Recent data suggest that the chemokine receptor CXCR3 is functionally involved in fibroproliferative disorders, including liver fibrosis. Neoangiogenesis is an important pathophysiological feature of liver scarring, but a functional role of angiostatic CXCR3 chemokines in this process is unclear. We therefore investigated neoangiogenesis in carbon tetrachloride (CCl4)‐induced liver fibrosis in Cxcr3−/− and wildtype mice by histological, molecular, and functional imaging methods. Furthermore, we assessed the direct role of vascular endothelial growth factor (VEGF) overexpression on liver angiogenesis and the fibroproliferative response using a Tet‐inducible bitransgenic mouse model. The feasibility of attenuation of angiogenesis and associated liver fibrosis by therapeutic treatment with the angiostatic chemokine Cxcl9 was systematically analyzed in vitro and in vivo. The results demonstrate that fibrosis progression in Cxcr3−/− mice was strongly linked to enhanced neoangiogenesis and VEGF/VEGFR2 expression compared with wildtype littermates. Systemic VEGF overexpression led to a fibrogenic response within the liver and was associated with a significantly increased Cxcl9 expression. In vitro, Cxcl9 displayed strong antiproliferative and antimigratory effects on VEGF‐stimulated endothelial cells and stellate cells by way of reduced VEGFR2 (KDR), phospholipase Cγ (PLCγ), and extracellular signal‐regulated kinase (ERK) phosphorylation, identifying this chemokine as a direct counter‐regulatory molecule of VEGF signaling within the liver. Accordingly, systemic administration of Cxcl9 led to a strong attenuation of neoangiogenesis and experimental liver fibrosis in vivo. Conclusion: The results identify direct angiostatic and antifibrotic effects of the Cxcr3 ligand Cxcl9 in a model of experimental liver fibrosis. The amelioration of liver damage by systemic application of Cxcl9 might offer a novel therapeutic approach for chronic liver diseases associated with increased neoangiogenesis. (HEPATOLOGY 2012)

BACKGROUND AND STUDY AIMS:Primary sclerosing cholangitis (PSC) is a rare, chronic cholestatic liver disease, which typically affects middle-aged men and is frequently associated with inflammatory bowel disease. Early recognition and accurate diagnosis remains a clinical challenge. Invasive diagnostic procedures, such as endoscopic retrograde cholangiography or liver biopsy are needed when magnetic resonance cholangiopancreatography remains inconclusive. As these procedures are associated with significant risks, the current study sought to determine whether endoscopic ultrasound (EUS) of the biliary tract is a useful diagnostic tool in cases of suspected PSC.PATIENTS AND METHODS:In a prospective pilot study, 138 patients presenting with chronic cholestatic hepatopathy were screened and 32 patients with possible PSC were evaluated further. In addition to all routine measures, EUS was included in the diagnostic work-up.  The following parameters were evaluated and compared with the definitive diagnosis: wall thickening ( ≥ 1.5  mm), irregular wall structure, significant changes of caliber of the common bile duct, and perihilar lymphadenopathy.RESULTS:In the 138 patients screened, a PSC prevalence of 13 % was found. Of the 32 patients included in the study, 17 had large-duct PSC diagnosed. When two of the aforementioned four parameters showed PSC-like features, sensitivity and specificity of predicting PSC were 76.4 % and 100 %, with positive and negative predictive values of 100 % and 79 %, respectively. In four patients presenting with strictly intrahepatic disease, EUS was not diagnostic.CONCLUSIONS:EUS proved to be a valuable tool in suspected PSC and accurately predicted extrahepatic disease. EUS should be evaluated further as an early procedure in routine diagnostic measurements. This approach promises a significant improvement in disease detection as well as a reduction in high risk invasive procedures.

