheme (C-WATCH) was derived to estimate the risk of complications from 3% to 86% with an area under the curve (AUC) of 0.723 in the development set and 0.704 in the validation set. In the validation set, no patient in the identified low-risk group (0-1 points), but 38.7% of patients in the high-risk group (≥ 2 points) reached the composite endpoint. Our easy-to-use scoring scheme is able to distinguish high-risk patients requiring urgent endoscopy, from low-risk cases who are suitable candidates for outpatient management or in whom endoscopy may be postponed. Based on our findings, a prospective validation of the C-WATCH score in different patient populations outside the university hospital setting seems warranted.

Stepwise combination of simple noninvasive fibrosis scoring systems increases diagnostic accuracy in nonalcoholic fatty liver disease.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease ranging from simple fatty liver to steatohepatitis, fibrosis, and cirrhosis. We aimed to analyze the diagnostic performance and clinical utility of simple noninvasive tests alone or in combination for the detection of advanced fibrosis in patients with NAFLD. DESIGN AND SUBJECTS: Data from 323 patients with biopsy-proven NAFLD/NASH who presented to the Clinic for Gastroenterology and Hepatology, University Hospital of Cologne between July 1998 and November 2009, were analyzed retrospectively. Sensitivity, specificity, positive predictive values, and negative predictive values were determined along with the area under receiver operating characteristic curves (AUROC) using published formulas for NAFLD, FIB-4, and BARD fibrosis scores. RESULTS: The area under receiver operating characteristic curves were as follows: NAFLD fibrosis score 0.96 [95% confidence interval (CI), 0.92-0.99], FIB-4 0.95 (95% CI, 0.91-1.00), BARD 0.82 (95% CI, 0.71-0.92) with negative predictive values for advanced fibrosis of 96%, 98%, and 96%, respectively. When applying the NAFLD, FIB-4, or BARD scoring systems 25%, 15%, or 26% of cases with advanced fibrosis would have been missed. Combining FIB-4 and BARD in a stepwise fashion, patients would have been correctly classified without biopsy in 67% of cases without missing a single case of advanced fibrosis. CONCLUSIONS: The FIB-4 and NAFLD fibrosis scores perform better than the BARD scoring system. Liver biopsy can securely be replaced only with a stepwise combination of simple noninvasive tests, otherwise the assessment of risk due to advanced fibrosis may be misleading in a clinically meaningful proportion of patients.

Telmisartan plus propranolol improves liver fibrosis and bile duct proliferation in the PSC-like Abcb4-/- mouse model.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease leading to cirrhosis and cholangiocellular carcinoma. Inhibitors of the renin-angiotensin system or the sympathetic nervous system delay liver fibrogenesis in animal models. AIMS: We investigated the antifibrotic potential of telmisartan, an angiotensin II type 1 receptor antagonist, and the ß-adrenoceptor blocker propranolol in the PSC-like Abcb4 knockout mouse model. METHODS: Sixty-five Abcb4 (-/-) mice were treated with telmisartan for 3 or 5 months (T) and with telmisartan plus propranolol for 3, 5, or 8 months (TP), or for 2 or 5 months starting with a delay of 3 months (TP delayed). Liver hydroxyproline content, inflammation, fibrosis, and bile duct proliferation were assessed; fibrosis-related molecules were analyzed by real-time polymerase chain reaction and Western blotting. RESULTS: Compared to controls, telmisartan monotherapy had no significant influence on hydroxyproline; however, telmisartan plus propranolol reduced hydroxyproline (TP 3 months, p = 0.008), fibrosis score (TP 3 months and TP 8 months, p = 0.043 and p = 0.008, respectively; TP delayed 8 months, p < 0.0005), bile duct proliferation (TP 8 months and TP delayed 8 months, p = 0.006 and p < 0.0005, respectively), and procollagen α1(I), endothelin-1, TIMP-1 and MMP3 mRNA as well as α-SMA, CK-19, and TIMP-1 protein. CONCLUSIONS: Telmisartan plus propranolol reduces liver fibrosis and bile duct proliferation in the PSC-like Abcb4 (-/-) mouse model, even when started at late stages of fibrosis, and may thus represent a novel therapeutic option for cholestatic liver diseases such as PSC.

Neighbor of Punc E 11: expression pattern of the new hepatic stem/progenitor cell marker during murine liver development.

