Publications by authors named "Natalia Rosso"

30 Publications

  • Page 1 of 1

Taste perception and expression in stomach of bitter taste receptor tas2r38 in obese and lean subjects.

Appetite 2021 11 7;166:105595. Epub 2021 Jul 7.

Department of Medical, Surgical and Health Sciences, University of Trieste, Strada di Fiume, 447, 34149, Trieste, Italy; Surgical Clinic Unit, Cattinara Hospital, ASUGI, Strada di Fiume, 447, 34149, Trieste, Italy.

Differences in taste perception have been related to eating behavior, nutritional status, and diseases. Recently, taste receptors have been identified in several extra-oral tissues, such as the gastrointestinal tract, where they seem to influence processes like digestion, sense of satiety as well as energy balance and intraluminal changes occurring in obesity. Our study aims to analyze differences in taste perception among 42 obese patients (OB) and 41 normal-weight subjects (LEAN). Polymorphisms in the gene codifying for the bitter taste receptor TAS2R38 and its expression on the surface of the gastric mucosa were tested and compared among OB and LEAN. Taste intensity of PROP (6-n-propylthiouracil), quinine, sucrose, citric acid and NaCl were measured on a labeled magnitude scale. DNA from peripheral whole blood was extracted and three polymorphisms in the TAS2R38 gene (rs713598, rs1726866, rs10246939) analyzed. Gastric biopsies were collected during bariatric surgery in OB and during endoscopy in LEAN. RNA was extracted and TAS2R38 gene expression assessed by RT-Real-Time qPCR. Anamnestic and anthropometric data were recorded in all participants during baseline visits. Logistic regression analysis showed that OB perceives sweet (sucrose) and bitter (PROP or 6-n-propylthiouracil) taste more intensely than LEAN (p-value = 0.02 and p-value = 0.005, respectively). While polymorphisms in TAS2R38 gene did not differ among OB and LEAN, we observed a significant increase of TAS2R38 mRNA levels in the stomach of OB compared to LEAN (p = 0.01). Our results provide new evidence of a link between obesity and altered taste perception as well as TAS2R38 expression in the stomach.
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http://dx.doi.org/10.1016/j.appet.2021.105595DOI Listing
November 2021

Diagnostic management of nonalcoholic fatty liver disease: a transformational period in the development of diagnostic and predictive tools-a narrative review.

Ann Transl Med 2021 Apr;9(8):727

Fondazione Italiana Fegato, ONLUS Area Science Park Basovizza, Trieste, Italy.

NAFLD is an emerging healthcare epidemic that is causing predictable adverse consequences for healthcare systems, societies and individuals. Whilst NAFLD is recognized as a multi-system disease with compound pathways that are both benign and pernicious in their unfolding; NASH is generally understood as a deleterious follow-on condition with path-specific tendencies that progress to cirrhosis, HCC and liver transplantation. Recent evidence is beginning to challenge this interpretation demanding more attention to the personalized nature of the disease and its pathogenesis across multiple different cohorts. This means that we need better diagnostic and prognostic tools not only to capture those 'at risk' disease phenotypes; but for better stratification and monitoring of patients according to their treatment strategies. With the advent of pipeline therapies for NASH underway, the medical profession looks to adopt more accurate non-invasive diagnostic tools that can help to delineate and eliminate NASH histology. This review looks at the search for the killer application revealing this particular moment in time as a transformational period; one that is pushing the boundaries of technology to integrate diverse panels of species through sensitive profiling and multi-omics approaches that cast wide, yet powerful diagnostic nets that have the potential to elucidate pathway specific biomarkers that are personalized and predictable.
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http://dx.doi.org/10.21037/atm-20-4723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106012PMC
April 2021

Optimization of Point-Shear Wave Elastography by Skin-to-Liver Distance to Assess Liver Fibrosis in Patients Undergoing Bariatric Surgery.

Diagnostics (Basel) 2020 Oct 7;10(10). Epub 2020 Oct 7.

Department of Medical, Surgical and Health Sciences, University of Trieste, 34149 Trieste, Italy.

Background: Obesity is a primary limiting factor in liver stiffness measurement (LSM). The impact of obesity has always been evaluated in terms of body mass index (BMI), without studying the effects of skin-to-liver distance (SLD) on LSM. We studied the impact of SLD on LSM in a cohort of obese patients undergoing bariatric surgery and intra-operatory liver biopsy.

Materials And Methods: 299 patients underwent LSM by point-shear wave elastography (ElastPQ protocol), with two different ultrasound machines. SLD was measured as the distance between the skin and the liver capsule, perpendicular to where the region of interest (ROI) was positioned. We used the following arbitrary cut-offs: <5.7 kPa, F0-1; 5.7-7.99 kPa, F2; ≥8 kPa, F3-4.

Results: We developed two logistic regression models using elastography-histology agreement (EHA) as the dependent variable and SLD as the independent variable. The model based on the second machine showed strongly more performant discriminative and calibration metrics (AIC 38.5, BIC 44.2, Nagelkerke Pseudo-R2 0.894, AUROC 0.90). The SLD cut-off value of 34.5 mm allowed a correct EHA with a sensitivity of 100%, a specificity of 93%, negative predictive value of 100%, positive predictive value of 87%, an accuracy of 96%, and positive likelihood ratio of 3.56.

Conclusion: The impact of SLD is machine-dependent and should be taken into consideration when interpreting LSM. We believe that our findings may serve as a reference point for appropriate fibrosis stratification by liver elastography in obese patients.
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http://dx.doi.org/10.3390/diagnostics10100795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601552PMC
October 2020

Modifications of IGF2 and EGFR plasma protein concentrations in NAFLD patients after bariatric surgery.

Int J Obes (Lond) 2021 02 17;45(2):374-382. Epub 2020 Sep 17.

Fondazione Italiana Fegato, Centro Studi Fegato, Area Science Park Basovizza Bldg. Q SS14 Km 163.5, Trieste, Italy.

Background: Nonalcoholic fatty liver disease (NAFLD) is strictly associated with the epidemic of obesity and is becoming the most prevalent liver disease worldwide. In severe obesity, bariatric surgery (BS) is the most effective treatment not only for obesity but also for the associated metabolic co-morbidities, NAFLD, among others. To date, noninvasive diagnostic/prognostic methods cannot evaluate hepatic improvements following surgery.

