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Background: The adoption of robotic technology in the treatment of prostate cancer (PCa) could lead to improvement in outcomes., Objective: To evaluate feasibility, to compare functional outcomes, and to assess the economic benefits of removing catheter on the postoperative day (POD) 3 versus POD 5 after robot-assisted radical prostatectomy (RARP)., Design, Setting, and Participants: From September 2016 to May 2017, patients selected to undergo RARP for clinically localized PCa at a high-volume center were prospectively randomized into group 1 (POD 3; n=72) versus group 2 (POD 5, n=74)., Intervention: All patients underwent RARP with anatomical posterior and anterior reconstruction., Outcome Measurements and Statistical Analysis: The primary endpoint was to compare acute urinary retention (AUR) and urinary leakage rate in the two groups. The secondary endpoints were early and mid-term postoperative functional outcomes assessed through questionnaires (ICIQ-MLUTS, IPSS), early continence rate, and postoperative pain/discomfort (visual analog scale score). The economic impact of early catheter removal was also assessed., Results and Limitations: AUR was reported in two (1.4%) cases, one for each study group (p=0.9). One case of vesicourethral leakage was reported (0.7%) in group 1. Urethral discomfort and pain at discharge was significantly higher in group 2 (p=0.03). In our clinical practice, POD 3 catheter removal approach would determine a saving of approximately €80 000 and 405 d of hospitalization yearly. The main limitation is the small sample size., Conclusions: Early catheter removal after RARP does not lead to an increase in perioperative complications. No negative effect on early and mid-term functional outcomes was observed. A significant impact on saving economic resources was reported., Patient Summary: We demonstrated that early catheter removal has no negative effect on spontaneous voiding, complications, or urinary continence recovery after robot-assisted radical prostatectomy., (Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Aim: The aim of this study was to analyze the economic efficiency of second-line diagnostic investigations in patients with undetermined lung nodules., Participants and Methods: A retrospective review of all surgical cases included in the DANTE trial from 2001 to 2006 for lung cancer screening was performed. Overall, 217 patients and 261 lung nodules were analyzed. The cohort was divided into patients investigated with PET and/or computed tomography (CT)-guided biopsy (PET-CTB protocol; N=100), compared with those assessed with serial low-dose CT scans (standard protocol; N=161). Outpatient's and inpatient's costs were expressed in euros and derived from the Italian National Health Service. Ineffective costs were defined as the cost of procedures that lead to avoidable surgical intervention., Results: The diagnostic accuracy of the two protocols was 91% for the standard (sensitivity 100%, specificity 91%, positive predictive value 26%, and negative predictive value 100%) and 90% for the PET-CTB protocol (sensitivity 98%, specificity 81%, positive predictive value 85%, and negative predictive value 97%). Average costs for outpatient's diagnostics were 694 and 1.462 euros, respectively, for the standard and PET-CTB protocol. Average inpatient's costs for both protocols were 12.121 euros. The two protocols showed comparable effectiveness in terms of outpatient's costs (94 and 90%, respectively; P=0.252). Inpatient's costs were effective in 36% of cases monitored according to the standard protocol compared with 85% of patients investigated with PET-CTB protocol. Ineffective costs corresponded to 64 and 15%, respectively (P<0.0001)., Conclusion: Despite a higher average cost for outpatient's diagnostics, the implementation of PET imaging with or without CT-guided needle biopsy in the workup of suspicious lung nodules results in reduced unnecessary harm and costs related to inpatient's procedures.

