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The aim of this study was to explore the intracellular signaling pathways involved in the stimulatory effects of estrogens on cholangiocyte proliferation. We investigated the tyrosine kinase-receptor pathway by evaluating the protein expression of total and phosphorylated mitogen-activated protein kinase (MAPK) isoform p44/p42 (e.g., extracellular signal-regulated kinase [ERK]1/2), the steroid-receptor coactivator Src and Shc (Src-homology/collagen protein). The study was performed in 3-week-old bile duct-ligated (BDL) rats, BDL rats treated with the antiestrogens, tamoxifen or Ici 182,780, and normal control rats. Proliferation was also evaluated in normal purified cholangiocytes treated with 17 beta estradiol in the presence or absence of tamoxifen, Ici 182,780, ERK, or Src inhibitors. After bile duct ligation, cholangiocyte proliferation was associated with a marked immunohistochemical nuclear positivity for phosphorylated (p)-ERK1/2, which was inhibited by in vivo treatment with tamoxifen or Ici 182,780. Protein expression of total and p-ERK1/2, and Shc in cholangiocytes isolated from BDL rats was markedly increased compared with controls and was inhibited by in vivo treatment with antiestrogens. In vitro, 17 beta estradiol-induced proliferation of isolated normal cholangiocyte was associated with increased (P

Background Triple therapy including Telaprevir or Boceprevir still represents in many European countries the standard of care for patients with Hepatitis C Virus genotype 1 infection. The number of patients who received this treatment resulted generally lower than expected. We investigated, among naive patients, number and characteristics of treatment candidates who were started on triple or dual therapy in comparison to those who were deferred. Patients and Methods 621 naive treatment candidates were prospectively evaluated at each center. Factors associated with decision to defer or treat with dual or triple therapy were investigated by univariate and multivariate analyses. Rates of Sustained Virological Response and safety profile were analysed. Results Of candidates to treatment, 33% did not received it. It was mostly due to high risk of Interferon-induced decompensation. Of 397 patients who were started on treatment, 266 (67%) received triple, 131 dual. Among patient receiving treatment, unfavorable IL28B, severe liver damage and higher albumin were independently associated with the physician decision to administer triple therapy. Sustained Virological Response after dual therapy was 66.4%, after triple 73.7% (p = 0.14). 142 patients received Telaprevir. The choice of Telaprevir-based therapy was associated with higher Body Mass Index and advanced liver disease. Sustained Virological Response rates were 71.1% after Telaprevir and 76.6% after Boceprevir. Conclusions Individualizing treatment with available regimens allows to maximize Sustained Virological Response and to reduce the number of patients who remain untreated. High proportion of patients with severe liver damage urgently need Interferon free treatment.

The activation of the biliary stem-cell signaling pathway hairy and enhancer of split 1/pancreatic duodenal homeobox-1 (Hes-1/PDX-1) in mature cholangiocytes determines cell proliferation. Neurogenin-3 (Ngn-3) is required for pancreas development and ductal cell neogenesis. PDX-1-dependent activation of Ngn-3 initiates the differentiation program by inducing microRNA (miR)-7 expression. Here we investigated the role Ngn-3 on cholangiocyte proliferation. Expression levels of Ngn-3 and miR-7 isoforms were tested in cholangiocytes from normal and cholestatic human livers. Ngn-3 was knocked-down in vitro in normal rat cholangiocytes by short interfering RNA (siRNA). In vivo, wild-type and Ngn-3-heterozygous (+/-) mice were subjected to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding (a model of sclerosing cholangitis) or bile duct ligation (BDL). In the liver, Ngn-3 is expressed specifically in cholangiocytes of primary sclerosing cholangitis (PSC) patients and in mice subjected to DDC or BDL, but not in normal human and mouse livers. Expression of miR-7a-1 and miR-7a-2 isoforms, but not miR-7b, was increased in DDC cholangiocytes compared to normal ones. In normal rat cholangiocytes, siRNA against Ngn-3 blocked the proliferation stimulated by exendin-4. In addition, Ngn-3 knockdown neutralized the overexpression of insulin growth factor-1 (IGF1; promitotic effector) observed after exposure to exendin-4, but not that of PDX-1 or VEGF-A/C. Oligonucleotides anti-miR-7 inhibited the exendin-4-induced proliferation in normal rat cholangiocytes, but did not affect Ngn-3 synthesis. Biliary hyperplasia and collagen deposition induced by DDC or BDL were significantly reduced in Ngn-3(+/-) mice compared to wild-type.Ngn-3-dependent activation of miR-7a is a determinant of cholangiocyte proliferation. These findings indicate that the reacquisition of a molecular profile typical of organ development is essential for the biological response to injury by mature cholangiocytes.

BACKGROUND: α-Fetoprotein (AFP) is a biomarker commonly used in the management of patients with hepatocellular carcinoma (HCC), although the possible determinants of its serum levels in these patients have not been adequately explored. For this study, the authors evaluated the relevance of demographic, clinical, and oncologic factors to the presence of elevated AFP levels in large cohort of patients with HCC. METHODS: In 4123 patients with HCC who were managed by the Italian Liver Cancer Group, AFP levels were assessed along with their association with demographic, biochemical, clinical, and oncologic characteristics. Patients were subdivided according to the presence of elevated AFP (ie, >10 ng/mL). RESULTS: AFP levels were elevated in 62.4% of patients with HCC. Multivariate logistic regression analysis indicated that being a woman (odds ratio [OR], 1.497; 95% confidence interval [95%CI], 1.250-1.793; P < .0001), the presence of cirrhosis (OR, 1.538; 95% CI, 1.050-2.254; P = .027), liver disease with viral etiology (OR, 1.900; 95% CI, 1.589-2.272; P < .0001), an elevated alanine aminotransferase level (OR, 1.878; 95% CI, 1.602-2.202; P < .0001), a low albumin level (OR, 1.301; 95% CI, 1.110-1.525; P = .012), an HCC tumor size >2 cm (OR, 1.346; 95% CI, 1.135-2.596; P = .001), multinodular HCC (OR, 1.641; 95% CI, 1.403-1.920; P < .0001), and the presence of vascular invasion (OR, 1.774; 95% CI, 1.361-2.311; P < .0001) were associated independently with elevated levels of AFP. Both the median AFP level and the proportion of patients who had elevated levels increased with decreasing degrees of HCC differentiation (P < .0001). CONCLUSIONS: Sex and features of chronic liver disease were identified as nontumor characteristics that influence serum AFP levels in patients with HCC. These findings should be taken into account as limitations in interpreting the oncologic meaning of this biomarker in clinical practice.

NAFLD is the most common liver disease worldwide but it is the potential evolution to NASH and eventually to hepatocellular carcinoma (HCC), even in the absence of cirrhosis, that makes NAFLD of such clinical importance. Aim: we aimed to create a mouse model reproducing the pathological spectrum of NAFLD and to investigate the role of possible co-factors in promoting HCC. Methods: mice were treated with a choline-deficient L-amino-acid-defined-diet (CDAA) or its control (CSAA diet) and subjected to a low-dose i.p. injection of CCl 4 or vehicle. Insulin resistance was measured by the euglycemic-hyperinsulinemic clamp method. Steatosis, fibrosis and HCC were evaluated by histological and molecular analysis. Results: CDAA-treated mice showed peripheral insulin resistance at 1 month. At 1-3 months, extensive steatosis and fibrosis were observed in CDAA and CDAA+CCl4 groups. At 6 months, equal increase in steatosis and fibrosis was observed between the two groups, together with the appearance of tumor. At 9 months of treatment, the 100% of CDAA+ CCl4 treated mice revealed tumor versus 40% of CDAA mice. Insulin-like Growth Factor-2 (IGF-2) and Osteopontin (SPP-1) were increased in CDAA mice versus CSAA. Furthermore, Immunostaining for p-AKT, p-c-Myc and Glypican-3 revealed increased positivity in the tumors. Conclusions: the CDAA model promotes the development of HCC from NAFLD-NASH in the presence of insulin resistance but in the absence of cirrhosis. Since this condition is increasingly recognized in humans, our study provides a model that may help understanding mechanisms of carcinogenesis in NAFLD. © 2014 De Minicis et al.