Background The chemokine CCL5 is involved in the recruitment of immune cells and a subsequent activation of hepatic stellate cells (HSC) after liver injury. We here investigate whether inhibition of CCL5 oligomerization and glycosaminoglycan binding by a mutated CCL5 protein (44AANA47-CCL5) has the potential to ameliorate liver cell injury and fibrosis in vivo. Methodology Liver injury was induced in C57BL/6 mice by intraperitoneal injection of carbon tetrachloride (CCl4) in an acute and a chronic liver injury model. Simultaneously, mice received either 44AANA47-CCL5 or vehicle. Liver cell necrosis and fibrosis was analyzed by histology, and measurement of serum transaminases and hydroxyproline. Intrahepatic mRNA expression of fibrosis and inflammation related genes were determined by quantitative RT-PCR and infiltration of immune cells was assessed by FACS analysis and immunocytochemistry. In vitro, HSC were stimulated with conditioned media of T-cell enriched splenocytes. Principal Findings 44AANA47-CCL5 treated mice displayed a significantly reduced degree of acute liver injury (liver cell necrosis, transaminases) and fibrosis (Sirus red positive area and hydroxyproline content) compared to vehicle treated mice. Ameliorated fibrosis by 44AANA47-CCL5 was associated with a decreased expression of fibrosis related genes, decreased α-smoth muscle antigen (αSMA) and a reduction of infiltrating immune cells. In the acute model, 44AANA47-CCL5 treated mice displayed a reduced immune cell infiltration and mRNA levels of TNF, IL-1 and CCL3 compared to vehicle treated mice. In vitro, conditioned medium of T-cell enriched splenocytes of 44AANA47-CCL5 treated mice inhibited the chemotaxis and proliferation of HSC. Conclusions The results provide evidence that inhibition of oligomerization and glycosaminoglycan binding of the chemokine CCL5 is a new therapeutic strategy for the treatment of acute and chronic liver injuries and represents an alternative to chemokine receptor antagonism.

Results: Most frequent etiology was non-alcoholic (63%).MELD score was 16 (6-31) and Child-Pugh 9±2.Fourteen (17.5%) patients developed HRS, all had LVDD grade 1 or 2. In the univariate analysis, 12 variables had predictive value for the development of HRS: portal pressure gradient, mean arterial pressure (MAP), cardiac output, ejection fraction, auricular and left ventricle diameter, LVDD parameters [early diastolic velocity at mitral annulus (Ea) and early diastolic velocity transmitral ratio (E/Ea)] and endogenous vasoactive system activity.Multivariate analysis showed that only MAP and the presence of LVDD expressed by Ea, were related to the appearance of HRS.Episodes of HRS-type1 developed in 9 (64%) patients with LVDD-2, and 5 (36%) with LVDD-1 (p < 0.001).Renal function normalization (complete response) happened in 2 (40%) with LVDD-1, and in 1 (11%) with LVDD-2.All 11 patients, whose renal function deteriorated, died.Multivariate analysis showed that the presence of LVDD-2 was associated independently with survival. Conclusion:The presence of LVDD in advanced cirrhosis is a risk factor for development HRS.Their severity is associated with poor prognosis in treatment response of HRS type1 and lower survival.

Background: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine which has been implicated in inflammatory diseases.Chronic inflammation also critically contributes to liver fibrosis.But the specific role of MIF in liver fibrosis has not yet been investigated.In our experiments we uncover an unexpected anti-fibrotic role of MIF in two independent fibrosis models in vivo and in vitro.Methods: Liver fibrosis was induced by CCl 4 or TAA (both 6 weeks) in Mif -/-, Cd74 -/-mice and wild-type mice.Fibrosis was analyzed by histology (sirius red staining), hydroxyproline content and intrahepatic mRNA expression of fibrosis-related genes (Col1a1, Timp1, Tgfb1 and Mmp2) in liver samples.In vitro, the effects of MIF on the proliferation (BrdU assay) and migration (Boyden chamber) of platelet-derived growth factor (PDGF)-stimulated stellate cells were analyzed and the role of AMPK and CD74 was evaluated in cell culture.Results: Constitutive MIF knockout mice (Mif -/-) unexpectedly presented a significantly increased degree of fibrosis as assessed by liver histology and hydroxyproline content in both models of chronic liver injury (CCl 4 and TAA).Pronounced liver fibrosis in Mif -/-mice was associated with strong alterations in fibrosisrelevant genes like Col1a1, Timp1, Mmp2 and Tgfb1.Recombinant Mif alone had no obvious effects on the migration or proliferation of stellate cells, but it strongly inhibited Pdgf-induced migration and proliferation of these cells.The inhibitory effects of Mif were mediated by CD74, which we detected as the most abundant MIF receptor on stellate cells.Recombinant Mif led to the phosphorylation of AMP-activated protein kinase (AMPK) in stellate cells.This mechanism was the basis for the inhibition of Pdgfinduced stellate cell activation by Mif, as shown by AMPK-specific inhibition.The crucial role of CD74 in MIF-mediated anti-fibrotic properties is further supported by augmented liver scarring in Cd74 -/-mice in vivo as assessed by histology and hepatic hydroxyproline content.Conclusions: We describe a novel and previously unexpected antifibrotic function of MIF which is mediated through CD74 and increased AMPK phosphorylation in stellate cells.These data imply MIF and its receptor CD74 as new targets for the treatment of fibrotic liver diseases.