The diagnosis and management of Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is still challenging. There is no definitive gold standard diagnostic test, which is made on patient history and with endoscopic and histological findings. This study analyzed serum proteins and fatty acids using mass spectrometry-based techniques. Quantitation of serum proteins was performed by depleting 14 high-abundance proteins, followed by tryptic digestion and LC-MS analysis, while fatty acids were analyzed using GC-MS. Bioinformatic tools were used to identify several new potential biomarkers for an early and non-invasive diagnosis of IBD, and to differentiate CD from UC. Moreover, the diagnostic power of the MS-identified biomarkers was also corroborated by Western Blot and ELISA assays. Hence, we identified the biological functions and pathways involved in the various subsets of IBD. Coagulation, fibrinolysis and acute phase response processes were found to be strongly involved in the condition. The involvement of several fatty acids, such as anti-inflammatory mediators, was also identified. Finally, proteomic and lipidomic data were integrated by using combinatorial and multivariate analyses to discover new combined biomarkers and to study the molecular pathways involved in IBD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Study Aims: Endoscopic submucosal dissection (ESD), a minimally invasive treatment for early gastrointestinal (GI) cancer, is considered challenging and risky in the colorectum. As such, most patients undergoing ESD are hospitalized due to the perceived increased risk of adverse events. The aim of this study was to compare the costs, safety and efficacy of colorectal-ESD in an outpatient vs inpatient setting in a tertiary level center., Methods: This is a retrospective study on consecutive patients admitted for colorectal-ESD. Patients were divided into outpatients (Group-A, same-day discharge), and inpatients (Group-B, admitted for at least one night). Data on overall costs, outcomes and adverse events were assessed for each group., Results: A total of 136 patients were considered. Fourteen were excluded because ESD was not performed due to intraprocedural suspicion of invasive cancer. Eighty-three patients were treated as outpatients (Group-A, 68%) and 39 (Group-B, 32%) were hospitalized. R0-rate was 90.4% in Group-A and 89.7% in Group-B(P = 0.98). One perforation occurred in Group-A (1.2%) and 2 in Group-B(5.1%, P = 0.2). Mean Length of stay (LOS) was 1 day for outpatients and 3.3 days for inpatients. Management of Group-A as outpatients produced a cost savings of 941€ on average per patient., Conclusions: Outpatient colorectal-ESD is a feasible, cost-effective strategy to manage superficial colorectal tumors with outcomes comparable to inpatient colorectal-ESD. By using proper selection criteria, outpatient ESD could be considered the first-line approach for most patients., (Copyright © 2018. Published by Elsevier Ltd.)

Arterial hypertension represents a common complication of immunosuppressive therapy after liver transplantation (LT). The aim of the study is to evaluate the prevalence and risk factors associated with hypertension after LT. From a cohort of 323 cirrhotic patients who underwent LT from 2008 to 2012, 270 patients were retrospectively evaluated, whereas 53 (16.4%) patients deceased. Hypertension was defined as blood pressure ≥140/90 mm Hg in at least two visits and/or the need for antihypertensive therapy. The prevalence of hypertension was 15% before LT and significantly increased up to 53% after LT (P < .001). Mean follow-up was 43 ± 19 months. In normotensive (NT) subjects at baseline, 35.9% developed sustained hypertension after LT, whereas 15.2% developed transient hypertension within the first month after LT, and then returned NT. The development of sustained hypertension after LT was related to the mammalian target of rapamycin inhibitor treatment (odds ratio [OR], 4.02; 95% confidence interval [CI], 1.26-13.48; P = .02), alcoholic cirrhosis before LT (OR, 3.38; 95% CI, 1.44-8.09; P = .005), and new-onset hepatic steatosis after LT (OR, 2.13; 95% CI, 1.10-4.11; P = .02). Tacrolimus, the etiology and severity of liver disease, and other immunosuppressive regimens were not related to the development of hypertension after LT. In our cohort, the prevalence of arterial hypertension has increased up to 53% after LT, and metabolic comorbidities and immunosuppressive treatment with mammalian target of rapamycin inhibitors are the risk factors for the development of hypertension after LT., (Copyright © 2018 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.)