Objectives: We aimed to measure plasma level of insulin-growth factor-2 protein (IGF2) and epithermal growth factor receptor (EGFR), and to assess their relationship with clinical and biochemical parameters during the 12 months follow-up.

Methods: Demographic, clinical-biochemical data, and plasma IGF2 and EGFR were measured in 69 patients preoperatively (T0) and 6 and 12 months (T6M and T12M, respectively) after BS. Liver biopsy was performed at T0. Relationships between IGF2, EGFR, and several biochemical parameters were performed using Pearson or Spearman correlation analysis.

Results: IGF2 plasma level increases during follow-up, passing from 2.5 (1.8-15.5) at baseline to 13.3 (8.6-19.1) at T12M, p < 0.001. Conversely, EGFR showed a not significant reduction. At T12M, the plasma level of both markers was comparable to those of lean subjects. The clinical-biochemical parameters (BMI, glycated hemoglobin, HOMA-IR) also return to the normal range at T12M. Correlation analysis demonstrated that IGF2 was significantly associated with total bilirubin, direct bilirubin, and albumin at T0 while with blood glucose, ALT, GGT, and AST/ALT ratio at T6M and T12M.

Conclusions: IGF2 plasma levels increase after bariatric surgery, and these changes are associated with the modification of hepatic biochemical parameters, even if other clinic or metabolic improvements cannot be excluded.
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http://dx.doi.org/10.1038/s41366-020-00687-0DOI Listing
February 2021

Occult hepatitis B virus infection predicts non-alcoholic steatohepatitis in severely obese individuals from Italy.

Liver Int 2020 07 24;40(7):1601-1609. Epub 2020 Apr 24.

Unit of Clinical and Molecular Hepatology, University Hospital of Messina, Messina, Italy.

Background & Aims: Obesity is associated with non-alcoholic fatty liver (NAFL), which may progress towards non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). Occult hepatitis B virus infection (OBI) may contribute to hepatic damage in patients with chronic liver disease of different aetiologies (eg HCV, alcohol). However, information on the prevalence and clinical impact of OBI in obese individuals is lacking. The aims of this study were to investigate NASH prevalence and risk factors in obese people who underwent bariatric surgery.

Methods: Two-hundred and twenty-six subjects (160 females; mean age 42.9 years ±10.8 SD) without evidence of any further cause of liver disease consecutively underwent bariatric surgery in two Italian liver centers. During surgery, all patients underwent liver biopsy for histological evaluation and molecular studies. Liver DNA extracts were tested for PNPLA3, TM6SF2, MBOAT7, IRGM polymorphisms and for OBI. Univariate and multivariate analyses were used to identify predictors of NASH.

Results: Histology showed NASH in 115 (50.9%) and NAFL in 111 cases (49.1%). Twenty-nine/226 (12.8%) cases had OBI, 24 (82.8%) of whom had NASH and 5 (17.2%) NAFL, whereas among the 197 OBI-negative cases, 91 (46.2%) had NASH and 106 (53.8%) NAFL (P = .0002). Multivariate analysis showed that older age (P = .03, OR 1.034), alanine aminotransferase values (P = .005, OR 1.023), insulin resistance/diabetes (P = .02, OR 2.257), TM6SF2 polymorphism (P = .04, OR 3.168) and OBI (P = .004, OR 5.503) were independent predictors of NASH.

Conclusion: NASH is highly prevalent in obese individuals undergoing bariatric surgery. OBI is one of the strongest risk factors of NASH in these patients.
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http://dx.doi.org/10.1111/liv.14473DOI Listing
July 2020

Differentiation between stages of non-alcoholic fatty liver diseases using surface-enhanced Raman spectroscopy.

Anal Chim Acta 2020 May 22;1110:190-198. Epub 2020 Feb 22.

Department of Engineering and Architecture, University of Trieste, Trieste, Italy. Electronic address:

Non-alcoholic fatty liver disease (NAFLD) is a chronic disorder progressing from an initial benign accumulation of fat (NAFL) towards steatohepatitis (NASH), a degenerative form that can lead to liver cirrhosis and cancer. The development of non-invasive, rapid and accurate method to diagnose NASH is of high clinical relevance. Surface-enhanced Raman spectroscopy (SERS) of plasma was tested as a method to distinguish NAFL from NASH. SERS spectra from plasma of female patients diagnosed with NAFL (n = 32) and NASH (n = 35) were obtained in few seconds, using a portable Raman spectrometer. The sample consisted of 5 μL of biofluid deposited on paper coated with Ag nanoparticles. The spectra show consistent differences between the NAFL and NASH patients, with the uric acid/hypoxanthine band area ratio statistically different (p-value <0.001) between the two groups. The average figures of merit for a diagnostic test based on these ratios, as derived from a repeated 4-fold cross-validation of a logistic regression model, are all between 0.73 and 0.79, with an average area under the curve of 0.81. We conclude that SERS may be a reliable and rapid method to discriminate NAFLD from NASH.
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http://dx.doi.org/10.1016/j.aca.2020.02.040DOI Listing
May 2020

Obeticholic acid and INT-767 modulate collagen deposition in a NASH in vitro model.

Sci Rep 2020 02 3;10(1):1699. Epub 2020 Feb 3.

Fondazione Italiana Fegato, AREA Science Park Basovizza, SS14 km 163.5, 34149, Trieste, Italy.

Pharmacological treatments for non-alcoholic steatohepatitis (NASH) are still unsatisfactory. Fibrosis is the most significant predictor of mortality and many anti-fibrotic agents are under evaluation. Herein, we assessed in vitro the effects of the FXR agonist obeticholic acid (OCA) and the dual FXR/TGR5 agonist INT-767 in a well-established co-culture NASH model. Co-cultures of human hepatoma and hepatic stellate (HSCs) cells were exposed to free fatty acids (FFAs) alone or in combination with OCA or INT-767. mRNA expression of HSCs activation markers and FXR engagement were evaluated at 24, 96 and 144 hours. Collagen deposition and metalloproteinase 2 and 9 (MMP2-9) activity were compared to tropifexor and selonsertib. FFAs induced collagen deposition and MMP2-9 activity reduction. Co-treatment with OCA or INT-767 did not affect ACTA2 and COL1A1 expression, but significantly reduced FXR and induced SHP expression, as expected. OCA induced a dose-dependent reduction of collagen and induced MMP2-9 activity. Similarly, INT-767 induced collagen reduction at 96 h and a slight increase in MMP2-9. Tropifexor and Selonsertib were also effective in collagen reduction but showed no modulation of MMP2-9. All tested compounds reduced collagen deposition. OCA exerted a more potent and long-lasting effect, mainly related to modulation of collagen turn-over and MMP2-9 activity.
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http://dx.doi.org/10.1038/s41598-020-58562-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997404PMC
February 2020

Silybin Modulates Collagen Turnover in an In Vitro Model of NASH.

