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We present the case of a 51 years-old woman who was referred to our Liver Unit for suspected dysmetabolic liver disease. She had a previous diagnosis of Systemic Sclerosis and Sjögren’s Syndrome and had altered hepatic enzymes with a positive anti-Nuclear Antibodies centromeric pattern, anti-Cardiolipin Antibodies and anti b2 Glycoprotein I Antibodies. Despite complete liver assessment and disease staging was negative, the clinical course was complicated by the development of anemia, due to esophageal varices bleeding associated with worsening splenomegaly. Liver biopsy was key for reaching the diagnosis as it showed portal tracts with fibrous expansion, ductular proliferation and focal lympho-granulocytic infiltrate, reduced caliber of portal vessels, hypoplastic portal tracts, focal herniation, aberrant microvasculature and positive endothelial CD34 staining. Having ruled out any other cause of portal hypertension such as cirrhosis, blood diseases, occlusion of the hepatic and portal veins, etc., we finally concluded that the portal hypertension was due to hepatoportal sclerosis associated with Systemic Sclerosis and Sjögren’s Syndrome. Keywords: Idiopathic Non-Cirrhotic Portal Hypertension (INCPH; Hepatoportal Sclerosis (HPS); Systemic sclerosis (SSc); Sjögren’s Syndrome (SS).

BACKGROUND Direct-acting antiviral agents (DAAs) are extremely effective in eradicating hepatitis C virus (HCV) in chronically infected patients. However, the protective role of the sustained virologic response (SVR) achieved by second- and third-generation DAAs against the onset of hepatocellular carcinoma (HCC) and mortality is less well established. AIM To examine the occurrence of HCC or death from any cause in a retrospective-prospective study of patients treated with DAAs. METHODS Patients were enrolled from a tertiary academic hospital center for liver disease management that collects subject data mainly from northeastern Italy. The study was conducted in 380 patients (age: 60 ± 13 years, 224 males, 32% with cirrhosis) treated with DAAs with or without SVR (95/5%), with a median follow up of 58 wk (interquartile range: 38-117). The baseline anthropometric features, HCV viral load, severity of liver disease, presence of extra-hepatic complications, coinfection with HIV and/or HBV, alcohol consumption, previous interferon use, alpha-fetoprotein levels, and renal function were considered to be confounders. RESULTS The incidence rate of HCC in patients with and without SVR was 1.3 and 59 per 100 person-years, respectively (incidence rate ratio: 44, 95%CI: 15-136, P < 0.001). Considering the combined endpoint of HCC or death from any cause, the hazard ratio (HR) for the SVR patients was 0.070 (95%CI: 0.025-0.194, P < 0.001). Other independent predictors of HCC or death were low HCV viremia (HR: 0.808, P = 0.030), low platelet count (HR: 0.910, P = 0.041), and presence of mixed cryoglobulinemia (HR: 3.460, P = 0.044). Considering SVR in a multi-state model, the independent predictors of SVR achievement were absence of cirrhosis (HR: 0.521, P < 0.001) and high platelet count (HR: 1.019, P = 0.026). Mixed cryoglobulinemia predicted the combined endpoint in patients with and without SVR (HR: 5.982, P = 0.028 and HR: 5.633, P = 0.047, respectively). CONCLUSION DAA treatment is effective in inducing SVR and protecting against HCC or death. A residual risk of HCC persists in patients with advanced liver disease or with complications, such as mixed cryoglobulinemia or renal failure.

Background: NAFLD (non-alcoholic fatty liver disease) and NASH (non-alcoholic steatohepatitis) are emerging conditions that sequentially affect hepatocyte metabolism. Fibroblast Growth Factor 21 (FGF21) is produced by the liver and has autocrine and paracrine actions on liver energy metabolism while serpinB3 (SCCA) is produced by damaged liver cells and is considered a marker of progression to cirrhosis and hepatocellular carcinoma. Aim of the study: A first aim of this study is to investigate expression profile of FGF21 and its receptor/coreceptor in liver biopsies of patients with NAFLD and NASH. A second aim is to assess whether circulating concentration of SCCA-IgM is increased in patients with NASH compared to patients with pure fatty liver. Materials and methods: Expression of FGF21 and its receptor was measured by real-time PCR in liver biopsies of 42 patients with NAFLD/NASH and 16 matched controls. Moreover in a separate group of patients with NAFLD (n = 39) and NASH (n = 45) serum concentration of SCCA-IgM was measured. Results: A significant lower expression of FGF21 (53±3.1 u.a. vs 27.2±1 u.a., p < 0.05), FGF21-R1 (4.7±0.2 u.a. vs 2.7±0.1 u.a., p < 0.02) and its coreceptor-klotho (107.6±6.4 u.a. vs 64.0±1 u.a., p < 0,03) was found in patients with NAFLD/NASH. SCCA-IgM serum concentration was significantly increased in patients with NASH compared with those with NAFLD (9.2±1.8 vs 31.0±7.2 u.a./mL, p = 0.007). Conclusions: Downregulation of FGF21, FGF21-R1 and-klotho suggests that both low hormonal levels and its membrane receptor and coreceptor might contribute to detrimental metabolic effect in hepatocytes of NAFLD/NASH patients. Moreover higher circulating levels of SCCA-IgM in patients with NASH compared to those with pure fatty liver suggest that this protein might represent a possible marker of hepatic damage before the progression to cirrhosis and hepatocellular carcinoma.