BACKGROUND The efficacy in renal protection of Everolimus (Evr)-based regimens compared to standard dosages of Tacrolimus (Tac) has recently become known. The purpose of this study was to retrospectively compare the renal function in the first year after LT, with a case-control approach, in patients treated with Tac monotherapy at sub-standard dose vs. an immunosuppressive regimen with Tac and Evr. MATERIAL/METHODS Following a 2: 1 case-control approach, 37 patients with an immunosuppression regimen based on Tac only (Tac Group) were retrospectively compared with 74 patients utilizing a combination of Evr and Tac (Evr-Tac Group), based on the following preoperative parameters: sex, age, MELD score (±3), and pre-LT chronic kidney disease (CKD) stage. RESULTS After a mean follow-up of 82.8 ± 24 vs. 42.4 ± 16.6 months (p < 0.001), overall survival in 1, 3, and 5 years was 97.3, 91.9, and 86.5% vs. 95.9, 81.1, and 69.5% (p = 0.10) for the Tac Group vs. the Evr-Tac Group, respectively. The trend of the estimated glomerular filtration rate (e-GFR) during the first year post-LT was similar between the 2 study groups (80.1 ± 21 vs. 73.3 ± 16 mL/min/1.73 m^2, for the Tac Group vs. the Evr-Tac Group, respectively, p = 0.23). The incidence of acute rejection histologically proven was 32.4% vs. 20% (p = 0.71) for the Tac Group vs. the Tac-Evr Group, respectively. The rate of CKD was also similar in the 2 study groups. CONCLUSIONS The early combination of Evr and Tac is an efficient immunosuppressant regimen and provided similar renal function at 1 year post-LT, compared to a minimization of the monotherapy dose of Tac. The combination therapy of Evr-Tac is subject to a higher rate of drugs discontinuation due to adverse effects of 1 of the 2 drugs.

Background: Splanchnic venous thrombosis (SVT) encompasses thrombosis in the mesenteric, splenic or portal veins (with or without hepatic veins involvement). Little is known about appropriate therapeutic interventions and long-term clinical outcome of SVT patients. Aim of this study was to identify the correct management of SVT and encourage a multidisciplinary approach by a team composed of hematologists, hepatologists, and infectivologists. Methods:We analyzed clinical, laboratory, therapeutic and outcome data of 127 patients with SVT that were recruited from 2000 to 2016. In patients with no active bleeding, anticoagulation treatment was started as soon as possible, according to platelet count. We administered intermediate or full therapeutic dose low-molecular-weight heparin (LMWH) and early initiation of vitamin-K antagonist (VKA; INR range 2-3 or 1.8-2.5 in patients with high bleeding risk) for a platelet count >50.000/μl, only half or prophylactic dose of LMWH for a platelet count >30.000 and < 50.000/μl and no treatment for a platelet count Results: Overall, 127 patients were included (median age 58 years; 74% males). The median follow-up of all patients was 11 months (1-212). Portal vein thrombosis was the most common site of thrombosis (50%), followed by multiple venous involvement (37%). Liver cirrhosis and solid neoplasms were the common underlying disease (72% and 36% respectively) while myeloproliferative neoplasms were identified in 8 patients (6.2%). Eighty-nine patients (70%) had esophageal varices (grade >2 in 55 patients) and 81 (64%) had thrombocytopenia (mean 72.000/μl range 28.000/μl-148.000/μl). Ninety-nine patients (78%) were treated with anticoagulant therapy: 36% with intermediate or full dose of LMWH, 40% with half or prophylactic dose of LMWH and 24% with VKA (TTR 76%). During a median duration therapy of 7 months, the incidence rate of thrombotic events was 1.1 per 100 pt-y while the incidence rate of major bleeding was 1.6 per 100 pt-y. At univariate analysis, esophageal varices (p=0.030), renal failure (p=0.001) and liver cirrhosis (p=0.05) significantly increased the risk of bleeding events. Moreover VKA exposure was associated with a significantly lower risk of bleeding events compared to LMWH (p=0.042). Fifty-six patients (44%) obtained vessel recanalization and the probability of recanalization of the occluded vessels was 50% at 18 months. At univariate analysis, factors associated with a lack of recanalization included liver cirrhosis (p=0.004) and solid tumor (p=0.010). Only one death was attributed to fatal bleeding whereas 31 patients died for causes not related to anticoagulation (cirrhosis, cancer). Conclusions: Our study suggests the effectiveness of anticoagulant therapy (especially VKA), leading to thrombus recanalization in 44% of patients with SVT. Notably, the anticoagulant treatment was associated with a very low bleeding incidence also in patients with major risk factors for bleeding (i.e. liver cirrhosis, cancer or esophageal varices). Treatment algorithm and therapeutic decisions were taken as a multidisciplinary team, able to adapt the individual approach and avoid fatal complications. Disclosures Offidani: Janssen: Honoraria; Celgene: Honoraria, Research Funding.

This study retrospectively evaluated 144 consecutive patients with unusual site thrombosis who referred to our Thrombosis Center between 2000 and 2016. All patients were classified as having either splanchnic venous thrombosis (SVT; n=127) or cerebral venous thrombosis (CVT; n=17). On the presence and type of provoking risk factors, then patients were categorized into three groups: unprovoked, those with possibly resolved provoking factors (PR), and those with persistent provoking factors (PP). Among the identified risk factors regarded as PP, we focused on Myeloproliferative Neoplasms (MPN) and performed a clinical comparison between MPN patients with SVT (MPN-SVT) and those with CVT (MPN-SVT). The characteristics of our cohort well reflects the clinical heterogeneity of clinical features commonly found in the routine clinical practice of diagnosing unusual site thrombosis. One hundred and twenty seven SVT patients were included: 7 unprovoked SVT, 10 SVT with PR, 110 SVT with PP; seventeen patients showed CVT, 5 unprovoked, 6 CVT with PR and 6 CVT with PP. Major risk factor for SVT with PP was liver cirrhosis (71.6%), whereas for CVT were MPN (5 patients, 29.4%). MPN was present in 8 patients (6.2%) of SVT (MPN-SVT vs MPN-CVT, p=0.009). For MPN-SVT, 3 patients showed the morphological features of polycytemia vera (PV), 2 of essential thrombocytemia (ET), 1 of primary myelofibrosis (PMF) and 2 fell into the MPN unclassified category (U-MPN); distribution of MPN subtypes for CVT was as follows: 1 PV, 2 ET and 2 U-MPN. Molecular analysis identified the JAK2V617F mutation respectively in 75% patients with MPN-SVT and in 60% patients with MPN-CVT; bone marrow histological features supported the diagnosis of MPN in all cases. Median age at MPN-SVT diagnosis was 46 years (range 17-78) vs 43 years (range 31-84) for MPN-CVT. Coexisting PR and thrombophilic abnormalities were identified in 75% and 20% of MPN-SVT and MPN-CVT respectively, so MPN are per se a strong risk for thrombosis and the leading systemic cause of CVT. In four of five cases (80%), CVT antedate the clinical phenotype of an overt MPN, whereas SVT occurred at MPN diagnosis in five patients and during MPN follow-up in three patients (median 164 months, range 88-215). At diagnosis MPN-SVT tended to have significantly lower platelet and white blood cell counts and haemoglobin concentration than MPN-CVT (respectively p Disclosures Offidani: Celgene: Honoraria, Research Funding; Janssen: Honoraria.