Molecules 2019 Apr 2;24(7). Epub 2019 Apr 2.

Fondazione Italiana Fegato, ONLUS, AREA Science Park Basovizza SS 14 km 163.5, 34149 Trieste, Italy.

Silybin has been proposed as a treatment for nonalcoholic steatohepatitis (NASH). In this study, we assessed the effect of Silybin in a well-established in vitro coculture model of early-stage NASH. LX2 and Huh7 cells were exposed to free fatty acid (FFA) and Silybin as mono- or coculture (SCC). Cell viability, LX2 activation, collagen deposition, metalloproteinase 2 and 9 (MMP2-9) activity, and ROS generation were determined at 24, 96, and 144 h. Exposure to FFA induced the activation of LX2 as shown by the increase in cell viability and upregulation of collagen biosynthesis. Interestingly, while cotreatment with Silybin did not affect collagen production in LX2, a significant reduction was observed in SCC. MMP2-9 activity was reduced in FFA-treated Huh7 and SCC and cotreatment with Silybin induced a dose-dependent increase, while no effect was observed in LX2. Silybin also showed antioxidant properties by reducing the FFA-induced production of ROS in all the cell systems. Based on these data, Silybin exerts its beneficial effects by reducing LX2 proliferation and ROS generation. Moreover, MMP2-9 modulation in hepatocytes represents the driving mechanism for the net reduction of collagen in this NASH in vitro model, highlighting the importance of hepatic cells interplay in NASH development and resolution.
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http://dx.doi.org/10.3390/molecules24071280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479571PMC
April 2019

Global epidemiology of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: What we need in the future.

Liver Int 2018 02;38 Suppl 1:47-51

Fondazione Italiana Fegato-Onlus, Trieste, Italy.

The estimated prevalence of non-alcoholic fatty liver disease (NAFLD) worldwide is approximately 25%. However, the real prevalence of NAFLD and the associated disorders is unknown mainly because reliable and applicable diagnostic tests are lacking. This is further complicated by the lack of consensus on the terminology of different entities such as NAFLD or nonalcoholic steatohepatitis (NASH). Although assessing fatty infiltration in the liver is simple by ultrasound, the gold standard for the assessment of fibrosis, the only marker of progression towards more severe liver disease is still liver biopsy. Although other non-invasive tests have been proposed, they must still be validated in large series. Because NAFL/NAFLD/NASH and related metabolic diseases represent an economic burden, finding an inexpensive method to diagnose and stage fatty liver is a priority. A translational approach with the use of cell and/or animal models could help to reach this goal.
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http://dx.doi.org/10.1111/liv.13643DOI Listing
February 2018

Effects of Oral Administration of Silymarin in a Juvenile Murine Model of Non-alcoholic Steatohepatitis.

Nutrients 2017 Sep 12;9(9). Epub 2017 Sep 12.

Fondazione Italiana Fegato ONLUS-Centro Studi Fegato, Area Science Park Basovizza Bldg, Q SS 14 Km 163,5, Basovizza, 34149 Trieste, Italy.

The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) in adolescents is challenging the global care system. No therapeutic strategies have been defined so far, and changes in the lifestyle remain the only alternative. In this study, we assessed the protective effects of silymarin in a juvenile non-alcoholic steatohepatitis (NASH) model and the in vitro effects on fat-laden human hepatocytes. C57Bl/6 mice were exposed to HFHC diet immediately after weaning. After eight weeks, animals showed histological signs of NASH. Silymarin was added to the HFHC diet, the treatment continued for additional 12 weeks and the effects on BMI, hepatomegaly, visceral fat, lipid profile, transaminases, HOMA-IR, steatosis, inflammation, fibrosis, oxidative stress, and apoptosis were determined. The switch from HFHC to control diet was used to mimic life style changes. In vitro experiments were performed in parallel in human hepatocytes. HFHC diet supplemented with silymarin showed a significant improvement in glycemia, visceral fat, lipid profile, and liver fibrosis. Moreover, it reduced (both in vitro and in vivo) ALT, hepatic inflammation, oxidative stress, and apoptosis. Lifestyle changes restored the control group parameters. The data presented show the beneficial effects of the oral administration of silymarin in the absence of changes in the dietary habits in a juvenile model of NASH.
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http://dx.doi.org/10.3390/nu9091006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622766PMC
September 2017

A simple in silico strategy identifies candidate biomarkers for the diagnosis of liver fibrosis in morbidly obese subjects.

Liver Int 2018 01 21;38(1):155-163. Epub 2017 Jul 21.

Fondazione Italiana Fegato, Centro Studi Fegato, Trieste, Italy.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disorder, tightly associated with obesity. The histological spectrum of the disease ranges from simple steatosis to steatohepatitis, with different stages of fibrosis, and fibrosis stage is the most significant predictor of mortality in NAFLD. Liver biopsy continues to be the gold standard for its diagnosis and reliable non-invasive diagnostic tools are unavailable. We investigated the accuracy of candidate proteins, identified by an in silico approach, as biomarkers for diagnosis of fibrosis.

Methods: Seventy-one morbidly obese (MO) subjects with biopsy-proven NAFLD were enrolled, and the cohort was subdivided according to minimal (F0/F1) or moderate (F2/F3) fibrosis. The plasmatic level of CD44 antigen (CD44), secreted protein acidic and rich in cysteine (SPARC), epidermal growth factor receptor (EGFR) and insulin-like growth factor 2 (IGF2) were determined by ELISA. Significant associations between plasmatic levels and histological fibrosis were determined by correlation analysis and the diagnostic accuracy by the area under receiver operating characteristic curves (AUROC).