Purpose: We investigated predictive factors of failure and performed a resource consumption analysis in patients who underwent active surveillance for nonmuscle invasive bladder cancer., Materials and Methods: This prospective observational study monitored patients with a history of pathologically confirmed stage pTa (grade 1-2) or pT1a (grade 2) nonmuscle invasive bladder cancer, and recurrent small size and number of tumors without hematuria and positive urine cytology. The primary end point was the failure rate of active surveillance. Assessment of failure predictive variables and per year direct hospital resource consumption analysis were secondary outcomes. Descriptive statistical analysis and Cox regression with univariable and multivariable analysis were done., Results: Of 625 patients with nonmuscle invasive bladder cancer 122 with a total of 146 active surveillance events were included in the protocol. Of the events 59 (40.4%) were deemed to require treatment after entering active surveillance. Median time on active surveillance was 11 months (IQR 5-26). Currently 76 patients (62.3%) remain under observation. On univariable analysis only time from the first transurethral resection to the start of active surveillance seemed to be inversely associated with recurrence-free survival (HR 0.99, 95% CI 0.98-1.00, p = 0.027). Multivariable analysis also revealed an association with age at active surveillance start (HR 0.97, 95% CI 0.94-1.00, p = 0.031) and the size of the lesion at the first transurethral resection (HR 1.55, 95% CI 1.06-2.27, p = 0.025). The average specific annual resource consumption savings for each avoided transurethral bladder tumor resection was €1,378 for each intervention avoided., Conclusions: Active surveillance might be a reasonable clinical and cost-effective strategy in patients who present with small, low grade pTa/pT1a recurrent papillary bladder tumors., (Copyright © 2018 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Robotic surgery is increasingly used to resect lung cancer. However costs are high. We compared costs and outcomes for robotic surgery, video-assisted thoracic surgery (VATS), and open surgery, to treat non-small cell lung cancer (NSCLC)., Methods: We retrospectively assessed 103 consecutive patients given lobectomy or segmentectomy for clinical stage I or II NSCLC. Three surgeons could choose VATS or open, the fourth could choose between all three techniques. Between-group differences were assessed by Fisher's exact, two-way analysis of variance (ANOVA), and Wilcoxon-Mann-Whitney test. P values <0.05 were considered significant., Results: Twenty-three patients were treated by robot, 41 by VATS, and 39 by open surgery. Age, physical status, pulmonary function, comorbidities, stage, and perioperative complications did not differ between the groups. Pathological tumor size was greater in the open than VATS and robotic groups (P=0.025). Duration of surgery was 150, 191 and 116 minutes, by robotic, VATS and open approaches, respectively (P<0.001). Significantly more lymph node stations were removed (P<0.001), and median length of stay was shorter (4, 5 and 6 days, respectively; P<0.001) in the robotic than VATS and open groups. Estimated costs were 82%, 68% and 69%, respectively, of the regional health service reimbursement for robotic, VATS and open approaches., Discussion: Robotic surgery for early lung cancer was associated with shorter stay and more extensive lymph node dissection than VATS and open surgery. Duration of surgery was shorter for robotic than VATS. Although the cost of robotic thoracic surgery is high, the hospital makes a profit., Competing Interests: Conflicts of Interest: G Veronesi is a consultant for ABI Medica SpA and Medtronic, other authors have no conflicts of interest to declare.