Background/Aims: Reactive oxygen species (ROS) induce HSCs activation, proliferation and collagen gene expression in vitro. Nitric oxide (NO) represents a reactive molecule that reacts with ROS, yielding peroxynitrite. We thus verified the effect of NO on ROS-induced HSCs proliferation in vitro and correlated iNOS expression and ROS formation to HSCs activation in the early phase of liver injury leading to hepatic fibrosis in vivo. Methods/Results: HSCs were incubated with iron ascorbate (FeAsc) in vitro, which induced ROS production, ERK1/2 phosphorylation and increased cell proliferation. This effect was significantly reduced by the presence of the NO donor S-nitroso-N-acetylpenicillamine. Liver injury was induced in vivo in rats by dimethylnitrosamine administration. HSCs activation started 6 h after DMN administration and peaked at 1 week. ROS generation and neutrophil infiltration were evident for at least 48 h after DMN treatment, showing an identical distribution pattern. Only a few inflammatory cells expressed iNOS 6 h after DMN administration. Conclusions: we have shown that NO acts as a ROS scavenger in vitro, thus inhibiting HSCs proliferation. ROS production by infiltrating neutrophils occurs in the early phase of liver fibrosis and can represent a stimulus to HSCs activation in vivo. The reduced iNOS expression may account for the low NO levels and the inability to prevent the ROS-induced HSC activation in vivo.

BACKGROUND/AIMS The involvement of a direct viral cytopathic effect or an immune-mediated mechanism in the progression of hepatic damage in chronic hepatitis C is controversial. The type of immune response is itself a matter of controversy, and histological data are lacking. The aim of this study was to identify the factors associated with the progression of liver injury in 30 HCV/RNA-positive untreated patients with chronic hepatitis. METHODS Necroinflammatory and architectural damage were evaluated using Ishak's score. Activated hepatic stellate cells (HSC) were visualized by immunohistochemistry for alpha-smooth muscle actin (alphaSMA) and quantitated by morphometry. Plasma HCV/RNA was evaluated using a competitive RT-PCR method. To study the type of immune response involved in the progression of liver injury, interferon gamma (IFNgamma)-positive cells (as expression of a Th1-like response) were evaluated by immunohistochemistry and quantitated by morphometry. RESULTS HSC were mostly detected close to areas of lobular necroinflammation or lining fibrotic septa. The alphaSMA- and Sirius Red-positive parenchyma correlated significantly with necroinflammatory and architectural scores. IFNgamma-positive cells were detected in periportal areas associated with the inflammatory infiltrates and significantly correlated with architectural damage. No relationship was found between the histological features of liver injury and viral load. CONCLUSIONS HSC activation and progression of liver injury are unrelated to viral load but associated with a Th1-like response, a plausible target for the treatment of chronic hepatitis C.

Insulin and insulin-like growth factor (IGF-1) are mitogenic for fibroblasts and smooth muscle cells. IGF-1 increases in inflamed and fibrotic tissues and induces proliferation of rat hepatic stellate cells (HSC). This study evaluates the potential roles of these hormones in the development of liver fibrosis. Insulin and IGF-1 receptor expression was evaluated by immunohistochemistry in both cultured human HSC and human liver tissue. Phosphorylation of both 70-kd S6 kinase and extracellular-regulated kinase (ERK), cell proliferation, type I collagen gene expression, and accumulation in HSC culture media were evaluated by Western blot, immunohistochemistry for bromodeoxyuridine (BrdU), Northern blot, and enzyme-linked immunosorbent assay, respectively. Insulin and IGF-1 receptors were detected in HSC in vitro and in liver sections from patients with chronic active hepatitis. Insulin and IGF-1 induced 70-kd S6 kinase phosphorylation in HSC, whereas IGF-1 only induced ERK phosphorylation. Insulin and IGF-1 stimulated HSC proliferation in a dose-dependent fashion, with IGF-1 being four to five times more potent than insulin. Cell exposure to specific inhibitors showed that both phosphatidylinositol 3-kinase (PI3-K) and ERK are involved in IGF-1-induced mitogenesis, whereas insulin stimulated mitogenesis through a PI3-K-dependent ERK-independent pathway. IGF-1 increased type I collagen gene expression and accumulation in HSC culture media through a PI3-K- and ERK-dependent mechanism. In conclusion, insulin and IGF-1, which stimulate HSC mitogenesis and collagen synthesis, may act in concert to promote liver fibrosis in vivo by a differential activation of PI3-K- and ERK1-dependent pathways.

Background/Aims: Oxidative stress is associated with liver fibrosis in vivo and with hepatic stellate cell (HSC) activation in vitro , but the intracellular mechanisms mediating these effects are mostly unknown. The Na + /H + exchanger plays a key role in regulating the cell cycle, and is involved in HSC proliferation. Its role in different HSC features, such as collagen accumulation, is still unknown. We thus evaluated if the Na + /H + exchanger modulates the fibrogenic effect of oxidative stress in rat HSC. Methods: HSC were incubated with 0.1 mM ferric nitrilotriacetate complex (FeNTA). Intracellular hydroperoxides and malonildialdehyde (MDA) levels in the culture media were measured by the dichlorofluorescein and TBARS method, respectively. Intracellular pH and Na + /H + exchanger activity were measured using the fluorescent dye BCECF. Cell proliferation was measured by immunohistochemistry for bromodeoxyuridine incorporation. Collagen type I accumulation in the culture media was measured by ELISA. Results: HSC incubation with FeNTA resulted in a significant production of intracellular hydroperoxides and MDA, associated with increased Na + /H + exchange activity and baseline intracellular pH (pHi). Exposure of HSC to FeNTA significantly enhanced the number of proliferating HSC and collagen type I levels in the culture medium. All these effects were reversed by the antioxidant resveratrol and by the Na + /H + exchanger inhibitor amiloride. Conclusions: This study indicates that the Na + /H + exchanger might represent a common mediator of the different effects induced by oxidative stress on HSC. The reduction in cell proliferation and collagen synthesis induced by amiloride could represent a new therapeutic challenge in liver fibrosis.

Background & Aims: The Na + /H + exchanger is the main intracellular pH regulator in hepatic stellate cells (HSCs), and its activity is increased by platelet-derived growth factor (PDGF). Amiloride, an Na + /H + exchange inhibitor, reduces PDGF-induced HSC proliferation, suggesting that the Na + /H + exchanger plays a role in regulating HSC proliferative response. The aim of this study was to characterize the intracellular pathways mediating activation of the Na + /H + exchanger by PDGF in HSCs. Methods: The activity of the Na + /H + exchanger and HSC proliferation rate were evaluated under control condition and after incubation with PDGF in the absence or presence of specific inhibitors of the main intracellular pathways of signal transduction. Na + /H + exchange protein expression was evaluated by means of Western blot. Results: PDGF induced a significant increase in the activity of the Na + /H + exchanger without modifying protein expression. Inhibition of the calcium/calmodulin– and protein kinase C–dependent pathways resulted in a significant inhibition of both Na + /H + exchange activity and of PDGF-induced HSC proliferation. The involvement of the two pathways was confirmed by showing that incubation of HSCs with both phorbol-12-myristate-13-acetate, a potent protein kinase C activator, and thapsigargin, which increases intracellular calcium levels, significantly increased both the Na + /H + exchanger activity and HSC proliferation rate. Inhibition of the protein kinase A pathway did not modify either PDGF-induced Na + /H + exchange activation or PDGF-induced HSC proliferation. On the contrary, inhibition of the mitogen-activated protein kinase– and of phosphatidylinositol 3-kinase–dependent pathways significantly reduced PDGF-induced HSC proliferation without affecting the activity of the Na + /H + exchanger. Conclusions: Activation of the Na + /H + exchanger by PDGF in HSCs is mediated by calcium/calmodulin- and protein kinase C–dependent pathways. PDGF-induced HSC proliferation is mediated by Na + /H + exchange–dependent and –independent pathways. GASTROENTEROLOGY 1999;116:1155-1166