Results: Eighty-two percentage of the subjects had F0/F1 and 18% with F2/F3 fibrosis. Plasmatic levels of IGF2, EGFR and their ratio (EGFR/IGF2) were associated with liver fibrosis, correlating inversely for IGF2 (P < .006) and directly (P < .018; P < .0001) for EGFR and EGFR/IGF2 respectively. The IGF2 marker had the best diagnostic accuracy for moderate fibrosis (AUROC 0.83), followed by EGFR/IGF2 ratio (AUROC 0.79) and EGFR (AUROC 0.71).

Conclusions: Our study supports the potential utility of IGF2 and EGFR as non-invasive diagnostic biomarkers for liver fibrosis in morbidly obese subjects.
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http://dx.doi.org/10.1111/liv.13505DOI Listing
January 2018

Hepatocyte-derived macrophage migration inhibitory factor mediates alcohol-induced liver injury in mice and patients.

J Hepatol 2017 11 22;67(5):1018-1025. Epub 2017 Jun 22.

Center for Liver Disease Research, Department of Pathobiology, Cleveland Clinic, Spain; Department of Molecular Medicine, Case Western Reserve University, USA. Electronic address:

Background & Aims: Macrophage migration inhibitory factor (MIF) is a multi-potent cytokine that contributes to the inflammatory response to injury. MIF is expressed by multiple cell types; however, the cellular source and actions of MIF in alcoholic liver disease (ALD) are not well known. Here we tested the hypothesis that non-myeloid cells, specifically hepatocytes, are an important cellular source of MIF in ALD.

Methods: MIF expression was measured in HuH7 and differentiated THP-1 cells in response to ethanol. Ethanol-induced liver injury was assessed in C57BL/6 (WT) and Mif bone marrow chimeras. MIF was measured in peripheral and suprahepatic serum, as well as visualized by immunohistochemistry in liver biopsies, from patients with alcoholic hepatitis (AH).

Results: HuH7 hepatocytes, but not THP-1 macrophages, released MIF in response to ethanol challenge in culture. In chimeric mice expressing MIF in non-myeloid cells (Mif→WT), chronic ethanol feeding increased ALT/AST, hepatic steatosis, and expression of cytokine/chemokine mRNA. In contrast, chimeric mice not expressing MIF in non-myeloid cells (WT→Mif) were protected from ethanol-induced liver injury. Immunohistochemical staining of liver biopsies from patients with AH revealed a predominant localization of MIF to hepatocytes. Interestingly, the concentration of MIF in suprahepatic serum, but not peripheral serum, was positively correlated with clinical indicators of disease severity and with an increased risk of mortality in patients with AH.

Conclusions: Taken together, these data provide evidence that hepatocyte-derived MIF is critical in the pathogenesis of ALD in mice and likely contributes to liver injury in patients with AH. Lay summary: Alcoholic liver disease is a major cause of preventable mortality worldwide, and lacks specific pharmacological therapies. Recent studies have recognized that macrophage migration inhibitor factor (MIF) has a critical role in the inflammatory response to liver damage. However, the cells that produce this protein are still unknown. Our present findings reveal that hepatocytes, the main cell type in the liver, are primarily responsible for MIF production in response to alcohol, which promotes liver injury. Our study suggests that drugs inhibiting MIF production could be beneficial in treating patients with liver disease due to excessive alcohol consumption.
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http://dx.doi.org/10.1016/j.jhep.2017.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5650516PMC
November 2017

Th17 involvement in nonalcoholic fatty liver disease progression to non-alcoholic steatohepatitis.

World J Gastroenterol 2016 Nov;22(41):9096-9103

Carla Melisa Chackelevicius, Sabrina Eliana Gambaro, Claudio Tiribelli, Natalia Rosso, Italian Liver Foundation, Area Science Park, Trieste, 34149 TS, Italy.

The nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. NAFLD encompasses a wide histological spectrum ranging from benign simple steatosis to non-alcoholic steatohepatitis (NASH). Sustained inflammation in the liver is critical in this process. Hepatic macrophages, including liver resident macropaghes (Kupffer cells), monocytes infiltrating the injured liver, as well as specific lymphocytes subsets play a pivotal role in the initiation and perpetuation of the inflammatory response, with a major deleterious impact on the progression of fatty liver to fibrosis. During the last years, Th17 cells have been involved in the development of inflammation not only in liver but also in other organs, such as adipose tissue or lung. Differentiation of a naïve T cell into a Th17 cell leads to pro-inflammatory cytokine and chemokine production with subsequent myeloid cell recruitment to the inflamed tissue. Th17 response can be mitigated by T regulatory cells that secrete anti-inflammatory cytokines. Both T cell subsets need TGF-β for their differentiation and a characteristic plasticity in their phenotype may render them new therapeutic targets. In this review, we discuss the role of the Th17 pathway in NAFLD progression to NASH and to liver fibrosis analyzing different animal models of liver injury and human studies.
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http://dx.doi.org/10.3748/wjg.v22.i41.9096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107591PMC
November 2016

An Animal Model for the Juvenile Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis.

PLoS One 2016 8;11(7):e0158817. Epub 2016 Jul 8.

Fondazione Italiana Fegato, AREA science Park, Trieste, Italy.

Non Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH) are the hepatic manifestations of the metabolic syndrome; worrisome is the booming increase in pediatric age. To recreate the full spectrum of juvenile liver pathology and investigate the gender impact, male and female C57Bl/6 mice were fed with high fat diet plus fructose in the drinking water (HFHC) immediately after weaning (equal to 3-years old human), and disease progression followed for 16 weeks, until adults (equal to 30-years old human). 100% of subjects of both genders on HFHC diet developed steatosis in 4weeks, and some degree of fibrosis in 8weeks, with the 86% of males and 15% of females presenting a stage 2 fibrosis at 16weeks. Despite a similar final liver damage both groups, a sex difference in the pathology progression was observed. Alterations in glucose homeostasis, dyslipidemia, hepatomegaly and obese phenotype were evident from the very beginning in males with an increased hepatic inflammatory activity. Conversely, such alterations were present in females only at the end of the HFHC diet (with the exception of insulin resistance and the hepatic inflammatory state). Interestingly, only females showed an altered hepatic redox state. This juvenile model appears a good platform to unravel the underlying gender dependent mechanisms in the progression from NAFLD to NASH, and to characterize novel therapeutic approaches.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158817PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938400PMC
July 2017

The Pros and the Cons for the Use of Silybin-Rich Oral Formulations in Treatment of Liver Damage (NAFLD in Particular).