NASH and hepatic steatosis using novel mass spectrometry-based technology in combination with murine models.Methods: C57BL/6 wild-type mice were fed either a high fat (HFAT) diet, a model of obesity and hepatic steatosis, a methionine and choline-deficient (MCD) diet, a model of severe NASH, or a control (CTL) diet (n = 5 in each group).After 7 weeks, plasma, adipose (epididymal) and liver tissue were collected.Levels of structurally specific oxidized amino acids (oxAA) that provide molecular fingerprints of the pathways responsible for their generation were measured using high-performance stable isotope dilution liquid chromatography-tandem mass spectrometry.Measured oxAA included chlorotyrosine (Cl-Tyr), bromotyrosine (Br-Tyr), nitrotyrosine (NO2-Tyr), meta-tyrosine (m-Tyr), and orthotyrosine (o-Tyr).Results: Of the oxAA measured, Cl-Tyr and Br-Tyr, products of myeloperoxidase (MPO)-and eosinophil peroxidase (EPO)catalyzed oxidation, respectively, were found to be markedly elevated in livers of mice with MCD-induced NASH compared to animals with hepatic steatosis fed HFAT diet or mice receiving CTL diet (Cl-Tyr: 65.7 vs. 13.8 vs. 14.2 umol/molTyr, p < 0.01; Br-Tyr: 57.2 vs. 18.8 vs. 18.3 umol/molTyr, p < 0.001).Levels of NO2-Tyr, a specific molecular fingerprint for protein modification by NOderived oxidants, were significantly elevated in adipose tissue of mice fed HFAT compared to CTL diet (276.3 vs. 147.8,p = 0.04), but unchanged in liver and plasma.Finally, levels of m-Tyr and o-Tyr, products of protein oxidation by metal-catalyzed hydroxyl radicallike species were unchanged in both HFAT-and MCD-fed animals compared to controls.Conclusions: These findings demonstrate significant tissue-specific increases in levels of specific molecular footprints of distinct oxidative pathways during the development of hepatic steatosis and NASH.This data supports the hypothesis that •NO-derived oxidants are involved in adipose tissue oxidative stress associated with hepatic steatosis, while MPO-and EPO-derived oxidants are involved in liver damage and disease progression to NASH.This concept has important implications for the development of novel treatment strategies for patients with this condition.

Background/Aims: Chemokines are small chemotactic molecules which regulate the infiltration of immune cells to sites of inflammatory injury. In recent years their contribution to the initiation and perpetuation of liver injury has been better defined. However, the role of chemokines in liver diseases related to the metabolic syndrome still needs to be elucidated in detail. Methods: Chemokines were mostly detected at the mRNA level in the liver and as proteins in the serum of patients with non-alcoholic steatohepatitis (NASH) or fatty liver. Animals with targeted deletion of chemokines have recently been subjected to NASH models to functionally dissect the role of chemokines in fatty liver diseases. Results: In human liver with features of NASH, different CC and CXC chemokines have been detected at elevated mRNA levels in comparison to healthy subjects. Some of these chemokines have also been associated with NASH by demonstrating higher serum levels in affected patients. Until now, only a few animal models have been analyzed with respect to the functional role of these molecules. However, data from CCL2 and CXCR3 knockout mice suggest that these pathways are important in liver injury. CCL2 seems to influence the infiltration of macrophages to adipose tissue and thereby modulate insulin resistance. Conclusions: The further elucidation of the pathophysiology of NASH will lead to new therapeutic options to halt or reverse progressive liver disease. In this respect, chemokines are attractive target molecules, as they influence immunologic and metabolic pathways and the first oral chemokine receptor antagonists have already been licensed for humans.