Oxidative stress is associated with liver fibrosis and with hepatic stellate cell (HSC) activation in vivo. However, it remains controversial whether oxidative stress contributes to HSC activation either directly or through a paracrine stimulation by damaged hepatocytes. A medium containing products released from cells undergoing oxidative stress was obtained after incubation of hepatocytes with (HCM/Fe) or without (HCM) 0.1 mmol/L ferric nitrilotriacetate complex (FeNTA). Exposure of HSC to HCM/Fe for 24 hours significantly increased the number of proliferating HSC compared with HCM and to controls at all dilutions tested. The simultaneous coincubation of HSC with HCM/Fe and desferrioxamine (50 micromol/L) did not reduce the observed increase in cell proliferation, thus excluding a role for eventually contaminating iron in HCM/Fe. HCM/Fe induced also a significant increase in collagen type I accumulation in HSC culture media. To study the cellular mechanism underlying HCM/Fe effects, we evaluated the activity of the Na+/H+ exchanger, which plays a role in regulating HSC proliferation. The incubation of HSC for 24 hours with HCM/Fe significantly increased baseline intracellular pH (pHi) and Na+/H+ exchanger activity, indicating a plausible role of this antiport in mediating cell response. In conclusion, hepatocytes undergoing oxidative stress release factors which are fibrogenic for HSC, thereby, confirming what has been only hypothesized in vivo. In addition, HSC proliferation is associated with changes in the Na+/H+ exchanger activity, thus providing a useful target for the evaluation of inhibitors of this pathway for the treatment of hepatic fibrosis.

Nonalcoholic fatty liver disease (NAFLD) may lead to hepatic fibrosis. Dietary habits affect gut microbiota composition, whereas endotoxins produced by Gram-negative bacteria stimulate hepatic fibrogenesis. However, the mechanisms of action and the potential effect of microbiota in the liver are still unknown. Thus, we sought to analyze whether microbiota may interfere with liver fibrogenesis. Mice fed control (CTRL) or high-fat diet (HFD) were subjected to either bile duct ligation (BDL) or CCl4 treatment. Previously gut-sterilized mice were subjected to microbiota transplantation by oral gavage of cecum content obtained from donor CTRL- or HFD-treated mice. Fibrosis, intestinal permeability, bacterial translocation, and serum endotoxemia were measured. Inflammasome components were evaluated in gut and liver. Microbiota composition (dysbiosis) was evaluated by Pyrosequencing. Fibrosis degree was increased in HFD+BDL versus CTRL+BDL mice, whereas no differences were observed between CTRL+CCl4 and HFD+CCl4 mice. Culture of mesenteric lymph nodes showed higher density of infection in HFD+BDL mice versus CTRL+BDL mice, suggesting higher bacterial translocation rate. Pyrosequencing revealed an increase in percentage of Gram-negative versus Gram-postive bacteria, a reduced ratio between Bacteroidetes and Firmicutes, as well as a dramatic increase of Gram-negative Proteobacteria in HFD+BDL versus CTRL+BDL mice. Inflammasome expression was increased in liver of fibrotic mice, but significantly reduced in gut. Furthermore, microbiota transplantation revealed more liver damage in chimeric mice fed CTRL diet, but receiving the microbiota of HFD-treated mice; liver damage was further enhanced by transplantation of selected Gram-negative bacteria obtained from cecum content of HFD+BDL-treated mice. Conclusions: Dietary habits, by increasing the percentage of intestinal Gram-negative endotoxin producers, may accelerate liver fibrogenesis, introducing dysbiosis as a cofactor contributing to chronic liver injury in NAFLD. (Hepatology 2014;59:1738–1749)

AIM: To assess the possible effect of two different types of preoperative transcatheter arterial chemoembolization (TACE) on recurrence-free survival after liver transplantation (LT) in patients with hepatocellular carcinoma (HCC) and to analyze the effects of TACE on tumor histology. METHODS: We retrospectively analyzed the histological features of 130 HCC nodules in 63 native livers removed at transplantation. Patients who received any other type of treatment such as radiofrequency tumor ablation, percutaneous ethanol ablation or who were not treated at all were excluded. All patients in the present study were within the Milan Criteria at the last imaging findings before transplantation. Doxorubicin-eluting bead TACE (DEB-TACE) was performed in 22 patients (38 nodules), and conventional TACE (c-TACE) in 16 (25 nodules). Patients’ and tumors’ characteristics were retrospectively reviewed. We performed a per-nodule analysis of the explanted livers to establish the mean percentage of necrosis of any nodule treated by TACE (conventional or DEB) and a per-patient analysis to establish the percentage of necrosis in the cumulative tumor area, including 21 nodules not reached by TACE. Inflammatory and fibrotic changes in the tissue surrounding the tumor nodule were analyzed and categorized as poor/absent, moderate and enhanced reaction. Uni- and multivariate analysis of risk factors for HCC-recurrence were performed. RESULTS: The number and diameter of the nodules, the time spent on the waiting list and the number of treatments were similar in the two groups. A trend towards higher appropriate response rates (necrosis ≥ 90%) was observed in the DEB-TACE group (44.7% vs 32.0%, P = 0.2834). The mean percentage of necrosis in the cumulative tumor area was 58.8% ± 36.6% in the DEB-TACE group and 50.2% ± 38.1% in the c-TACE group (P = 0.4856). Fibrotic and inflammatory reactions surrounding the tumor nodule were markedly more common in the DEB-TACE group (P < 0.0001, for both the parameters). The three-year recurrence-free survival was higher in DEB-TACE-treated patients than in conventionally treated patients (87.4% vs 61.5%, P = 0.0493). Other factors affecting recurrence-free survival included viable tumor beyond Milan Criteria on histopathological examination, the percentage of necrosis on CTA ≤ 50% and a pre-transplant serum α-fetoprotein level greater than 70 ng/mL. On multivariate analysis, the lack of treatment with DEB-TACE, high levels of α-fetoprotein and viable tumor beyond Milan Criteria at histology examination were identified as independent predictors of tumor recurrence. CONCLUSION: DEB-TACE can effectively promote tumor necrosis and improves recurrence-free survival after LT in HCC.