Curr Med Chem 2015 ;22(25):2954-71

Fondazione Italiana Fegato, Area Science Park Basovizza, SS14 km 163.5, 34149 Trieste, Italy.

The increasing prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD) worldwide is becoming a challenge for the modern global care system. The lipotoxic process is characterized by an oxidative stress followed by a burst of the inflammatory response, prompting the wound healing process (fibrosis), which can ultimately lead to the development of cirrhosis and the subsequent complications. There is no consensus concerning an effective pharmacological treatment. Therefore, there is a need for effective therapeutic compounds. Silibinin the major active compound of Milk Thistle may be a potential candidate mainly due to its anti-oxidant, anti-inflammatory, and anti-fibrotic properties. In spite of the large number of data obtained in experimental models, the translation of the evidence in clinical setting is far to be conclusive. The aim of this paper is to critically review the aspects of the use of the different formulations of Silibinin in several experimental and clinical settings and to provide hints on the needed future studies.
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http://dx.doi.org/10.2174/0929867322666150729114235DOI Listing
May 2016

The interplay between hepatic stellate cells and hepatocytes in an in vitro model of NASH.

Toxicol In Vitro 2015 Oct 14;29(7):1753-8. Epub 2015 Jul 14.

Centro Studi Fegato (CSF) - Liver Research Center, Fondazione Italiana Fegato, Bldg Q AREA Science Park, Basovizza Campus SS 14 km 163.5, 34149 Trieste, Italy. Electronic address:

Background & Aim: A complex interplay exists between hepatocytes and hepatic stellate cells (HSC) in hepatic fibrogenesis. The activation of HSCs after liver injury leads to production of extracellular matrix (ECM). Co-culture models could be useful to mimic the liver microenvironment. This study evaluates the effect of free fatty acids (FFA) on HSC cells and the interplay with hepatocytes via both soluble-mediator and cell-cell contact.

Methods: The human hepatocyte cell line (HuH7) and HSC cells (LX2) were exposed to FFA for 24 h in 3 different experimental set-ups: (A) monoculture of HSC; (B) Transwell® system (effect of soluble mediators); and (C) Simultaneous Co-Culture (SCC) (cell-to-cell connections). Intracellular FFA accumulation was assessed qualitatively (microscopy) and quantitatively (flow cytometry); the activation of HSC (alpha smooth muscle actin, α-SMA) expression of ECM components were quantified by RT-PCR.

Results: FFA exposure induces intracellular fat accumulation in all the experimental set-up but the expression of α-SMA was significantly increased only in SCC. On the contrary, the expression of ECM was substantially decreased in the transwell® system.

Conclusions: The HSC activation is independent of FFA accumulation but requires cell-to-cell interaction with hepatocyte. On the contrary, the gene regulation of ECM components seems to occur through paracrine mediators.
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http://dx.doi.org/10.1016/j.tiv.2015.07.010DOI Listing
October 2015

The importance of the interaction between hepatocyte and hepatic stellate cells in fibrogenesis induced by fatty accumulation.

Exp Mol Pathol 2015 Feb 20;98(1):85-92. Epub 2014 Dec 20.

Fondazione Italiana Fegato, Centro Studi Fegato, Area Science Park Basovizza Bldg. Q SS14 Km 163.5, 34149 Trieste, Italy. Electronic address:

Background & Aims: Non-alcoholic fatty liver disease is characterized by an initial accumulation of triglycerides that can progress to non-alcoholic steatohepatitis, which can ultimately evolve to cirrhosis and hepatocellular carcinoma. Hepatic stellate cells play a key role in liver fibrogenesis by an increased activation and an altered profile of genes involved in the turnover of extracellular matrix components. To reproduce in-vitro the functional cell connections observed in vivo it is essential to consider cell-to-cell proximity and interaction. The aim of this study was to determine the response to free fatty acids in a simultaneous co-culture model of hepatocytes and hepatic stellate cells.

Methods: Simultaneous co-culture model and monoculture of each cell type (control) were exposed to FFA for 24 up to 144 h. Quantification of steatosis; stellate cell activation; assessment of fibrogenic response; expression and activity of metalloproteinases as well as collagen biosynthesis were evaluated.

Results: Free fatty acids induced comparable steatosis in simultaneous co-culture and monoculture. However, the activation of the stellate cells assessed by alpha-smooth muscle actin expression is greater when cells were in close contact. Furthermore, a time-dependent increment of tissue inhibitor metalloproteinase-2 protein was observed, which was inversely correlated with protein expression and activity of matrix-metalloproteinases, suggesting enhanced collagen biosynthesis. This behavior was absent in cell monoculture.

Conclusions: These data indicate that cell-to-cell proximity between hepatocytes and stellate cells is necessary for the initiation of the fibrotic process.
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http://dx.doi.org/10.1016/j.yexmp.2014.12.006DOI Listing
February 2015

Translational approaches: from fatty liver to non-alcoholic steatohepatitis.

World J Gastroenterol 2014 Jul;20(27):9038-49

Natalia Rosso, Claudio Tiribelli, Stefano Bellentani, Norberto C Chavez-Tapia, Fondazione Italiana Fegato, 34149 Trieste, Italy.

Over the past few decades, non-alcoholic fatty liver disease (NAFLD) has become one, if not the most common, cause of chronic liver disease affecting both adults and children. The increasing number of cases at an early age is the most worrying aspect of this pathology, since it provides more time for its evolution. The spectrum of this disease ranges from liver steatosis to steatohepatitis, fibrosis and in some cases, hepatocellular carcinoma. NAFLD may not always be considered a benign disease and hepatologists must be cautious in the presence of fatty liver. This should prompt the use of the available experimental models to understand better the pathogenesis and to develop a rational treatment of a disease that is dangerously increasing. In spite of the growing efforts, the pathogenesis of NAFLD is still poorly understood. In the present article we review the most relevant hypotheses and evidence that account for the progression of NAFLD to non-alcoholic steatohepatitis (NASH) and fibrosis. The available in vitro and in vivo experimental models of NASH are discussed and revised in terms of their validity in translational studies. These studies must be aimed at the discovery of the still unknown triggers or mediators that induce the progression of hepatic inflammation, apoptosis and fibrosis.
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http://dx.doi.org/10.3748/wjg.v20.i27.9038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112858PMC
July 2014

Kinetics of the inflammatory response induced by free fatty acid accumulation in hepatocytes.

