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Background.Reproducible structural analysis was made on cirrhotic human liver samples in order to reveal potential connections between morphological and laboratory parameters.Material and methods.Large histological samples were taken from segment VII of 56 cirrhotic livers removed in connection with liver transplantation. Picro Sirius red and immunohistochemically (smooth muscle actin [SMA], cytokeratin 7 [CK7], Ki-67) stained sections were digitalized and morphometric evaluation was performed.Results.The Picro Sirius-stained fibrotic area correlated with the average thickness of the three broadest septa, extent of SMA positivity, alkaline phosphatase (ALP) values and it was lower in the viral hepatitis related cirrhoses than in samples with non-viral etiology. The extent of SMA staining increased with the CK7-positive ductular reaction. The proliferative activity of the hepatocytes correlated positively with the Ki-67 labeling of the ductular cells and inversely with the septum thickness. These data support the potential functional connection among different structural components, for example, myofibroblasts, ductular reaction and fibrogenesis but challenges the widely proposed role of ductular cells in regeneration.Conclusion.Unbiased morphological characterization of cirrhotic livers can provide valuable, clinically relevant information. Similar evaluation of routine core biopsies may increase the significance of this ‘Gold Standard’ examination. [ABSTRACT FROM AUTHOR]

Background. Reduced glutathione (γ-glutamylcysteinylglycine), GSH, is essential when protecting cells from oxidative stress and also an indicator of disease risk. Reported concentrations of GSH and its oxidized form, glutathione disulfide (GSSG), varies considerably, primarily due to the instability of GSH and various analytical methods. Methods. We designed a sensitive method to analyze GSH and GSSG in porcine hepatocytes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after stabilization with N-ethylmaleimide (NEM). This method includes stable isotope labeled internal standards and simple synthesis of labeled GSSG which commercial sources rarely offer. GSH and GSSG were analyzed in porcine liver biopsies giving a reference interval based on a large number of samples (26 pigs; 3 parallels). Results. The LC-MS/MS results revealed excellent linearity for both GSH and GSSG, with interday coefficient of variation (%CV) for GSH-NEM and GSSG <10 %. Accuracy for recovery tests was between 95.6% and 106.7% ( n = 3) for GSH and between 92.3% and 107.7% ( n = 3) for GSSG. The limits of quantification were 0.1 μM for GSH-NEM and 0.08 μM for GSSG. The mean concentration of GSH was 3.5 (95% CI = 1.5-8.1) mmol/liter and of GSSG 0.0023 (95% CI = 0.0003-0.019) mmol/liter. Conclusion. For the first time GSH and GSSG are analyzed in porcine hepatocytes by LC-MS/MS yielding a reference level of GSH and GSSG. The method is reproducible in any laboratory with LC-MS/MS service and will probably be applicable in all soft tissues and cell suspensions, essentially with no modification. [ABSTRACT FROM AUTHOR]

Objective. The remarkable ability of liver to regenerate after insults has been harnessed by surgeons when designing techniques for liver resection or transplantation. However, the underlying mechanisms of liver regeneration are not fully clarified. On the other hand, aquaporins (AQPs) are small transmembrane proteins with unexpected physiological roles in addition to water transport. For example, they play pivotal roles in cell migration, angiogenesis, and cell proliferation, events that are also occurred during liver regeneration. We thus examined the possible involvement of AQPs in this regenerative process. Material and methods. A two-thirds partial hepatectomy (PH) rat model was employed. The temporal expression of various AQPs in the liver following PH was determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The localization of AQPs was evaluated by immunohistochemistry. Results. As anticipated, AQP0, 8, 9, and 11 were detected mainly in hepatocytes; unexpectedly, Kupffer cells were observed to express AQP8 during a specific period of time in the regenerative process. AQP9 protein was shown to be expressed in a progressively enhanced pattern at early time points after PH. A transient expression of AQP11 in the nucleus of hepatocytes was observed. Conclusion. These findings suggest the possibility that AQP might be involved in the PH-induced liver regeneration. [ABSTRACT FROM AUTHOR]