Interferon gamma (IFN-gamma) inhibits in vitro the activation of hepatic stellate cells (HSC), the primary extracellular matrix-producing cells in liver fibrosis. This study was undertaken to determine in vivo the effect of IFN-gamma in the rat model of liver fibrosis induced by dimethylnitrosamine (DMN), where HSC activation represents an early response to cell injury. Rats were killed after 1 or 3 weeks of treatment with DMN, IFN-gamma, DMN + IFN-gamma, or saline. Immunohistochemistry was used to identify proliferating (desmin- positive/bromodeoxyuridine (BrdU)-positive cells) and activated (alpha- smooth-muscle actin [alpha-SMA]-positive cells) HSCs. Collagen deposition was determined colorimetrically and by morphometry. The parenchymal extension of desmin- and actin-positive cells and of fibrotic tissue was measured by point-counting technique and expressed as a percentage of area. Western blot was used to determine laminin and fibronectin accumulation. The levels of messenger RNA (mRNA) for procollagen type I, fibronectin, and laminin were evaluated by Northern blot. No differences were observed in rats treated with either saline or IFN-gamma alone. IFN-gamma reduced HSC activation induced by liver injury, as shown by the decreased number of proliferating HSC and the reduction of parenchymal area occupied by alpha-SMA-positive cells observed in DMN + IFN-gamma-treated animals compared with the DMN group. This was associated with reduced collagen, laminin, and fibronectin accumulation and lower levels of mRNA for procollagen type I, fibronectin, and laminin in the DMN + IFN-gamma group. Thus, this study indicates that IFN-gamma reduces extracellular matrix deposition in vivo by inhibition of HSC activation. (Hepatology 1996 May;23(5):1189-99)

Hepatocarcinogenesis is a multistep process involving different genetic alterations that ultimately lead to malignant transformation of the hepatocyte. The liver is one of the main targets for different metastatic foci, but it represents an important and frequent locus of degeneration in the course of chronic disease. In fact, Hepatocellular carcinoma (HCC) represents the outcome of the natural history of chronic liver diseases, from the condition of fibrosis, to cirrhosis and finally to cancer. HCC is the sixth most common cancer in the world, some 630,000 new cases being diagnosed each year. Furthermore, about the 80% of people with HCC, have seen their clinical history developing from fibrosis, to cirrhosis and finally to cancer. The three main causes of HCC development are represented by HBV, HCV infection and alcoholism. Moreover, metabolic disease [starting from Non Alcoholic Fatty Liver Disease (NAFLD), Non Alcoholic Steatohepatitis (NASH)] and, with reduced frequency, some autoimmune disease may lead to HCC development. An additional rare cause of carcinogenetic degeneration of the liver, especially developed in African and Asian Countries, is represented by aflatoxin B1. The mechanisms by which these etiologic factors may induce HCC development involve a wide range of pathway and molecules, currently under investigation. In summary, the hepatocarcionogenesis results from a multifactorial process leading to the common condition of genetic changes in mature hepatocytes mainly characterized by uncontrolled proliferation and cell death. Advances in understanding the mechanism of action are fundamental for the development of new potential therapies and results primarily from the association of the research activities coming from basic and clinical science. This review article analyzes the current models used in basic research to investigate HCC activity, and the advances obtained from a basic and clinical point of view.

Transforming growth factor beta 1 (TGF beta 1) is involved in promoting cell death by apoptosis in the liver, whereas the activation of Na+/H+ exchanger has been related to cell proliferation. The aim of this study was to gain information on the effects of TGF beta 1 on intracellular pH and Na+/H+ exchange activity in isolated periportal (PP) and perivenular (PV) rat hepatocytes using the pH-sensitive dye BCECF in a perfused subconfluent hepatocyte monolayer. Steady-state intracellular pH (pHi) in a bicarbonate-free solution (HEPES) were 7.17 +/- 0.031 in PP and 7.15 +/- 0.041 in PV cells. Treatment with TGF beta 1 (120 pmol/L) for 7 hours increased the number of apoptotic bodies by 25% and 38%, and decreased steady-state pHi to 7.11 +/- 0.018 (P = .05) and to 7.07 +/- 0.021 (P < .02), respectively, in PP and PV hepatocytes. In HEPES, cells recovered from an acid load, extruding protons at a rate (JH) of 4.85 +/- 1.01 mmol/L/min in PP cells and of 4.91 +/- 0.99 mmol/L/min in PV hepatocytes. This recovery appeared amiloride inhibitable (1 mmol/L). Culture with TGF beta 1 for 7 hours induced (in HEPES) a decrease of pHi recovery rate from an acid load more in PV (by 46%) than in PP hepatocytes (by 35%, P < .05). Acute administration of epidermal growth factor (EGF) (10 to 100/mL) induced an increase in Na+/H+ exchange activity by 32% and 27%, respectively, in PP and PV cells compared with controls. In contrast, in cells cultured for 7 hours with 120 pmol/L TGF beta 1, the acute administration of EGF slightly increased Na+/H+ exchange activity (by 18%, P < .05) only in PP cells. This study demonstrates that pHi and Na+/H+ exchange activity are decreased by TGF beta 1, which increases the number of apoptotic bodies in periportal and perivenular rat hepatocyte primary cultures.

Hepatocellular carcinoma and cholangiocarcinoma are primary liver cancers, both represent a growing challenge for clinicians due to their increasing morbidity and mortality. In the last few years a number of in vivo models of hepatocellular carcinoma and cholangiocarcinoma have been developed. The study of these models is providing a significant contribution in unveiling the pathophysiology of primary liver malignancies. They are also fundamental tools to evaluate newly designed molecules to be tested as new potential therapeutic agents in a pre-clinical set. Technical aspects of each model are critical steps, and they should always be considered in order to appropriately interpret the findings of a study or its planning. The purpose of this review is to describe the technical and experimental features of the most significant rodent models, highlighting similarities or differences between the corresponding human diseases. The first part is dedicated to the discussion of models of hepatocellular carcinoma, developed using toxic agents, or through dietary or genetic manipulations. In the second we will address models of cholangiocarcinoma developed in rats or mice by toxin administration, genetic manipulation and/or bile duct incannulation or surgery. Xenograft or syngenic models are also proposed.

Background & Aims Cholangiocyte proliferation plays a role in the progression of cholangiopathies, in particular in primary sclerosing cholangitis. The mechanisms regulating cholangiocyte proliferation are still undefined. Pancreatic Duodenal Homeobox protein 1 (PDX-1) is expressed by reactive cholangiocytes. In the adult pancreas, PDX-1 regulates the proliferative response to injury of ductal cells. Its effects can be counteracted by Hairy and enhancer of split 1 (Hes-1). We aimed at studying whether PDX-1/Hes-1 interactions regulate cholangiocyte proliferation in response to injury. Methods The effect of the loss of PDX-1 on cholangiocyte proliferation was studied in vitro . In vivo PDX-1 -heterozygous (+/−) mice were subjected to either DDC feeding (a model of sclerosing cholangitis) or to bile duct ligation (BDL). PDX-1/Hes-1 interactions on cell proliferation were determined by exposure to All-trans Retinoic Acid (At-RA), an inductor of Hes-1. Results In vitro , cholangiocyte proliferation was undetectable in cells pre-treated with PDX-1 siRNA. In vivo , increases in bile duct mass and collagen deposition observed after DDC feeding or BDL were significantly reduced in PDX-1 +/− mice. Hes-1 expression is reduced in proliferating cholangiocytes; At-RA induced a dose-dependent increase in Hes-1 and a decrease in PDX-1 expression. At-RA neutralized the increases in PDX-1 expression and cell proliferation, both in vitro and in vivo in DDC mice. PDX-1 is overexpressed and Hes-1 downregulated in cholangiocytes isolated from PSC livers. Conclusions Hes-1 downregulation allows PDX-1 to act as a major determinant of cholangiocyte proliferation in response to cholestatic injury. These findings provide novel mechanistic insights into the pathophysiology of cholangiopathies.