Ann Hepatol 2013 Jan-2014 Feb;13(1):113-20

Centro Studi Fegato, Fondazione Italiana Fegato, Trieste, Italy; Department of Medical Sciences, University of Trieste, Italy.

BACKGROUND. The information available on time and dose effects of the exposure of hepatocytes to free fatty acids (FFA) in vitro is controversial, and very few studies have assessed the hepatocyte inflammatory response in an in vitro model. AIM. To analyze the effect of treatment with FFA on cell viability and on the kinetics of cytokine expression using hepatic cell lines. MATERIAL AND METHODS. Hepatic cell lines, IHH and HuH7, were cultured for 3 h, 6 h, 12 h and 24 h in an enriched medium with palmitic and oleic acids. The cytotoxicity of the FFA was assessed by the MTT test and the intracellular fat content determined cytofluorimetrically and by fluorescence microscopy using Nile Red staining. The expression of mRNA for interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α was assessed by real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS. Treatment with 600 μM FFA did not affect the viability of either cell line despite a significant increase in the intracellular content of lipid droplets already evident after 3 h of treatment. A time- and dose-dependent upregulation of the expression of IL-6 and IL-8 mRNA was observed during the treatment at 3 and 24 h. In contrast, TNF-α mRNA expression was highly upregulated at 3 h after FFA exposure but returned to control values at 24 h. In conclusion, hepatocytes exposed in vitro for a short time to low FFA concentrations showed a significant upregulation of IL-6 and IL-8 to,and a rapid but transitory elevation of TNF-α.
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September 2014

Alterations in the cell cycle in the cerebellum of hyperbilirubinemic Gunn rat: a possible link with apoptosis?

PLoS One 2013 1;8(11):e79073. Epub 2013 Nov 1.

Fondazione Italiana Fegato (Italian Liver Foundation, Centro Studi Fegato), Trieste, Italy.

Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001). This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose) polymerase (54%, p < 0.01) and active Caspase3 (two fold, p < 0.01). These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0079073PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815147PMC
August 2014

Alcohol, liver and genes: an intricate puzzle.

Ann Hepatol 2013 Nov-Dec;12(6):860

Liver Research Center, The Italian Liver Foundation and Department of Medical Sciences, University of Trieste. Italy.

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August 2014

Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking.

JIMD Rep 2012 6;2:59-69. Epub 2011 Sep 6.

Regional Coordinator Centre for Rare Disease, University Hospital "Santa Maria della Misericordia", Piazzale "S. Maria della Misericordia", 15, 33100, Udine, Italy.

Niemann Pick type C (NPC) disease is an autosomal recessive disorder characterized by the lysosomal/late endosomal (LE) accumulation of unesterified cholesterol and other lipids due to a defect in the intracellular lipid trafficking. About 95% of patients present mutations in the NPC1 gene. Among the 290 mutations reported in the NPC1 gene, about 70% are missense. However, little information is available regarding the impact of missense mutations on NPC1 protein stability and function. In this study, we in vitro characterized the pathogenic effect of 7 NPC1 missense mutations. In all cases, the basal levels of mutant NPC1 expression were reduced with respect to wild type. Treatment of fibroblasts carrying NPC1 missense mutations in homo or hemizygosity, with the proteasome inhibitor MG132 or glycerol 10%, a chemical chaperone known to stabilize misfolded proteins, resulted in a significant increase of NPC1 protein levels in all cell lines, indicating that these mutants are subjected to proteasomal degradation due to protein misfolding The increment of NPC1 mutant protein induced by the proteasome inhibitor was associated with a localization of NPC1 protein within lysosomal/LE compartment. In cell lines carrying mutations p.N1156S, p.L1191F, p.V1165M, and p.I1061T, the increment of NPC1 mutant protein resulted in an improvement of the intracellular trafficking of cholesterol and GM1. These findings showed that it is possible to correct the NPC cellular phenotype by increasing the amount of endogenous NPC1 mutated protein, suggesting that at least some NPC1 mutations might be potentially rescued by small molecules-based chaperone therapy.
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http://dx.doi.org/10.1007/8904_2011_49DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509841PMC
February 2013

Gene and functional up-regulation of the BCRP/ABCG2 transporter in hepatocellular carcinoma.

BMC Gastroenterol 2012 Nov 15;12:160. Epub 2012 Nov 15.

Centro Studi Fegato, Fondazione Italiana Fegato, Bld Q AREA Science Park Basovizza, Trieste, Italy.

Background: The Breast Cancer Resistance Protein (BCRP/ABCG2) is one member of ABC transporters proteins super family responsible of drug resistance. Since data on ABCG2 expression in liver malignances are scanty, here we report the expression of ABCG2 in adult human hepatocellular carcinoma (HCC) in both in vivo and in vitro models with different degree of malignancy.

Methods: In cell lines derived from human hepatocellular carcinoma, ABCG2 gene expression was assessed by reverse transcription quantitative real time PCR and function by Hoechst 33342 efflux assay; protein content was assessed by SDS-PAGE Western blot.

Results: ABCG2 expression was found to be highest in the most undifferentiated cell lines, and this was related with a higher functional activity. ABCG2 expression was sensitive to antineoplastic drugs since exposure to 5 μM doxorubicin for 24 hours resulted in significant up-regulations of ABCG2 in all cell lines, particularly in those lines with low basal ABCG2 expression (p<0.01). The gene expression was also investigated in 51 adult liver tissues with HCC and related cirrhosis; normal liver tissue was used as control. ABCG2 gene expression was higher in HCC than both cirrhotic paired tissue and normal tissue. This up-regulation was greater (p<0.05) in pathological poorly differentiated grade G3/G4 than in well-differentiated G1/G2 HCC.