Background: The application of artificial intelligence to extract predictors of Gambling disorder (GD) is a new field of study. A plethora of studies have suggested that maladaptive personality dispositions may serve as risk factors for GD., New Method: Here, we used Classification and Regression Trees algorithm to identify multivariate predictive patterns of personality profiles that could identify GD patients from healthy controls at an individual level. Forty psychiatric patients, recruited from specialized gambling clinics, without any additional comorbidity and 160 matched healthy controls completed the Five-Factor model of personality as measured by the NEO-PI-R, which were used to build the classification model., Results: Classification algorithm was able to discriminate individuals with GD from controls with an AUC of 77.3% (95% CI 0.65-0.88, p<0.0001). A multidimensional construct of traits including sub-facets of openness, neuroticism and conscientiousness was employed by algorithm for classification detection., Comparison With Existing Method(s): To the best of our knowledge, this is the first study that combines behavioral data with machine learning approach useful to extract multidimensional features characterizing GD realm., Conclusion: Our study provides a proof-of-concept demonstrating the potential of the proposed approach for GD diagnosis. The multivariate combination of personality facets characterizing individuals with GD can potentially be used to assess subjects' vulnerability in clinical setting., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In an increasing proportion of cases, hepatocellular carcinoma (HCC) develops in patients with nonalcoholic fatty liver disease (NAFLD). Mutations in telomerase reverse transcriptase (hTERT) are associated with familial liver diseases. The aim of this study was to examine telomere length and germline hTERT mutations as associated with NAFLD-HCC. In 40 patients with NAFLD-HCC, 45 with NAFLD-cirrhosis and 64 healthy controls, peripheral blood telomere length was evaluated by qRT-PCR and hTERT coding regions and intron-exon boundaries sequenced. We further analyzed 78 patients affected by primary liver cancer (NAFLD-PLC, 76 with HCC). Enrichment of rare coding mutations (allelic frequency <0.001) was evaluated by Burden test. Functional consequences were estimated in silico and by over-expressing protein variants in HEK-293 cells. We found that telomere length was reduced in individuals with NAFLD-HCC versus those with cirrhosis (P = 0.048) and healthy controls (P = 0.0006), independently of age and sex. We detected an enrichment of hTERT mutations in NAFLD-HCC, that was confirmed when we further considered a larger cohort of NAFLD-PLC, and was more marked in female patients (P = 0.03). No mutations were found in cirrhosis and local controls, and only one in 503 healthy Europeans from the 1000 Genomes Project (allelic frequency = 0.025 vs. <0.001; P = 0.0005). Mutations with predicted functional impact, including the frameshift Glu113Argfs*79 and missense Glu668Asp, cosegregated with liver disease in two families. Three patients carried missense mutations (Ala67Val in homozygosity, Pro193Leu and His296Pro in heterozygosity) in the N-terminal template-binding domain (P = 0.037 for specific enrichment). Besides Glu668Asp, the Ala67Val variant resulted in reduced intracellular protein levels. In conclusion, we detected an association between shorter telomeres in peripheral blood and rare germline hTERT mutations and NAFLD-HCC., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Nonalcoholic fatty liver disease (NAFLD) represents an emerging cause of hepatocellular carcinoma (HCC), especially in non-cirrhotic individuals. The rs641738 C > T MBOAT7/TMC4 variant predisposes to progressive NAFLD, but the impact on hepatic carcinogenesis is unknown. In Italian NAFLD patients, the rs641738 T allele was associated with NAFLD-HCC (OR 1.65, 1.08-2.55; n = 765), particularly in those without advanced fibrosis (p < 0.001). The risk T allele was linked to 3'-UTR variation in MBOAT7 and to reduced MBOAT7 expression in patients without severe fibrosis. The number of PNPLA3, TM6SF2, and MBOAT7 risk variants was associated with NAFLD-HCC independently of clinical factors (p < 0.001), but did not significantly improve their predictive accuracy. When combining data from an independent UK NAFLD cohort, in the overall cohort of non-cirrhotic patients (n = 913, 41 with HCC) the T allele remained associated with HCC (OR 2.10, 1.33-3.31). Finally, in a combined cohort of non-cirrhotic patients with chronic hepatitis C or alcoholic liver disease (n = 1121), the T allele was independently associated with HCC risk (OR 1.93, 1.07-3.58). In conclusion, the MBOAT7 rs641738 T allele is associated with reduced MBOAT7 expression and may predispose to HCC in patients without cirrhosis, suggesting it should be evaluated in future prospective studies aimed at stratifying NAFLD-HCC risk.