BACKGROUND AND AIM The role of insulin resistance in predicting virological response to therapy of chronic hepatitis C is debated. We assessed the association between basal (defined as homeostasis model assessment of insulin resistance (HOMA IR)>2) and post load insulin resistance (as oral glucose insulin sensitivity index

In many tumor types serum lactate dehydrogenase (LDH) levels is an indirect marker of tumor hypoxia, neo-angiogenesis and worse prognosis. However data about hepatocellular carcinoma (HCC) are lacking in the clinical setting of patients undergoing transarterial-chemoembolization (TACE) in whom hypoxia and neo-angiogenesis may represent a molecular key to treatment failure. Aim of our analysis was to evaluate the role of LDH pre-treatment levels in determining clinical outcome for patients with HCC receiving TACE. One hundred and fourteen patients were available for our analysis. For all patients LDH values were collected within one month before the procedure. We divided our patients into two groups, according to LDH serum concentration registered before TACE (first: LDH≤450 U/l 84 patients; second: LDH>450 U/l 30 patients). Patients were classified according to the variation in LDH serum levels pre- and post-treatment (increased: 62 patients vs. decreased 52 patients). No statistically significant differences were found between the groups for all clinical characteristics analyzed (gender, median age, performance status ECOG, staging systems). In patients with LDH values below 450 U/l median time to progression (TTP) was 16.3 months, whereas it was of 10.1 months in patients above the cut-off (p = 0.0085). Accordingly median overall survival (OS) was 22.4 months and 11.7 months (p = 0.0049). In patients with decreased LDH values after treatment median TTP was 12.4 months, and median OS was 22.1 months, whereas TTP was 9.1 months and OS was 9.5 in patients with increased LDH levels (TTP: p = 0.0087; OS: p

Alcoholic and, to a lesser extent, nonalcoholic patients with liver disease have serum antibodies to acetaldehyde-protein adducts produced in vitro. These antibodies presumably reflect the presence of adducts in the liver, but the protein that triggers this immune response has not been identified. To study this, we measured the reactivity of cytosolic proteins to rabbit IgG developed against a P-450 2E1—acetaldehyde adduct, isolated from alcohol-fed rats, that recognizes acetaldehyde-modified epitopes in proteins. Adducts were determined on Western blots by scanning densitometry of antibody-linked alkaline phosphatase activity in 4 normal livers and in needle biopsy specimens from subjects with liver disease, 17 alcoholic and 14 nonalcoholic. In all livers, except for a normal one, we found a reactive protein of at least 200 kD, similar to the collagen-acetaldehyde adduct we reported to be markedly increased in rats with experimentally induced cirrhosis. The immunostaining intensity in the alcoholic patients with liver disease was eightfold (p

BACKGROUND: Survival of hepatic stellate cells (HSCs) is a hallmark of liver fibrosis, while the induction of HSC apoptosis may induce recovery. Activated HSC are resistant to many pro-apoptotic stimuli. To this issue, the role of Endoplasmic Reticulum (ER) stress in promoting apoptosis of HSCs and consequently fibrosis resolution is still debated. AIM: To evaluate the potential ER stress-mediated apoptosis of HSCs and fibrosis resolution METHODS: HSCs were incubated with the ER stress agonists, tunicamycin or thapsigargin. In vivo, HSC were isolated from normal, bile duct-ligated (BDL) and bile duct-diverted (BDD) rats. RESULTS: In activated HSC, the specific inhibitor of ER stress-induced apoptosis, calpastatin, is significantly increased vs. quiescent HSCs. Calpain is conversely reduced in activated HSCs. This pattern of protein expression provides HSCs resistance to the ER stress signals of apoptosis (apoptosis-resistant phenotype). However, both tunicamycin and thapsigargin are able to induce apoptosis in HSCs in vitro, completely reversing the calpain/calpastatin pattern expression. Furthermore, in vivo, the fibrosis resolution observed in rat livers subjected to bile duct ligation (BDL) and subsequent bile duct diversion (BDD), leads to fibrosis resolution through a mechanism of HSCs apoptosis, potentially associated with ER stress: in fact, BDD rat liver shows an increased number of apoptotic HSCs associated with reduced calapstatin and increased calpain protein expression, leading to an apoptosis-sensible phenotype. CONCLUSIONS: ER stress sensitizes HSC to apoptosis both in vitro and in vivo. Thus, ER stress represents a key target to trigger cell death in activated HSC and promotes fibrosis resolution.

High-fat dietary intake and low physical activity lead to insulin resistance, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Recent studies have shown an effect of glucagon-like peptide-1 (GLP-1) on hepatic glucose metabolism, although GLP-1 receptors (GLP-1r) have not been found in human livers. The aim of this study was to investigate the presence of hepatic GLP-1r and the effect of exenatide, a GLP-1 analogue, on hepatic signalling.The expression of GLP-1r was evaluated in human liver biopsies and in the livers of high-fat diet-treated rats. The effect of exenatide (100 nM) was evaluated in hepatic cells of rats fed 3 months with the high-fat diet.GLP-1r is expressed in human hepatocytes, although reduced in patients with NASH. Similarly, in rats with NASH resulted from 3 months of the high-fat diet, we found a decreased expression of GLP-1r and peroxisome proliferator-activated receptor γ (PPARγ), and reduced peroxisome proliferator-activated receptor α (PPARα) activity. Incubation of hepatocytes with exenatide increased PPARγ expression, which also exerted an insulin-sensitizing action by reducing JNK phosphorylation. Moreover, exenatide increased protein kinase A (PKA) activity, Akt and AMPK phosphorylation and determined a PKA-dependent increase of PPARα activity.GLP-1 has a direct effect on hepatocytes, by activating genes involved in fatty acid β-oxidation and insulin sensitivity. GLP-1 analogues could be a promising treatment approach to improve hepatic insulin resistance in patients with NAFLD/NASH.

OBJECTIVE: To study the mechanism(s) linking insulin resistance (IR) to hepatic fibrosis and the role of the epithelial component in tissue repair and fibrosis in chronic hepatitis C (CHC). DESIGN: Prospective observational study. SETTING: Tertiary care academic centre. PATIENTS: 78 consecutive patients with CHC. MAIN OUTCOME MEASURES: IR, calculated by the oral glucose insulin sensitivity during oral glucose tolerance test; necroinflammatory activity and fibrosis, defined according to Ishak's score; steatosis, graded as 0 (66%). To evaluate the role of the epithelial component in tissue repair and fibrosis, the expansion of the ductular reaction (DR) was calculated by keratin-7 (CK7) morphometry. Nuclear expression of Snail, downregulation of E-cadherin and expression of fibroblast specific protein-1 (FSP1) and vimentin by CK7-positive cells were used as markers of epithelial-mesenchymal transition in DR elements. RESULTS: IR, the degree of necroinflammation and expansion of the DR (stratified as reactive ductular cells (RDCs), hepatic progenitor cells and intermediate hepatobiliary cells according to morphological criteria) were all associated with the stage of fibrosis. Nuclear Snail expression, E-cadherin downregulation and vimentin upregulation were observed in RDCs. By dual immunofluorescence for CK7 and FSP1, the number of RDCs undergoing epithelial-mesenchymal transition progressively increased together with the necroinflammatory score. By multivariate analysis, total inflammation and insulin resistance were the only factors significantly predicting the presence of advanced fibrosis (Ishak score ≥3) and the expansion of RDCs. CONCLUSION: This study indicates that IR is associated with the degree of necroinflammatory injury in CHC and contributes to hepatic fibrosis by stimulating the expansion of RDCs that express epithelial-mesenchymal transition markers.

Summary Increased acetaldehyde levels have been found in non-alcoholic liver diseases and an acetaldehyde-collagen adduct has been reported in rats with CCl 4 -induced cirrhosis. In cytosol and microsomes of rats with cirrhosis produced by N -nitrosodimethylamine, a similar acetaldehyde-protein adduct of approximately 200 kD was recognized by rabbit IgG raised against either an in vitro produced hemocyanin-acetaldehyde adduct or an in vivo occurring P4502E1-acetaldehyde adduct isolated from alcohol-fed rats, as well as by anti-rat collagen (I) IgG. Its immune complexes contained 3 proteins that reacted with the anti-collagen IgG and were digested by collagenase: 2 proteins with molecular weights similar to procollagens α1 and α2, and a β1,2(I)-like protein which was readily produced by in vitro modification of cytosol with acetaldehyde.