Conclusions: Our results suggest a correlation of ABCG2 gene expression and differentiation stage both in human and HCC derived cell lines. The rapid up-regulation of ABCG2 to exposure to doxorubicin emphasizes the importance of this transporter in accounting for drug resistance in liver tumors.
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http://dx.doi.org/10.1186/1471-230X-12-160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538657PMC
November 2012

Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease.

BMC Gastroenterol 2012 Mar 1;12:20. Epub 2012 Mar 1.

Fondazione Italiana Fegato- Centro Studi Fegato, AREA SCIENCE Park Basovizza, Bldg Q, Trieste, Italy.

Background: In vitro exposure of liver cells to high concentrations of free fatty acids (FFA) results in fat overload which promotes inflammatory and fibrogenic response similar to those observed in patients with Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH). Since the mechanisms of this event have not been fully characterized, we aimed to analyze the fibrogenic stimuli in a new in vitro model of NASH.

Methods: HuH7 cells were cultured for 24 h in an enriched medium containing bovine serum albumin and increasing concentrations of palmitic and oleic acid at a molar ratio of 1:2 (palmitic and oleic acid, respectively). Cytotoxic effect, apoptosis, oxidative stress, and production of inflammatory and fibrogenic cytokines were measured.

Results: FFA induces a significant increment in the intracellular content of lipid droplets. The gene expression of interleukin-6, interleukin-8 and tumor necrosis factor alpha was significantly increased. The protein level of interleukin-8 was also increased. Intracellular lipid accumulation was associated to a significant up-regulation in the gene expression of transforming growth factor beta 1, alpha 2 macroglobulin, vascular endothelial growth factor A, connective tissue growth factor, insulin-like growth factor 2, thrombospondin 1. Flow cytometry analysis demonstrated a significant increment of early apoptosis and production of reactive oxygen species.

Conclusions: The exposure of hepatocytes to fatty acids elicits inflammation, increase of oxidative stress, apoptosis and production of fibrogenic cytokines. These data support a primary role of FFA in the pathogenesis of NAFLD and NASH.
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http://dx.doi.org/10.1186/1471-230X-12-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3313845PMC
March 2012

Effects of E2F1-cyclin E1-E2 circuit down regulation in hepatocellular carcinoma cells.

Dig Liver Dis 2011 Dec 9;43(12):1006-14. Epub 2011 Aug 9.

Department of Medical, Technological and Translational Sciences, University Hospital of Cattinara, Trieste, Italy.

Background: No effective therapy is available for hepatocellular carcinoma. To identify novel therapeutic strategies, we explored the effects of the depletion of E2F1, cyclin E1-E2 whose inter-relationships in hepatocellular carcinoma cell proliferation have never been defined.

Methods: siRNA-mediated depletion of the targets was studied in the hepatocellular carcinoma cells HepG2, HuH7 and JHH6 characterized by high, medium and low hepatocyte differentiation grade, respectively; a model of normal human hepatocytes was also considered.

Results: The depletion of each target mRNA reduced the levels of the other two mRNAs, thus demonstrating a close regulatory control, also confirmed by over-expression experiments. At the protein level, an exception to this trend was observed for cyclinE1 whose amount increased upon cyclin E2 (HepG2, HuH7, JHH6) and E2F1 (HepG2) depletion. In HepG2, reduced cyclinE1 proteolysis accounted for this observation. Additionally, cyclin E1-E2-E2F1 targeting decreased the levels of cyclin A2 mRNA and of the hyper-phosphorylated form of pRb thus leading to an S-phase cell decrease; migration was impaired as well. Finally, the model of human hepatocytes used was clearly less affected by target mRNAs depletion than hepatocellular carcinoma cells.

Conclusion: Our data provide novel mutual relationships amongst cyclin E1-E2-E2F1 and indicate their role in sustaining hepatocellular carcinoma cell proliferation/migration, validating the concept of an anti-cyclin E1-E2-E2F1 therapeutic approach for hepatocellular carcinoma.
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http://dx.doi.org/10.1016/j.dld.2011.07.007DOI Listing
December 2011

Hepatic cancer stem cells and drug resistance: Relevance in targeted therapies for hepatocellular carcinoma.

World J Hepatol 2010 Mar;2(3):114-26

Caecilia HC Sukowati, Natalia Rosso, Claudio Tiribelli, Centro Studi Fegato, AREA Science Park Basovizza, Trieste 34012, Italy.

Hepatocellular carcinoma (HCC) is one of most common malignancies in the world. Systemic treatments for HCC, particularly for advanced stages, are limited by the drug resistance phenomenon which ultimately leads to therapy failure. Recent studies have indicated an association between drug resistance and the existence of the cancer stem cells (CSCs) as tumor initiating cells. The CSCs are resistant to conventional chemotherapies and might be related to the mechanisms of the ATP Binding Cassette (ABC) transporters and alterations in the CSCs signaling pathways. Therefore, to contribute to the development of new HCC treatments, further information on the characterization of CSCs, the modulation of the ABC transporters expression and function and the signaling pathway involved in the self renewal, initiation and maintenance of the cancer are required. The combination of transporters modulators/inhibitors with molecular targeted therapies may be a potent strategy to block the tumoral progression. This review summarizes the association of CSCs, drug resistance, ABC transporters activities and changes in signaling pathways as a guide for future molecular therapy for HCC.
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http://dx.doi.org/10.4254/wjh.v2.i3.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998960PMC
March 2010

Galectin-1 and its involvement in hepatocellular carcinoma aggressiveness.

Mol Med 2010 Mar 21;16(3-4):102-15. Epub 2009 Dec 21.

CEINGE Biotecnologie Avanzate, Naples, Italy.