Background. Both non-alcoholic steatohepatitis (NASH) and polycystic ovary syndrome (PCOS) are associated with metabolic syndrome (MS) and insulin resistance (IR). Except for a few case reports, there are no systematic assessments of NASH in PCOS patients. Aim. To determine the prevalence of NASH and independent factors associated with NASH in a cohort of patients with documented PCOS. Methods. Patients with established diagnosis of PCOS and matched controls (matched for gender, age, and body mass index (BMI)) were included in the study. Causes of other liver diseases were systematically excluded by clinical and laboratory tests. Excessive alcohol use was defined as alcohol consumption of greater than 10 g/day. All liver biopsies were read by a single pathologist blinded to the clinical data. Histologic NASH was defined as steatosis with lobular inflammation and ballooning degeneration of hepatocytes with or without Mallory--Denk bodies or pericellular fibrosis. Univariate and multivariate analyses with logistic regression were performed to compare PCOS to matched controls. Results. Sixty-six patients were included in the study (34 PCOS and 32 matched controls). Of PCOS patients, 73% had a liver biopsy while 78% of the matched controls had a liver biopsy. In comparing PCOS patients to the matched controls, clinical (BMI, waist circumference, type 2 diabetes, MS, or its components, any alcohol consumption in the prior year, ethnic background, age, gender, etc.) and laboratory data (aminotransferases, ferritin, glucose, etc.) were not significantly different ( p > 0.05). However, PCOS patients tended to have more histologic NASH on their liver biopsies (44.0% vs. 20.8%, p == 0.08). Independent predictors of histologic NASH in PCOS patients were elevated aspartate aminotransferase (AST), high triglycerides and small amounts of alcohol consumption ( p == 0.019, 10-fold cross-validated AUC == 0.80, 95% CI == 0.56--0.94). Although about half of PCOS patients did not report any alcohol consumption, 50% did report rare alcohol use. In fact, PCOS patients with histologic NASH tended to report higher alcohol consumption per week than PCOS without NASH (3.80 ±± 6.16 vs. 1.11 ±± 1.87 g/week, p == 0.1). Nevertheless, these amounts of alcohol consumption were quite minimal. Conclusions. Despite similar clinical and laboratory profiles to the matched controls, PCOS patients seem to have more histologic NASH. Although alcohol consumption was rare for both PCOS and controls, even rare alcohol consumption in PCOS patients was independently associated with histologic NASH. [ABSTRACT FROM AUTHOR]

Objective. The novel adipokines omentin, chemerin, and adipsin are associated with insulin resistance and the components of the metabolic syndrome. We assayed circulating levels of these molecules and examined their association with clinical, biochemical, and histological phenotypes in patients with nonalcoholic fatty liver disease (NAFLD). Material and methods. Serum levels of omentin, chemerin, and adipsin were assayed by enzyme-linked immunosorbent assay in 99 patients with biopsy-proven NAFLD and 75 control subjects. We analyzed associations between adipokines and the characteristics of patients with NAFLD using multivariable linear regression models. Results. Adipsin levels did not differ between patients and controls, whereas both omentin and chemerin levels were significantly higher in patients with biopsy-proven NAFLD than in controls (both p values <0.001). Serum omentin levels were significantly associated with C-reactive protein (r= 0.29, p < 0.01) and the degree of hepatocyte ballooning (r= 0.27, p < 0.01), whereas chemerin showed a modest association with liver fibrosis (r = 0.22, p = 0.04). After stepwise linear regression analysis adjusting for potential confounders, serum omentin levels retained their independent significance as a predictor of hepatocyte ballooning in patients with NAFLD (β = 1.42; t- 2.79, p < 0.01). Conclusions. Our results suggest that serum omentin levels are raised in patients with NAFLD regardless of potential confounders and represent an independent predictor of hepatocyte ballooning. [ABSTRACT FROM AUTHOR]

Objective. Hepatic stellate cells only produce fibronectin isoforms in disease states. The isoform-defining domains can be detected in the blood circulation. This study examines whether circulating levels of fibronectin isoforms show a relationship with liver fibrosis on histology in patients with chronic hepatitis C. Material and methods. In a prospective study, 50 patients with chronic hepatitis C who underwent a liver biopsy were compared to 50 matched controls and 35 patients with other liver conditions. Results. Circulating levels of the fibronectin isoforms were significantly higher in patients with chronic hepatitis C compared to healthy controls [oncofetal fibronectin (oFN) 2.45 ± 0.17 versus 1.76 ± 0.16 mg/l, P < 0.005; extra domain-A (EDA) 1.05 ± 0.06 versus 0.86 ± 0.06 mg/l, P < 0.05; and extra domain-B (EDB) 14.55 ± 0.74 versus 9.31 ± 0.58 mg/l, P < 0.001], even though total fibronectin was lower (198.9 ± 3.5 versus 343.6 ± 14.5 mg/l, P < 0.001). A correlation with the fibrosis score was found for both oFN ( r = 0.46, P < 0.005) and EDA ( r = 0.51, P < 0.001). The combination of an elevation in both markers (oFN and EDA) in the upper quartile was associated with a specificity of > 99% for predicting significant fibrosis (stages 2–4) and 95% for predicting advanced fibrosis (stages 3–4). A combination of decreased values in the lowest tertile for both markers had a specificity of 94% for excluding significant fibrosis. Based on these findings, 30% of the patients scheduled for a liver biopsy could be correctly classified as having or not having significant fibrosis. The remainder would have to proceed with a biopsy. Conclusion. Circulating fibronectin isoforms produced by activated stellate cells represent a viable marker for the presence of significant fibrosis or a lack thereof. [ABSTRACT FROM AUTHOR]