More data about TACE and pTACE seem necessary to better define the global treatment strategy for HCC. Aim of our analysis was to evaluate the role of TACE, either with lipiodol (traditional) or drug-eluting microspheres in terms of response rate (RR), time to progression (TTP), overall survival (OS) and toxicity in HCC. Patients with HCC undergoing traditional TACE or pTACE (either alone or in combination with other treatment options) were eligible One hundred and fifty patients were analyzed. In the global patient population median OS was 46 months for lipiodol TACE and 19 months for pTACE (p < 0.0001), TTP was 30 months versus 16 months for patients receiving TACE or pTACE respectively (p = 0.003). These results were confirmed also among the group of patients who received exclusive TACE or pTACE. Neither RR nor toxicity was different between TACE or pTACE. At multivariate analysis, age, the Okuda stage, type of TACE and number of TACE proved to be independent prognostic factors influencing overall survival. In our experience, lipiodol TACE showed a better OS and TTP over pTACE, without difference in toxicity profile and RR. Among the staging systems analyzed only the Okuda stage seemed able to reliably predict patients outcome.

Liver disease has become the second most common cause of death of HIV-infected patients,1 and the risk of liver-related death increases with progressive HIV-associated immunodeficiency.2 This observation allows for the possibility that the pathogenic mechanisms which promote T cell depletion may also promote liver injury. In HIV-infected patients, liver disease occurs most commonly from hepatitis C virus (HCV) co-infection, active hepatitis B virus (HBV) infection, non-alcoholic steatohepatitis (NASH)3 as well as alcohol-associated steatohepatitis.4 However, due to common modes of transmission, HCV infection is the most frequent cause of liver disease found in HIV-infected persons. It has been shown that the overall prevalence of HCV infection among HIV-infected individuals is ∼30–50%, with rates of co-infection as high as 90% in intravenous drug users and almost 100% in haemophiliacs.5 To date it is known that the HIV genome encodes structural, regulatory and accessory proteins: the 160 kDa Env protein (gp160) is cleaved by cellular proteases to generate gp41 and gp120.6 In turn, gp120 envelope protein, expressed in tissue, on the surface of virions or even as a free protein, may act on specific target cells. However, it is still unclear whether and how gp120 may interfere with fibrosis acting on hepatic stellate cells (HSCs), one of the subpopulation involved in …

The metabolic syndrome encompasses metabolic and cardiovascular risk factors which predict diabetes and cardiovascular disease (CVD) better than any of its individual components. Nonalcoholic fatty liver disease (NAFLD) comprises a disease spectrum which includes variable degrees of simple steatosis (nonalcoholic fatty liver, NAFL), nonalcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is the hepatic manifestation of the metabolic syndrome, with insulin resistance as the main pathogenetic mechanism. Recent data indicate that hyperinsulinemia is probably the consequence rather than cause of NAFLD and NAFLD can be considered an independent predictor of cardiovascular disease. Serum free fatty acids derived from lipolysis of visceral adipose tissue are the main source of hepatic triglycerides in NAFLD, although hepatic de novo lipogenesis and dietary fat supply contribute to the pathogenesis of NAFLD. Approximately 10-25% NAFLD patients develop NASH, the evolutive form of hepatic steatosis. Presumably in a genetically predisposed environment, this increased lipid overload overwhelms the oxidative capacity and reactive oxygen species are generated, leading to lipid peroxidation, cytokine induction, chemoattraction of inflammatory cells, hepatic stellate cell activation and finally fibrogenesis with extracellular matrix deposition. No currently available therapies for NAFLD and NASH exist. Recently nuclear receptors have emerged as key regulators of lipid and carbohydrate metabolism for which specific pharmacological ligands are available, making them attractive therapeutic targets for NAFLD and NASH.

Nonadherence to immunosuppressive regimens among solid organ transplantation to range has been estimated from 15% to 55%. This problem has been identified as a leading cause of preventable graft loss. Tacrolimus once daily Advagraf has been developed to provide a more convenient dosing regimen to improve adherence. The aim of this study was to analyze the safety of a 1:1 dose conversion from twice-daily tacrolimus (Prograf) to Advagraf in 36 stable liver transplant recipients. The tacrolimus whole blood trough level at T0 was 6.7 +/- 2.9 ng/mL with a daily dose of 3.7 +/- 1.8 mg. The mean tacrolimus blood trough levels at T1 (7 days) and T2 (14 days) were 5.8 +/- 2.5 and 5.8 +/- 1.8 ng/mL with mean daily doses of 3.9 +/- 1.9 and 4.1 +/- 1.8 mg, respectively. There was no significant difference between T0, T1, and T2, either for tacrolimus blood trough levels or for tacrolimus daily dosages. Liver and renal function tests remained stable; no episodes of acute rejection were encountered after the conversion. A switching policy using a dose ratio of 1:1 from twice-daily tacrolimus to once-daily prolonged-release tacrolimus was safely applied to stable liver transplant recipients.

The human liver is a complex tissue consisting of epithelial, endothelial, hematopoietic, and mesenchymal elements that probably derive from multiple lineage-committed progenitors, but no comprehensive study aimed at identifying and characterizing intrahepatic precursors has yet been published. Cell suspensions for this study were obtained by enzymatic digestion of liver specimens taken from 20 patients with chronic liver disease and 13 multiorgan donors. Stem and progenitor cells were first isolated, amplified, and characterized ex vivo according to previously validated methods, and then optimized flow cytometry was used to assess their relative frequencies and characterize their immunophenotypes in the clinical specimens. Stem and progenitor cells committed to hematopoietic, endothelial, epithelial, and mesenchymal lineages were clearly identifiable in livers from both healthy and diseased subjects. Within the mononuclear liver cell compartment, epithelial progenitors [epithelial cell adhesion molecule (EpCAM)(+)/CD49f(+)/CD29(+)/CD45(-)] accounted for 2.7-3.5% whereas hematopoietic (CD34(+)/CD45(+)), endothelial [vascular endothelial growth factor-2 (KDR)(+)/CD146(+)/CD45(-)], and mesenchymal [CD73(+)/CD105(+)/CD90 (Thy-1)(+)/CD45 (-)] stem cells and progenitors accounted for smaller fractions (0.02-0.6%). The patients' livers had higher percentages of hematopoietic and endothelial precursors than those of the donors. In conclusion, we identified and characterized precursors committed to four different lineages in adult human liver. We also optimized a flow cytometry approach that will be useful in exploring the contribution of these cells to the pathogenesis of liver disease.

Hepatic fibrosis represents the wound-healing response process of the liver to chronic injury, independently from aetiology. Advanced liver fibrosis results in cirrhosis that can lead to liver failure, portal hypertension and hepatocellular carcinoma. Currently, no effective therapies are available for hepatic fibrosis. After the definition of hepatic stellate cells (HSCs) as the main liver extracellular matrix-producing cells in the 1980s, the subsequent decade was dedicated to determine the role of specific cytokines and growth factors. Fibrotic progression of chronic liver diseases can be nowadays considered as a dynamic and highly integrated process of cellular response to chronic liver injury. The present review is dedicated to the novel mechanisms of cellular response to chronic liver injury leading to hepatic myofibroblasts' activation. The understanding of the cellular and molecular pathways regulating their function is crucial to counteract therapeutically the organ dysfunction caused by myofibroblasts' activation.