Hepatocellular carcinoma is one of the most common cancers worldwide. Despite several efforts to elucidate hepatocellular carcinoma molecular pathogenesis, it is still not fully understood. To acquire further insights into the molecular mechanisms of hepatocellular carcinoma, we performed a systematic functional genomic approach on human HuH-7 and JHH-6 cells. The subsequent analysis of the differentially expressed genes in human specimens revealed a molecular signature of 11 genes from which we selected the LGALS1 gene, which was overexpressed in hepatocellular carcinoma. The expression analysis in humans of Galectin-1 (Gal-1), the protein encoded by LGALS1, showed a Gal-1 preferential accumulation in the stromal tissue around hepatocellular carcinoma tumors. Moreover, a significant association between increased expression of Gal-1 in hepatocellular carcinoma and the presence of metastasis was observed. Interestingly, Gal-1 overexpression resulted in an increase of cell migration and invasion. In conclusion, this study provides a portfolio of targets useful for future investigations into molecular marker-discovery studies on a large number of patients and functional assays. In addition, our data provide evidence that Gal-1 plays a role in hepatocellular carcinoma cell migration and invasion, and we suggest that further studies should be conducted to fully establish the role of Gal-1 in hepatocellular carcinoma pathogenesis and evaluate Gal-1 as a potential molecular therapeutic target.
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http://dx.doi.org/10.2119/molmed.2009.00119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829614PMC
March 2010

Molecular and functional analysis of the HEXB gene in Italian patients affected with Sandhoff disease: identification of six novel alleles.

Neurogenetics 2009 Feb 29;10(1):49-58. Epub 2008 Aug 29.

Unità di Malattie Metaboliche, IRCCS Burlo Garofolo, Trieste, Italy.

We report the molecular characterization of 12 unrelated Italian patients affected with Sandhoff disease (SD), a recessively inherited disorder caused by mutations in HEXB gene. We identified 11 different mutations of which six are novel: one large deletion of 2,406 nt, (c.299+1471_408del2406), one frameshift mutation c.965delT (p.I322fsX32), one nonsense c.1372C>T (p.Q458X), and three splicing mutations (c.299G>T, c.300-2A>G and c.512-1G>T). One allele was only characterized at the messenger RNA (mRNA) level (r = 1170_1242del). Real-time polymerase chain reaction analysis of the HEXB mRNA from fibroblasts derived from patients carrying the novel point mutations showed that the presence of the premature termination codon in the transcript bearing the mutation c.965delT triggers the nonsense-mediated decay (NMD) pathway, which results in the degradation of the aberrant mRNA. The presence of the c.299G>T mutation leads to the degradation of the mutated mRNA by a mechanism other than NMD, while mutations c.300-2A>G and c.512-1G>T cause the expression of aberrant transcripts. In our group, the most frequent mutation was c.850C>T (p.R284X) representing 29% of the alleles. Haplotype analysis suggested that this mutation did not originate from a single genetic event. Interestingly, the common 16-kb deletion mutation was absent. This work provides valuable information regarding the molecular genetics of SD in Italy and provides new insights into the molecular basis of the disease.
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http://dx.doi.org/10.1007/s10048-008-0145-1DOI Listing
February 2009

Subcellular localization of APE1/Ref-1 in human hepatocellular carcinoma: possible prognostic significance.

Mol Med 2007 Jan-Feb;13(1-2):89-96

Centro Studi Fegato, AREA Science Park and University of Trieste, Trieste, Italy.

APE1/Ref-1, normally localized in the nucleus, is a regulator of the cellular response to oxidative stress. Cytoplasmic localization has been observed in several tumors and correlates with a poor prognosis. Because no data are available on liver tumors, we investigated APE1/Ref-1 subcellular localization and its correlation with survival in 47 consecutive patients undergoing hepatocellular carcinoma (HCC) resection. APE1/Ref-1 expression was determined by immunohistochemistry in HCC and surrounding liver cirrhosis (SLC) and compared with normal liver tissue. Survival probability was evaluated using Kaplan-Meier curves (log-rank test) and Cox regression. Cytoplasmic expression of APE1/Ref-1 was significantly higher in HCC than in SLC (P = 0.00001); normal liver showed only nuclear reactivity. Patients with poorly differentiated HCC showed a cytoplasmic expression three times higher than those with well-differentiated HCC (P = 0.03). Cytoplasmic localization was associated with a median survival time shorter than those with negative cytoplasmic reactivity (0.44 compared with 1.64 years, P = 0.003), and multivariable analysis confirmed that cytoplasmic APE1/Ref-1 localization is a predictor of survival. Cytoplasmic expression of APE1/Ref-1 is increased in HCC and is associated with a lower degree of differentiation and a shorter survival time, pointing to the use of the cytoplasmic localization of APE1/Ref-1 as a prognostic marker for HCC.
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http://dx.doi.org/10.2119/2006-00084.dimasoDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1869623PMC
July 2007

Gene expression analysis in HBV transgenic mouse liver: a model to study early events related to hepatocarcinogenesis.

Mol Med 2006 Apr-Jun;12(4-6):115-23

Sezione di Gastroenterologia, Dipartimento di Emergenza e di Trapianto d'Organo, Università di Bari, Bari, Italia.

Hepatitis B virus (HBV) is one of the major etiological factors responsible for the development of hepatocellular carcinoma (HCC). We used a transgenic mouse, containing HBV sequences, as a model system to unravel the molecular mechanisms of hepatocarcinogenesis induced by HBV. We chose this animal model because it consistently develops liver cancer after intermediate steps that mimic the natural history of HBV infection in humans. In this study, we focus our attention on the early events leading to liver cancer. We compared the gene expression profile of 3-month-old transgenic mice with that of 3-month-old wild-type (wt) animals. In the transgenic mouse, microarray data analysis showed a total of 45 significantly differentially expressed genes, 25 highly expressed (fold change > or =2; P = 0.0025), and 20 downregulated (fold change < or =0.5; P = 0.0025). These genes belong to several different functional categories such as the regulation of immunological response, transcription, intracellular calcium ion mobilization, regulation of cell cycle and proliferation, NF-kappab signal transduction cascades, and apoptosis. In particular, the upregulation of the antiapoptotic gene NuprI and the downregulation of the proapoptotic gene Bnip3 were found. This observation was supported by an in vitro apoptosis assay that showed downregulation of apoptosis in hepatocytes of HBV transgenic mouse compared with wt mice treated with staurosporine. In conclusion, our experimental approach allowed identification of new genes modulated by HBV and showed that the apoptotic process was deregulated in transgenic mouse hepatocytes. These data shed light on one possible mechanism by which HBV induces hepatocarcinogenesis.
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http://dx.doi.org/10.2119/2006-00015.BaroneDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1578771PMC
December 2006
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