We have previously identified Neighbor of Punc E 11 (Nope) as a specific cell surface marker of stem/progenitor cells in the murine fetal liver that is also expressed in hepatocellular carcinoma. Here, we focus on the differential expression pattern of Nope during murine fetal and postnatal liver development as well as in a normal and regenerating adult liver including oval cell activation. In the fetal liver, Nope shows a constantly high expression level and is a useful surface marker for the identification of Dlk, E-cadherin, and CD133-positive hepatoblasts by flow cytometry. Postnatally, Nope expression declines rapidly and remains barely detectable in the adult liver as shown by quantitative real-time reverse-transcriptase polymerase chain reaction and western blot analyses. Immunohistochemically, costainings for Nope- and epithelial-specific markers (E-cadherin), markers of early hepatoblasts (alpha-fetoprotein), and biliary marker proteins (CK19) demonstrate that Nope is initially expressed on bipotent hepatoblasts and persists thereafter on commited hepatocytic as well as cholangiocytic progenitor cells during late fetal liver development. Postnatally, Nope loses its circular expression pattern and is specifically directed to the sinusoidal membrane of early hepatocytes. While Nope is only weakly expressed on cholangiocytes in the normal adult liver, activated stem/progenitor (oval) cells clearly coexpress Nope together with the common markers A6, EpCAM, and CD24 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model. In conclusion, Nope should be most useful in future research to define the differentiation stage of hepatic-specified cells of various sources and is a promising candidate to identify and isolate hepatic stem cells from the adult liver.

ß-Adrenoceptor blockade in sclerosing cholangitis of Mdr2 knockout mice: antifibrotic effects in a model of nonsinusoidal fibrosis.

Primary sclerosing cholangitis (PSC) is a cholestatic liver disease with high propensity to develop into cholangiocarcinoma. The hepatobiliary disorder of PSC is due to progressive fibrosis surrounding the intra- and extrahepatic bile ducts. Until now, no effective medical therapy exists. To study the progression of sclerosing cholangitis after inhibition of the sympathetic nervous system by blockade of the ß-adrenoceptors, we used the Mdr2(-/-) mouse model, which develops periportal fibrosis similar to human PSC. Liver tissues of Mdr2(-/-) mice untreated or treated with the ß-adrenoceptor antagonist propranolol were analyzed for inflammation and fibrosis progression at different time points by histological scoring and immunostaining for α-smooth muscle actin (α-SMA), CD45 and S100A4. Transaminases and hydroxyproline contents were determined. Expression of angiotensinogen, endothelin-1, TGF-ß, TNF-α, CTGF and procollagen 1A1 was studied by real-time PCR on laser-microdissected areas of acinar zones I and II-III. After 3 months, periportal fibrosis had developed in Mdr2(-/-) mice, but immunostaining revealed no sinusoidal and only minor periportal contribution of myofibroblasts with prominent fibroblasts. Propranolol treatment of Mdr2(-/-) mice improved liver architecture. Additionally, inflammation and fibrosis were significantly reduced. After 3 months of treatment, the antifibrotic effect of the ß-blockade was most obvious. The transcript levels of procollagen 1A1, TNF-α, TGF-ß, CTGF and endothelin-1 were markedly repressed in the portal areas of treated mice. Taken together, these data show that propranolol efficiently delays progression of sclerosing cholangitis. Therefore, the blockade of ß-adrenoceptors is a promising option to support future therapeutic strategies in the treatment of human PSC.

Liver biopsy in primary biliary cirrhosis: clinicopathological data and stage.

The purpose of the present study was to characterize histopathological lesions in primary biliary cirrhosis (PBC) and to assess the relationship between histopathological lesions and biochemistry. Liver biopsies of 252 patients with PBC were investigated. A laboratory database was established. Histopathological characterization was performed. Relationships between detailed histopathological features and biochemistry were calculated statistically. Combining the data, a PBC group, consisting of an anti-mitochondrial antibody (AMA)-positive and -negative subgroup, and an overlap group were defined, with a female preponderance of >90% and higher activity of aspartate aminotransferase (AST) in the overlap group. Histopathological changes were characteristic in >80%. Periductal concentric fibrosis, lobular granuloma formation and steatosis were frequently remarkable. Correlations were found between alanine aminotransferase activity and modified hepatitis activity index in the overlap group and the AMA-positive group. A positive significant relationship was demonstrated between mean AST activity and portal fibrosis for the AMA-positive group. A highly significant positive link was seen between mean concentration of bilirubin and stage of fibrosis. Biochemistry reflects only in part the degree of severity of histopathological lesions in PBC. Histopathology indicates comorbidity in a high percentage of patients.

Hepatocyte expression of angiotensin II type 1 receptor is downregulated in advanced human liver fibrosis.

BACKGROUND: The renin-angiotensin system plays an important role in fibrosis. Angiotensin II regulates key steps in tissue remodelling processes through angiotensin II type 1 receptor (AT1R). In bile duct-occluded rats, AT1R expression is significantly decreased in advanced liver fibrosis. Therefore, we studied the AT1R expression in human liver tissue during different stages of fibrosis caused by chronic hepatitis C. METHODS: Liver biopsy specimens from 85 patients were analysed. Real-time reverse transcription polymerase chain reaction was used to quantify AT1R mRNA. Immunohistochemical labelling of AT1R and double staining for AT1R, CD31, CD68, CD3 and fibulin-2 were performed. RESULTS: AT1R mRNA was significantly reduced in human liver tissue with end-stage cirrhosis compared with early fibrosis. In liver cirrhosis, immunohistochemistry revealed a decreased expression of AT1R on hepatocytes, together with an increased staining intensity on myofibroblasts, vascular endothelium and bile duct epithelium. CONCLUSION: In conclusion, AT1R expression is downregulated in human liver cirrhosis specimens because of the reduced expression levels on hepatocytes. Therefore, antifibrogenic therapy with AT1R blockers may be most promising if initiated during early stages of fibrosis.