Objective. To assess the production of the endogenous IL-1 modulators IL-1 receptor antagonist (IL-1Ra), type I and II soluble IL-1 receptors (IL-1sRI and II) in patients with chronic liver disease (CLD). Material and methods. Plasma levels of IL-1beta (IL-1β) and IL-1 modulators were assessed in 126 CLD patients and 39 healthy controls. IL-1sRII was also measured in the supernatants of primary hepatocyte cultures. Results. Plasma IL-1sRI and IL-1Ra levels were significantly higher in cirrhotic CLD patients than in non-cirrhotic CLD patients and in controls. Levels did not depend on the etiology of CLD. Likewise, plasma IL-1β levels were elevated in CLD patients compared with those in controls. In contrast, IL-1sRII levels did not differ between CLD patients and controls. Cultures of human primary hepatocytes showed that IL-1sRII is induced by IL-1β, but not IL-6. Conclusions. In cirrhotic CLD patients elevated plasma IL-1β is not counteracted by endogenous levels of IL-1sRII, whereas high IL-1sRI is expected to neutralize the naturally occurring antagonist IL-1Ra, resulting in a dysregulation of the IL-1 system that might enhance pro-inflammatory activity of IL-1. [ABSTRACT FROM AUTHOR]

Objective. In infection with hepatitis C virus (HCV), spontaneous clearance of the virus occurs in 30-40% of cases. By contrast, in chronic infection, this is rare. The basis for viral clearance in acute disease is unknown. Whereas cellular immune responses have been studied in detail, few data exist on the role of viral structural proteins, such as the core protein. The purpose of this study was to investigate the effects of core produced de novo within adult mouse hepatocytes by using a new transgenic mouse line in which expression of HCV core is regulated by tetracycline (tet-off). Material and methods. In this work, transgenic mice with conditional HCV core were created, to study the acute expression of HCV core protein in the context of the mature liver. The subcellular distribution of the core, hepatocellular oxidative stress and apoptosis were monitored. Results. Core protein is readily detectable and strongly associated with cytoplasmic lipid vesicles, endoplasmic reticulum and mitochondria. Mitochondrial oxidative stress was evidenced by a reduction in thioredoxin-2 (trx2). Concurrently, caspase-3 activity and TUNEL increased and, over time, the level of core protein in the liver declined. Conclusions. Mice that are conditionally transgenic for HCV core protein, which is readily detected and morphologically associated with steatosis in individual hepatocytes, were developed. Acute expression of core protein causes mitochondrial stress, as demonstrated by a reduction in trx2 and in the apoptosis of core-positive hepatocytes. We speculate that these events could be involved in the clearance of virus during acute hepatitis C, by both reducing the burden of virus in the liver and effectively priming the immune response. [ABSTRACT FROM AUTHOR]

Objective. Nicotinamide has been shown to inhibit proliferation and induce apoptosis in a variety of cells. Moreover, nicotinamide treatment attenuates collagen accumulation and fibrogenesis in the bleomycin model of lung fibrosis. We hypothesized that nicotinamide may be useful as an antifibrotic agent in liver fibrosis and we investigated the in vitro effect of nicotinamide on hepatic stellate cells proliferation, apoptosis and collagen I expression. Material and methods. Transforming growth factor β1 (TGF-β1) was used for activation of the rat HSC-T6 cell line. Apoptosis was determined by fluorescence activated cell sorter (FACS) analysis after propidium iodide staining and by immunohistochemistry showing presence of the active form of caspase 3. Expression of activation marker α-smooth muscle actin (α-SMA), apoptotic and cell cycle markers cyclin D1, P53 and caspase 3 was determined by Western blotting. Collagen I expression was assessed by Northern blotting. Results. Nicotinamide inhibits hepatic stellate cell proliferation and induces apoptosis with caspase-3 activation. There is no effect of nicotinamide on the levels of cell cycle stimulator cyclin D1. Expression of p53 is induced in the presence of nicotinamide. Nicotinamide reduces activation marker α-SMA and decreases both basal and TGFβε-induced collagen I expression. Moreover, in TGFβ-activated cells, nicotinamide reduces expression of pro-inflammatory and pro-fibrotic cytokines TGFβ2, IL-1β, TNFα and macrophage chemotactic protein-1. Conclusions. The in vitro effect of nicotinamide on activation and proliferation of hepatic stellate cells suggests that nicotinamide may have a potential beneficial role in attenuation of liver fibrogenesis. [ABSTRACT FROM AUTHOR]

Examines the effect of rebamipide on the expression of hepatocyte growth factor (HGF), c-met, cyclooxygenase-2 (Cox- 2) and subtype of the prostaglandin E2 receptor in acetic acid-induced gastric ulcer, a model of human ulcer. Major mechanisms of rebamipide; Clinical use of rebamipide as a potent anti-ulcer agent; Indications.