Alcohol-induced endothelial changes might contribute to an increase in blood pressure in regular alcohol consumers. Some antihypertensive drugs affect oxidative stress and endothelial function and might counteract the effects of alcohol at the cellular level. The aim of this study was to investigate in vitro the effects of three different types of antihypertensive agents on alcohol-induced endothelial responses and oxidative stress. Cultured human endothelial cells were exposed to increasing concentrations (1, 10, 60 micromol/L) of zofenoprilat, carvedilol, and lacidipine in the absence and in the presence of ethanol (140 mmol/L). Concentrations of endothelin (ET) and nitric oxide (NO) were measured in the culture media as markers of endothelial function, and malondialdehyde (MDA) and intracellular glutathione (GSHi) were measured as markers of oxidative stress. Exposure to alcohol increased the levels of ET, NO, and MDA, and decreased GSHi. Carvedilol and zofenoprilat were more effective than lacidipine in counteracting the effects of alcohol on ET production. Alcohol-induced NO production was enhanced by carvedilol, whereas zofenoprilat and lacidipine did not have a significant effect. The alcohol-induced increase in MDA concentrations was blunted by all three drugs, but only carvedilol restored a normal response. All three drugs increased GSHi levels, with the effect being greater for carvedilol and lacidipine than zofenoprilat. Carvedilol is more effective than zofenoprilat and lacidipine in counteracting alcohol-induced endothelial responses in vitro and in decreasing oxidative stress. These effects might be particularly beneficial in patients with alcohol-related hypertension.

Apoptosis plays an important role within the endocrine system, particularly in the thyroid gland, although little is known about its regulation in normal thyroids. Because thyrotropin (TSH) regulates many thyroid-specific functions and cell proliferation, we investigated whether TSH can influence such mechanisms. To induce apoptosis we used UV-C radiation. The FRTL-5 rat thyroid cell strain, a cloned strain of differentiated and untransformed cells that reproduces many of the characteristics of the normal thyroid was chosen for this study. The FRTL-5 cells are a particularly suitable model because they actively proliferate when cultured in the presence of TSH (6H medium), while in TSH-free medium (5H medium) cells remain in a physiologic quiescent state for a long period of time. FRTL-5 cells in both culture conditions were irradiated with UV-C radiation (254 nm wavelength). At 48 hours after radiation, 6H cultured cells showed the characteristic signs of apoptosis. However, 5H cultured cells did not present macroscopic signs of damage, DNA fragmentation, or detectable apoptosis. Furthermore, the expression of 23 apoptosis-related genes was compared. Results indicate that Bcl2 and caspase-2 expression is enhanced, while bax, GADD45 and mdm-2 expression is reduced in irradiated cells. These data confirm that TSH plays a major role in regulating UV-induced apoptosis in FRTL-5 cells.