Cholangiocarcinoma is a strongly aggressive malignancy with a very poor prognosis. Effective therapeutic strategies are lacking because molecular mechanisms regulating cholangiocarcinoma cell growth are unknown. Furthermore, experimental in vivo animal models useful to study the pathophysiologic mechanisms of malignant cholangiocytes are lacking. Leptin, the hormone regulating caloric homeostasis, which is increased in obese patients, stimulates the growth of several cancers, such as hepatocellular carcinoma. The aim of this study was to define if leptin stimulates cholangiocarcinoma growth. We determined the expression of leptin receptors in normal and malignant human cholangiocytes. Effects on intrahepatic cholangiocarcinoma (HuH-28) cell proliferation, migration, and apoptosis of the in vitro exposure to leptin, together with the intracellular pathways, were then studied. Moreover, cholangiocarcinoma was experimentally induced in obese fa/fa Zucker rats, a genetically established animal species with faulty leptin receptors, and in their littermates by chronic feeding with thioacetamide, a potent carcinogen. After 24 weeks, the effect of leptin on cholangiocarcinoma development and growth was assessed. Normal and malignant human cholangiocytes express leptin receptors. Leptin increased the proliferation and the metastatic potential of cholangiocarcinoma cells in vitro through a signal transducers and activators of transcription 3–dependent activation of extracellular signal-regulated kinase 1/2. Leptin increased the growth and migration, and was antiapoptotic for cholangiocarcinoma cells. Moreover, the loss of leptin function reduced the development and the growth of cholangiocarcinoma. The experimental carcinogenesis model induced by thioacetamide administration is a valid and reproducible method to study cholangiocarcinoma pathobiology. Modulation of the leptin-mediated signal could be considered a valid tool for the prevention and treatment of cholangiocarcinoma. [Cancer Res 2008;68(16):6752–61]

Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryogenesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis.

Non-alcoholic steatohepatitis (NASH), a cause of cirrhosis and hepatocellular carcinoma, is characterized by fatty infiltration of the liver, inflammation, hepatocellular damage and fibrosis. Progress has been made in understanding the molecular and cellular mechanisms implicated in the pathogenesis of this condition, therefore, we here review recent developments regarding the basic mechanisms of NASH development. Accumulation of triglycerides in the hepatocytes is the result of increased inflow of free fatty acids and de novo lipogenesis. Steatosis leads to lipotoxicity, which causes apoptosis, necrosis, generation of oxidative stress and inflammation. The resulting chronic injury activates a fibrogenic response that leads eventually to end-stage liver disease. A better understanding of these mechanisms is crucial for the design of novel diagnostic and therapeutic strategies.

BACKGROUND: Insulin resistance is a significant risk factor for hepatic fibrosis in patients with both non-alcoholic fatty liver disease (NAFLD) and chronic hepatitis C (CHC), either directly or by favouring hepatic steatosis. Several methods are available to assess insulin resistance, but their impact on this issue has never been evaluated. AIMS: To determine the relative contribution of steatosis, metabolic abnormalities and insulin resistance, measured by different basal and post-load parameters, to hepatic fibrosis in CHC and in NAFLD patients. METHODS: In 90 patients with CHC and 90 pair-matched patients with NAFLD, the degree of basal insulin resistance (by the homeostasis model assessment, (HOMA)) and post-load insulin sensitivity (by the oral glucose insulin sensitivity (OGIS) index) was assessed, together with the features of the metabolic syndrome according to Adult Treatment Panel III definition. Data were correlated with hepatic histopathology. RESULTS: The prevalence of basal insulin resistance (HOMA values >75th percentile of normal) was 23.3% in CHC patients and 57.8% in NAFLD, but it increased to 28.8 and 67.8% when measured by post-load insulin resistance (OGIS

Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease worldwide in association with oxidative stress and insulin resistance. Steatosis can evolve to steatohepatitis (NASH) by lipotoxicity, inflammation, fibrosis and cell death leading to cirrhosis and hepatocellular carcinoma. No valid therapy has been developed so far to control inflammatory and fibrogenic pathways of NAFLD/NASH. α-Tocopherol (α-TOH) is an antioxidant that partially reverts the histopatological spectrum of NASH. However, the biological mechanism that may support a preventive or therapeutic role of vitamin E in NASH and chronic liver diseases remain elusive. In this study we explored in vitro and in vivo the metabolism of vitamin E in liver cells and in the whole liver exposed to some pathogenic stimuli associated with the progression of NAFLD/NASH. Protein levels of CYP4F2, the cytochrome P450 isoenzyme involved in the metabolism of vitamin E increased in HepG2 cells incubated with α-TOH. This effect increased with a synergic action by the treatment with the fatty acids oleic acid or palmitic acid, or with fructose, which are well-known NASH stimuli. In mice fed control or NAFLD/NASH diets, e.g. high-fat (HFD) or HFD plus fructose diet, the abnormal lipid metabolism of liver tissue was associated with a lowered expression of CYP4F2 protein. The same finding was observed in animals treated with CCl4, a free radical-generating hepatotoxic xenobiotic leading to hepatic fibrosis, in the presence of lowered levels of liver α-TOH. In conclusion, either acute or chronic challenges with NAFLD/NASH stimuli influence the liver expression of CYP4F2. The observed acute transcriptional upregulation and the in vivo downregulation of this protein, point to a role for CYP4F2 in the inflammatory and lipotoxicity signaling of NASH. Transcriptional effects of vitamin E on this gene may help to explain earlier and positive clinical findings, which are worth of further investigation.

Background and Aims: The inflammasomes are cytoplasmic multiprotein complexes that are responsible for the activation of inflammatory reactions. They mediate the cleavage and activation of caspase-1 and IL-1b that in turn leads to a complex network of cellular reactions that at the end initiate local and systemic inflammation [1]. We have shown that the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) is virtually absent in untreated cultured hepatocytes while stimulation with lipopolysaccharides (LPS) results in strong activation of NLRP3 expression in these cells [2]. In the present study we investigated the impact of NF-uB signalling on NLRP3 activation in primary hepatocytes. Methods: Murine primary hepatocytes were isolated according to the collagenase method [3]. The NF-uB activation inhibitor QNZ and the adenoviral expression vector Ad5-CMV-IuB(S32A/S36A) expressing a hemagglutinin-tagged human superrepressor of NF-uB [4] were used to block NF-uB signalling. After LPS challenge, the expression of TNF-a, IL-1b and NLRP3 was analysed by qRTPCR, Western blot and immunohistochemistry. The influence of NF-uB signalling on NLRP3 expression was further investigated in a novel murine hepatocytic cell system that can be Cre-dependently depleted for the NF-uB essential modulator (NEMO). Results: We found that QNZ blocks LPS-induced expression of TNF-a, IL-1b and NLRP3 as demonstrated by qRT-PCR, Western blot and immunohistochemistry. Even more, the basal expression that was observed in uninfected control cells or in cells that were infected with a control virus (Ad5-CMV-Luc) was abrogated. Likewise, the superrepressor of NF-uB prevents expression of NLRP3. In cells that were depleted for NEMO (and Caspase 8), we could confirm that there is a close link of NF-uB activity and NLRP3 expression. As a consequence, the expression of Lipocalin 2 representing a biomarker of hepatic inflammation was blunted suggesting that NF-uB activity plays an important role in mediating inflammasome activity. Conclusions: We conclude that NF-uB signalling is a necessary prerequisite for proper activation of the NLRP3 inflammasome in primary hepatocytes. Our data further suggests that targeting the NF-uB pathway will be potentially therapeutic useful to block hepatic inflammasome activity.

s / Digestive and Liver Disease 47S (2015) e1–e18 e7 (months; 95% CI): CPT 0 62 (52.9–71.1), A 44 (41.6–46.4), B 22 (19.7–24.3), C 9 (6.6–11.3), p