POSTERSrespectively.Cell survival was assayed by cytotoxicity assays and the expression of c-Jun and its potential target genes was analyzed.Results: Chemical induction of ER stress resulted in a rapid and robust expression of c-Jun in both, human hepatoma cells and mouse primary hepatocytes, but surprisingly, no major differences in ER stress-mediated cell death were observed in cells lacking c-Jun.However, massive cytoplasmic vacuolization occurred upon ER stress in primary knock-out hepatocytes, most likely due to expansion of the ER.A similar phenotype was observed in primary hepatocytes in which Jun N-terminal kinases, important regulators of c-Jun activity, were inhibited pharmacologically.This vacuolization correlated with increased expression of ER stress marker genes such as gadd153, bip and spliced xbp-1 and subsequent expression of several chemokines including CXCL1 and CXCL2.Recent findings suggest that ER expansion is tightly controlled by self-degradation of these organelles in a process called autophagy.Interestingly, autophagy appeared to be impaired in the absence of c-Jun.Conclusions: These findings suggest that c-Jun, although not essential for hepatocyte survival, tightly controls the hepatocellular response to ER stress possibly by interacting with the autophagy machinery.

Liver fibrosis results from chronic liver damage and is characterized by scarring of the liver parenchyma. Liver fibrosis can occur in all chronic liver diseases and shows progression towards liver cirrhosis in 20-40% of cases. The clinical presentation of liver fibrosis is usually unspecific. Therefore, most patients with liver fibrosis are identified by elevated liver enzymes during other medical examinations. The gold standard for quantification of liver fibrosis is percutaneous liver biopsy, but non-invasive markers (e. g. serum markers, transient elastography) have recently been evaluated to identify individuals with significant fibrosis. In case of fibrosis detection, medical therapies aim at stabilizing liver scarring or even at inducing the regression of fibrosis. Primarily this is achieved by etiology specific therapies of chronic liver diseases (e. g. antiviral therapy, immunosuppressive therapy etc.). However, in cases of failure of these specific therapies, non-specific interventions for fibrosis regression are actively being investigated. These treatment options are based on the growing molecular knowledge of fibrogenesis but are not yet available for routine clinical use.

Celiac disease is an immune-mediated enteropathy characterized by mucosal inflammation and villous atrophy of the small bowel upon exposure to ingested gluten. Refractory celiac disease (RCD), defined as persisting villous atrophy with crypt hyperplasia despite strict gluten-free diet, is a rare form of celiac disease with poor prognosis due to a higher rate of severe complications such as life-threatening malnutrition or the development of intestinal T-cell lymphoma. The cavitating mesenteric lymph node syndrome (CMLNS) represents a rare complication of celiac disease with unknown pathogenesis which is associated with but not restricted to RCD and not necessarily associated with a malignant course. We here report a 64-year-old patient who was referred to us with a history of refractory celiac disease. During further diagnostic work-up multiple intraabdominal cystic structures were detected by a computed tomography scan and magnetic resonance imaging. A laparotomy was performed to exclude T-cell lymphoma. Histology of the intraabdominal cysts revealed the diagnosis of cavitating mesenteric lymph node syndrome as the underlying cause of the masses. Steroid therapy was initiated which led to complete regression of diarrhoea but did not induce a diminution of mesenteric lymph nodes. Three years after the diagnosis of CMLNS, the patient presented with an acute abdomen due to a small bowel perforation caused by an enteropathy associated T-cell lymphoma. We discuss the differential diagnoses of intraabdominal masses in celiac disease and review the current literature on CMLNS.