Alteration of the redox potential has been proposed as a mechanism influencing gene expression. Reduced glutathione (GSH) is one of the cellular scavengers involved in the regulation of the redox potential. To test the role that GSH may play in thyroid cells, we cultured a differentiated rat thyroid cell strain (FRTL-5) in the presence of L-buthionine-(S,R)-sulfoximine (BSO). BSO affects GSH synthesis by irreversibly inhibiting gamma-glutamylcysteine synthetase (EC 6.3.2.2), a specific enzyme involved in GSH synthesis. BSO-treated FRTL-5 cells show a great decrease in the GSH level, whereas malondialdehyde increases in the cell culture medium as a sign of lipid peroxidation. In these conditions the activity of two thyroid-specific promoters, thyroglobulin (Tg) and thyroperoxidase (TPO), is strongly reduced in transient transfection experiments. As both Tg and TPO promoters depend upon the thyroid-specific transcription factors, thyroid-specific transcription factor-1 (TTF-1) and Pax-8 for full transcriptional activity, we tested whether reduction of GSH concentration impairs the activity of these transcription factors. After BSO treatment of FRTL-5 cells, both transcription factors fail to trans-activate the respective chimerical targets, C5 and B-cell specific activating protein promoters, containing, respectively, multimerized TTF-1- or Pax-8-binding sites only as well as the Tg and TPO natural promoters. Northern analysis revealed that endogenous Tg messenger RNA (mRNA) expression is also reduced by BSO treatment, whereas endogenous TPO expression is not modified. Furthermore, the Pax-8 mRNA steady state concentration does not change in BSO-treated cells, whereas TTF-1 mRNA slightly decreases. Immunoblotting analysis of FRTL-5 nuclear extracts does not show significant modification of the Pax-8 concentration in BSO-treated cells, whereas a decrease of 25% in TTF-1 protein is revealed. Furthermore, BSO treatment decreases the DNA-binding activity to the respective consensus sequence of both transcription factors. Finally, different mechanisms seem to act on TTF-1 and Pax-8 functional impairment in BSO-treated cells. Indeed, with a lowered GSH concentration, the overexpressed Pax-8 still activates transcription efficiently, whereas, on the contrary, the overexpressed TTF-1 does not recover its transactivation capability when the respective chimerical target sequences are used (C5 and BSAP). When the natural Tg and TPO promoter sequences are used, overexpression of Pax-8 parallels the effect on both promoters observed using the chimeric target sequences, whereas overexpression of TTF-1 increases TPO promoter transcriptional activity only.

Hepatocellular carcinoma (HCC) is often diagnosed after the tumor manifests clinical signs and symptoms. Early diagnosis is usually performed thanks to HCC screening programs for patients affected by liver cirrhosis or chronic viral hepatopathies using ultrasound and serum alfa-fetoprotien. In most HCC cases, clinical signs and symptoms of this tumor may occur several months after development, when therapy can not be curative, given the advanced tumor stage and underlying liver disease, which preclude curative options, such as ablation, resection, or liver transplantation.

Nonalcoholic fatty liver disease (NAFLD) is strongly associated to oxidative stress, metabolic syndrome, and cardiovascular risk. Hepatocytes overloaded with fatty acids (FA) could generate substances that interfere with endothelial function, providing a potential explanation for this association. We have investigated the response of cultured human hepatoblastoma cells (Hep-G2) that were exposed to FA by measuring markers of oxidative stress and thrombosis and expression of the insulin receptor.Hep-G2 cells were conditioned with a mixture of FA with or without N-acetyl-L-cysteine (NAC), glutathione (GSH), or adiponectin (ADN). After 7 days, we measured intracellular GSH (iGSH), nitric oxide (NO), malondialdehyde (MDA), and tissue plasminogen inhibitor-1 (PAI-1). Real-time polymerase chain reaction (PCR) was used to determine gene expression of inducible nitric oxide synthase (iNOS) and insulin receptor (INS-R).Exposure to FA decreased iGSH and NO levels in Hep-G2 cells and increased MDA and PAI-1 production. Gene expression of iNOS and INS-R in Hep-G2 cells was decreased by exposure to FA. Co-incubation with NAC and GSH prevented the change of iNOS mRNA levels, but not of INS-R; co-incubation with ADN restored the gene expression of INS-R, but not of i-NOS. ADN prevented also the FA-induced increase in MDA in cultured human endothelial cells.Exposure to FA activates oxidative stress and production of prothrombotic markers and decreases expression of insulin receptors in cultured human hepatocytes. These effects of FA are partially prevented by ADN and might contribute to the increased cardiovascular risk in patients with NAFLD and metabolic syndrome.

Combination therapy with interferon-α and ribavirin is considered the treatment of choice for chronic hepatitis C. However, interferon-α may induce severe depression. It has been suggested that interferon-α is able to modify cytochrome P450 (CYP) 1A2 and 2D6 activity. We therefore decided to study the effects of the interferon-α-2b pegylated derivative on fluoxetine disposition in patients receiving combination chemotherapy for chronic hepatitis C. After approval by the institutional ethics committee, 20 adult patients with chronic hepatitis C, but with no history of other liver diseases, were prospectively admitted to the study, which included phenotyping by means of a dextromethorphan test and evaluation of fluoxetine and norfluoxetine pharmacokinetic parameters (the area under the serum concentration-time curve, maximum serum concentration, time to reach the maximum serum concentration and terminal elimination half-life) before and after 2 months of continuous peginterferon-α-2b therapy. The only statistically significant difference we observed was a significant reduction in the terminal elimination half-life of fluoxetine (from 47.30 to 33.23 hours; p =0.014) after peginterferon-α-2b treatment. These data suggest that interferon-α may induce, rather than inhibit, the biotransformation of fluoxetine.