Use of robot-assisted techniques is growing fast in several surgical disciplines, now including thoracic surgery. The paper reviews experience of robotic surgery to resect lung cancer and in particular analyzes data on the costs of these procedures in comparison to open surgery and video-assisted thoracoscopic surgery (VATS). Retrospective studies published over 14 years show that robotic surgery for lung cancer has the advantages of minimally invasive surgery for patients, and some advantages over VATS for the surgeon. Limited data indicate that oncological outcomes are comparable with those of VATS and open surgery, while lymph node dissection may be more radical. Other studies indicate that robotic surgery for lung cancer offers no advantages either in terms of costs or outcomes. The high costs of purchase, maintenance and consumables are a concern and continue to limit uptake of robot systems in thoracic surgery. Most studies-but not all-indicate that robotic surgery for lung cancer is more expensive than VATS and open surgery. However limited data also indicate that hospitals can make a profit from robotic thoracic surgery, as costs seem to be lower than reimbursements from paying bodies. Nevertheless robotic thoracic surgery is still too expensive for many public hospitals, particularly in low income countries. Entry of new surgical robot manufacturers onto the market will bring much-needed competition that may also lead to cost reduction., Competing Interests: Conflicts of Interest: G Veronesi is a consultant for ABI Medica SpA and Medtronic. And other authors have no conflicts of interest to declare.

Chronic hepatitis C is a systemic disease inducing metabolic alterations leading to extrahepatic consequences. In particular, hepatitis C virus (HCV) infection seems to increase the risk of incident type 2 diabetes mellitus in predisposed individuals, independently of liver disease stage. The mechanisms through which hepatitis C induces T2DM involve direct viral effects, insulin resistance, pro-inflammatory cytokines and other immune-mediated processes. Many studies have reported the clinical consequences of type 2 diabetes mellitus on hepatitis C outcome, but very few studies have addressed the issue of microangiopathic complications among patients with hepatitis C only, who develop type 2 diabetes mellitus. Moreover, clinical trials in HCV-positive patients have reported improvement in glucose metabolism after antiviral treatment; recent studies have suggested that this metabolic amelioration might have a clinical impact on type 2 diabetes mellitus-related complications. These observations raise the question as to whether the HCV eradication may also have an impact on the future morbidity and mortality due to type 2 diabetes mellitus. The scope of this review is to summarise the current evidence linking successful antiviral treatment and the prevention of type 2 diabetes mellitus and its complications in hepatitis C-infected patients., (Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: The combination of non-invasive markers for the detection of fibrosis in patients with chronic liver diseases is still a matter of debate., Aims: To test the performance of cytokeratin18-Aspartate396 alone or in combination with transient elastography as a marker of fibrosis, compared to liver biopsy as gold standard., Methods: In 259 prospectively enrolled patients with chronic liver diseases, clinical, biochemical, and histological features were assessed. Serum cytokeratin18-Aspartate396 and Fibroscan were performed within 6 months prior to liver biopsy., Results: Cytokeratin18-Aspartate396 levels predicted both significant and advanced fibrosis in non-alcoholic fatty liver disease group, correctly identifying 83.7% and 80.8% of cases, respectively. Liver stiffness performed best in predicting severe fibrosis in patients with chronic viral infection, correctly identifying 78.7% of chronic hepatitis B and 88.6% of chronic hepatitis C subjects. The combination of cytokeratin18-Aspartate396 and liver stiffness improved their diagnostic performance for the detection of significant and advanced fibrosis in non-alcoholic fatty liver disease group, only (sensitivity=78.3%, specificity=90.7%; sensitivity=91.7%, specificity=71.6%, respectively)., Conclusion: Cytokeratin18-Aspartate396 and liver stiffness can improve the non-invasive prediction of significant and advanced fibrosis in patients with non-alcoholic fatty liver disease, while in hepatitis B and C virus infected patients their combined use had no advantage over the diagnostic accuracy of transient elastography