Background and study aims: Narrow-band imaging (NBI) has been developed as a new technique to differentiate tissue patterns in vivo. The aim of this study was to evaluate the diagnostic accuracy of NBI endoscopy with and without high magnification for the differentiation of neoplastic from non-neoplastic colorectal polyps.Patients and methods: Among 200 colorectal polyps from 131 patients, 100 lesions were classified according to vascular patterns by NBI endoscopy with high optical magnification and 100 lesions by high-definition endoscopy without high magnification. Additionally, the clarity of the vessel network was assessed. Histologic analysis was performed on all lesions.Results: NBI endoscopy with high magnification resulted in a sensitivity of 92.1% and a specificity of 89.2% for the differentiation of neoplastic versus non-neoplastic lesions. This performance was statistically comparable to high-definition NBI endoscopy without high magnification, which showed a sensitivity of 87.9% and specificity of 90.5%. However, vessel network was significantly better visualized by NBI endoscopy with optical magnification compared with high-definition NBI endoscopy without high magnification. In comparison with NBI endoscopy, white-light endoscopy, with or without magnification, resulted in inferior discrimination between neoplastic and non-neoplastic polyps.Conclusion: The results demonstrate that the superior visibility of capillary vessels by the NBI technique allows the evaluation of colorectal lesions - based on the vascular patterns - with high diagnostic accuracy. In clinical routine, high-definition NBI endoscopy without high magnification may be used to sufficiently predict colorectal polyp histology, and high magnification can additionally facilitate visualization of vascular networks.

POSTERSto frequent administration have limited its potential clinical use.Therefore, we hypothesized that targeting of IFNg to HSCs might be an interesting approach to improve its efficacy and reduces its side effects.Since it is well known that PDGF-receptor expression is up-regulated in HSCs in liver injury, we used pPB, a cyclic peptide against PDGF-receptor to target IFNg to HSCs.Methods: We chemically conjugated IFNg to pPB using a PEG linker to improve the pharmacokinetics of IFNg.IFNg-PEG-pPB was characterized using Western blot analysis.To determine whether IFNg conjugate retained its biological activity after chemical conjugation, we performed Nitric Oxide (NO) release assay in RAW cells.The specific binding of conjugate to the PDGF-receptor was determined in LX2 (human HSCs).In-vitro, we evaluated the antifibrotic effects of IFNg-PEG-pPB by immuno-cytochemistry and quantitative-PCR.In-vivo, we investigated the anti-fibrotic effects of targeted IFNg conjugate in acute and chronic CCl 4 -induced liver fibrosis in mice.Results: IFNg-PEG-pPB conjugate was synthesised, characterized and showed similar biological activity as unmodified IFNg.We found that IFNg-PEG-pPB specifically bound to the PDGF-receptor on LX2 as confirmed by anti-PDGF-receptor blocking.In-vitro, IFNg-conjugate showed significant reduction of TGFb-induced a-SMA, collagen expression and increased collagen degradation (MMP13/TIMP1 ratio).In contrast, unmodified IFNg did not show any effect.In-vivo in CCl 4 -induced acute and chronic liver injury, treatment with IFNg-PEG-pPB significantly inhibited several fibrotic parameters (a-SMA and collagen) and substantially enhanced collagen degradation.Importantly, IFNg-PEG-pPB induced significant improvement in body weights and WBC counts (elevated in CCl 4 animals) and did not show IFNg-related side effects e.g.low body temperature, high triglyceride content and reduced platelets.Conclusions: Cell-specific targeting of IFNg to HSC's through the PDGF-receptor significantly increases its efficacy and reduces its adverse effects.

Results: After 7 days, BDL rats showed elevated serum levels of GGT, AST, ALT, LDH, and bilirubin, bile duct proliferation and fibrosis, compared to SO group.TUDCA treatment did not significantly alter these parameters, but the iNOS inhibitor SMT ameliorated hepatocellular injury, as demonstrated by lower levels of circulating hepatic enzymes and bilirubin, and a decreased grade of bile duct proliferation and fibrosis.Both TUDCA and SMT treatments reversed Mrp2 canalicular pump expression to control levels.However, only SMT treatment significantly lowered the increased levels of plasma NO and S-nitrosation (S-nitrosylation) of liver proteins in BDL rats.Moreover, BDL resulted in a reduction of the S-nitrosoglutathione reductase (GSNOR/Adh5) enzymatic activity and a down-regulation of the GSNOR/Adh5 mRNA expression that was reverted by SMT, but not TUDCA, treatment.Twenty five liver proteins, including S-adenosyl methionine synthetase, betainehomocysteine S-methyltransferase, Hsp90 and protein disulfide isomerase, were found to be S-nitrosated in BDL rats.Conclusions: The inhibition of NO production during induced cholestasis ameliorates hepatocellular injury.This effect is in part mediated by the improvement of cell proficiency in maintaining S-nitrosothiol homeostasis.