Background Epidemiological investigations have demonstrated a close association between heavy alcohol consumption and hypertension. The mechanisms of this association, however, remain elusive. We studied the effects of alcohol withdrawal on blood pressure, hormonal parameters, and circulating markers of endothelial activity. Methods In 14 hypertensive heavy alcohol consumers (> 200 g/day) who agreed to participate in a hospital withdrawal programme we monitored, for 30 days, blood pressure, plasma levels of renin, aldosterone, cortisol, endothelin, and plasminogen activator inhibitor 1 (PAI-1), and urinary levels of catecholamines. Patients in the withdrawal group were compared with eight hypertensive heavy drinkers who refused to participate in the programme and maintained regular alcohol consumption and 11 normotensive teetotalers. Results By the third day after withdrawal, blood pressure was significantly decreased and the normalization of levels was obtained in 13 of 14 patients by the end of the study. Alcohol withdrawal significantly decreased plasma aldosterone and cortisol levels, but did not affect levels of active renin and fractionated urinary catecholamines. At baseline, plasma endothelin and PAI-1 levels were significantly higher in alcoholic individuals than in teetotalers, and after the cessation of alcohol intake decreased progressively, reaching levels different from baseline within 1 week. A significant correlation was found between changes in endothelin and PAI-1, and blood pressure variations during alcohol abstinence that remained significant only for endothelin with the multivariate approach. Conclusion Hypertension is rapidly reversible in the majority of heavy drinkers after the withdrawal of alcohol consumption. In these patients, hypertension is associated with an increased release of endothelial factors that might contribute to the increase in blood pressure.

Context: The recently discovered hormone resistin is linked to the development of insulin resistance, but direct evidence of resistin levels in humans with nonalcoholic fatty liver disease (NAFLD) is lacking. Methods:Weconductedthisstudytoassesstherelationshipbetween serum resistin and NAFLD. We measured serum resistin and biochemical, hormonal, and histological correlates in 28 NAFLD patients,33controls,and30obesepatients[bodymassindex(BMI),30 kg/m 2 ] without NAFLD. Results: Resistin and adiponectin expression were measured in sc adipose tissue by quantitative RT-PCR. Resistin was higher in NAFLD patients compared with controls (5.87 0.49 vs. 4.30 0.20 ng/ml; P 0.002) and obese patients (4.37 0.27 ng/ml; P 0.002). Increased resistin mRNA was also found in the adipose tissue of NAFLD patients compared with controls and obese subjects. Conclusions: Both NAFLD and obese patients had lower adiponectin levels, whereas leptin was increased only in the obese group. No correlation was found between resistin and high-sensitivity C-reactive protein, BMI, homeostasis model assessment, insulin, glucose, transaminases, and lipid values. A positive correlation was found between resistin and histological inflammatory score. These data report increased resistin in NAFLD patients that is related to the histological severity of the disease, but do not support a link between resistin and insulin resistance or BMI in these patients. (J Clin Endocrinol Metab 91: 1081–1086, 2006)

Background: Although heavy alcohol drinkers are at an increased risk of developing cardiovascular events, moderate alcohol intake is associated with reduced incidence of cardiovascular death. This paradox might reflect a dose-related effect of different alcohol intakes on endothelial function and this, in turn, might depend on changes in oxidative stress Methods: We tested the effects of alcohol withdrawal in heavy alcohol consumers and compared the plasma levels of endothelin-1, nitric oxide, plasminogen activator inhibitor-1, von Willebrand factor, malondialdehyde, and intracellular glutathione with those of alcoholics that did not modify their alcohol intake and teetotalers. In human endothelial cells that had been cultured for 2 weeks in the presence of different concentrations of ethanol, we assessed the same parameters after withdrawal of ethanol exposure Results: Alcohol increased the levels of endothelin-1, nitric oxide, and plasminogen activator inhibitor-1 and decreased the levels of von Willebrand factor both in vivo and in vitro. These changes were dose dependent, rapidly reversed after withdrawal of exposure, and associated with the presence of increased oxidative stress as indicated by increased levels of both malondialdehyde and intracellular glutathione. Blockade of oxidative stress by incubation of endothelial cells in the presence of oxidants’ scavengers prevented the alcohol-induced functional modifications of the endothelium Conclusions: Alcohol affects endothelial function with an effect that is mediated by an activated oxidative stress and is rapidly reversed after withdrawal. Dose-related endothelial responses to different alcohol intakes might translate in either vascular protection or vascular damage.