Ribavirin priming in patients with chronic hepatitis C and normal ALT: a pilot study.

BACKGROUND/AIMS: Ribavirin has favorable immune-modulating effects in patients with hepatitis C. Therefore, a ribavirin monotherapy preceding the combination of peginterferon plus ribavirin may be beneficial. We conducted a pilot study with a sequential regimen using a reduced dosage of peginterferon-alfa 2b in patients with chronic hepatitis C and normal transaminases. METHODOLOGY: Twenty patients (17 genotype 1, 3 genotype 2/3) were treated with ribavirin for 4 wk followed by ribavirin plus peginterferon-alfa 2b 100 microg for 4 wk and ribavirin plus peginterferon-alfa 2b 50 microg for 44 wk. TH1-cytokines interferon-gamma and interleukin-2, and TH2-cytokines interleukin-4 and interleukin-10 were measured in the supernatant from PHA-stimulated lymphocytes. RESULTS: Sustained viral response defined as negative HCV-RNA after follow up was 50%, 41%, and 100% for all patients, genotype 1, and genotype 2/3, resp. The increase in interferon-gamma following ribavirin monotherapy was significantly higher for patients with sustained viral response. CONCLUSIONS: The sustained viral response rate of this pilot study using ribavirin priming and reduced peginterferon dosage is in line with previous trials using standard treatment regimens for chronic hepatitis C. Our data stress the positive impact of the ribavirin-induced TH2-TH1 cytokine shift at least in patients with normal ALT. Larger clinical studies with ribavirin priming seem warranted.

New cell surface markers for murine fetal hepatic stem cells identified through high density complementary DNA microarrays.

UNLABELLED: Isolation of hepatic stem cells from the adult liver (AL) has not yet been achieved due to the lack of specific cell surface markers. To identify new surface markers for hepatic stem cells, we analyzed differences in the gene expression profile of embryonic day (ED) 13.5 fetal liver stem/progenitor cells (FLSPC) versus AL by complementary DNA (cDNA) microarray technology. Using FLSPC purified to >90% by immunomagnetic selection for E-cadherin and high density (27k) mouse cDNA microarrays, we identified 474 genes that are more strongly expressed in FLSPC (FLSPC-up genes) and 818 genes that are more strongly expressed in AL (AL-up genes). The most highly overrepresented gene ontology (GO) categories for FLSPC-up genes are nucleus, cellular proliferation, and cell cycle control. AL-up genes are overrepresented for genes in metabolic pathways for specific hepatic functions. We identified 24 FLSPC-up gene surface markers and 69 AL-up gene surface markers. Western blot studies confirmed the expression of the FLSPC-up gene neighbor of Punc E11 (Nope) in fetal liver, but expression was not detectable in AL. Immunohistochemistry, confocal microscopy, and fluorescence-activated cell sorting (FACS) analysis of fetal liver demonstrated that Nope is specifically expressed on the surface of FLSPC within the fetal liver. CONCLUSION: This is the first microarray study to analyze the specific gene expression profile of purified murine FLSPC. Our analysis identified 24 new/potential cell surface markers for murine fetal hepatic stem cells, of which Nope may be particularly useful in future studies to identify, characterize and isolate hepatic stem cells from the AL.

Expression of angiotensin II receptor type 1 is reduced in advanced rat liver fibrosis.

In this study, we assessed the hypothesis that the expression of angiotensin II receptor type 1 (AGTR1) in liver tissue changes with increasing fibrosis, which would influence the antifibrotic efficacy of AGTR1 blockers. Rats were treated with candesartancilexetil (CAN) initiated 8 or 15 days after bile duct occlusion (BDO). Four weeks after BDO, AGTR1 mRNA and protein were decreased compared to those in sham-operated animals depending on the amount of fibrosis. Starting CAN early, but not late, reduced mRNA of profibrotic TGF-beta, MMP2, and Smad2. However, CAN had no significant effect on collagen I, fibrosis, or intrahepatic resistance. In conclusion, progression of liver fibrosis reduces AGTR1 expression. Therefore, in our model, antifibrotic effects of CAN are insufficient to improve fibrosis or intrahepatic resistance. However, if AGTR1 blockade is started early, a decrease in essential profibrotic molecules is achieved. Hence, early initiation of therapy with AGTR1 blockers may be crucial for the prevention of cirrhosis.