APE/Ref-1 is a multifunctional protein possessing both redox and DNA repair functions. Through its redox activity, APE/Ref-1 controls the DNA-binding function of several transcriptional regulators (AP1, NF-kappaB, p53, Pax proteins). We have previously shown that APE/Ref-1 upregulates the transcriptional activity of the thyroid-specific transcription factor Pax8. In thyroid cells, APE/Ref-1 can be detected both in the nuclear and cytoplasmatic compartments. In this study regulatory mechanisms acting on APE/Ref-1 were revealed using the FRTL-5 cell line. TSH induces both cytoplasm-to-nucleus translocation and neosynthesis of APE/Ref-1 protein. Interestingly, only neosynthesis is dependent on cAMP signalling. In contrast, the cytoplasm-to-nucleus translocation is dependent on redox-mediated mechanisms. Based upon the data shown in this study and in others, a bimodal control of APE/Ref-1 by TSH can be delineated.

Vascular endothelial cells play an important role in coagulation regulation of vascular tone and in a variety of synthetic and metabolic functions. Endothelial cells also have a pivotal role in immunological diseases atherogenesis and tumor angiogenesis. Endothelial cells are often used as system to study the pathophysiology of late complications in diabetes mellitus atherosclerotic damages and leukocyte adhesion in inflammatory diseases. Most of the studies have been performed on primary arterial and venous endothelial cell cultures with problems such as availability of autoptic material and reproducibility of cell cultures. We have isolated and characterized a novel system of proliferating long-term cultures of human aortic endothelial cells that maintain their differentiated characteristics for many generations in vitro. They produce antithrombotic and thrombotic factors such as t-PA and PAI-1 and respond to TNFalpha, an important factor correlated with the inflammatory process by modifying growth characteristics by producing cytokines such as GM-CSF by expressing ICAM-1 on the surface and by producing large amounts of nitric oxide and endothelin. This new system may be very useful to understand and study the molecular mechanisms involved in many vascular alteration pathologies and in the aging process.

It has been reported that glucose may autooxidize generating free radicals which have been hypothesized to induce important cellular abnormalities. To investigate the cell damage induced by glucose-dependent oxidative stress, the FRTL5 cell strain was incubated in 10 or 20 mM glucose, either alone or in the presence of buthionine-sulfoximine, a transition state inhibitor that blocks glutathione synthesis. We found indeed that buthionine-sulfoximine greatly inhibited glutathione production and increased malondialdehyde (a marker of oxidative cell damage) levels, especially in 20 nM glucose. We also found that, when glutathione production was inhibited, 10 mM glucose induced apoptosis and 20 mM glucose induced necrosis. These data show that the glucose-dependent cell damage is a function of glutathione production. They also show that such glucose-dependent free radical production may be critical for determining cell damage, even for small variations as the ones we tested (from 10 to 20 mM glucose).

The natural resources are currently overexploited and since 1992 the Conference of Rio de Janeiro has focused on sustainable development to safeguard our planet for future generations. The Fungi kingdom includes producers of goods and services for ecosystems and organisms widely used in the food industry. Besides, macrofungi are recognized as nontimber forest products and could be utilized as agents of environmental management through weed biocontrol and environmental improvement. Moreover, the cultivation of fungi, in particular truffles, can provide an important income in agroecosystems, especially in marginal areas, along with the development of new technologies to produce novel products from fungi. © 2013 Societá Botanica Italiana.

Hypogeous fungi are common in arid and semi-arid areas of the Mediterranean basin and, in particular, they are found in Italy, Spain, northern African countries (especially in Morocco, Algeria, and Tunisia), and in the Middle East. These fungi and, in particular, some species belonging to the genus Tuber and the desert truffles (Terfezia spp. and Tirmania spp.) form ascomata of considerable economic value. In this review, a panorama of hypogeous fungal diversity and information on their ecology in Mediterranean arid and semi-arid ecosystems have been reported; their economical importance and cultivation potential are also underlined.