alone., (Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Surrogate indexes of insulin resistance and insulin sensitivity are widely used in nonalcoholic fatty liver disease (NAFLD), although they have never been validated in this population. We aimed to validate the available indexes in NAFLD subjects and to test their ability to predict liver damage also in comparison with the NAFLD fibrosis score. Surrogate indexes were validated by the tracer technique (6,6-D2 -glucose and U-(13) C-glucose) in the basal state and during an oral glucose tolerance test. The best-performing indexes were used in an independent cohort of 145 nondiabetic NAFLD subjects to identify liver damage (fibrosis and nonalcoholic steatohepatitis). In the validation NAFLD cohort, homeostasis model assessment of insulin resistance, insulin to glucose ratio, and insulin sensitivity index Stumvoll had the best association with hepatic insulin resistance, while peripheral insulin sensitivity was most significantly related to oral glucose insulin sensitivity index (OGIS), insulin sensitivity index Stumvoll, and metabolic clearance rate estimation without demographic parameters. In the independent cohort, only oral glucose tolerance test-derived indexes were associated with liver damage and OGIS was the best predictor of significant (≥F2) fibrosis (odds ratio = 0.76, 95% confidence interval 0.61-0.96, P = 0.0233) and of nonalcoholic steatohepatitis (odds ratio = 0.75, 95% confidence interval 0.63-0.90, P = 0.0021). Both OGIS and NAFLD fibrosis score identified advanced (F3/F4) fibrosis, but OGIS predicted it better than NAFLD fibrosis score (odds ratio = 0.57, 95% confidence interval 0.45-0.72, P < 0.001) and was also able to discriminate F2 from F3/F4 (P < 0.003)., Conclusion: OGIS is associated with peripheral insulin sensitivity in NAFLD and inversely associated with an increased risk of significant/advanced liver damage in nondiabetic subjects with NAFLD., (© 2015 by the American Association for the Study of Liver Diseases.)

Contrasting data exist on the effect of gender and menopause on the susceptibility, development and liver damage progression in non-alcoholic fatty liver disease (NAFLD). Our aim was to assess whether menopause is associated with the severity of liver fibrosis in individuals with NAFLD and to explore the issue of ovarian senescence in experimental liver steatosis in zebrafish. In 244 females and age-matched males with biopsy-proven NAFLD, we assessed anthropometric, biochemical and metabolic features, including menopausal status (self-reported); liver biopsy was scored according to 'The Pathology Committee of the NASH Clinical Research Network'. Young and old male and female zebrafish were fed for 24 weeks with a high-calorie diet. Weekly body mass index (BMI), histopathological examination and quantitative real-time PCR analysis on genes involved in lipid metabolism, inflammation and fibrosis were performed. In the entire cohort, at multivariate logistic regression, male gender [odds ratio (OR): 1.408, 95% confidence interval (95% CI): 0.779-2.542, P=0.25] vs women at reproductive age was not associated with F2-F4 fibrosis, whereas a trend was observed for menopause (OR: 1.752, 95% CI: 0.956-3.208, P=0.06). In women, menopause (OR: 2.717, 95% CI: 1.020-7.237, P=0.04) was independently associated with F2-F4 fibrosis. Similarly, in overfed zebrafish, old female fish with failing ovarian function [as demonstrated by extremely low circulating estradiol levels (1.4±0.1 pg/µl) and prevailing presence of atretic follicles in the ovaries] developed massive steatosis and substantial fibrosis (comparable with that occurring in males), whereas young female fish developed less steatosis and were totally protected from the development of fibrosis. Ovarian senescence significantly increases the risk of fibrosis severity both in humans with NAFLD and in zebrafish with experimental steatosis., (© 2015. Published by The Company of Biologists Ltd.)

The top three leading causes of death in patients with nonalcoholic fatty liver disease (NAFLD) in descending order are cardiovascular disease, cancer, and liver disease. It is clear now that the increased risk of metabolic and macro- and microvascular complications in NAFLD stems from the associated features of metabolic syndrome. However, NAFLD itself may contribute to the spectrum of risk factors associated with insulin resistance. The primary focus of this review is to summarize the main systemic associations of NAFLD, as well as to discuss the mechanisms that link them to NAFLD. Hepatic lipid accumulation in NAFLD impairs hepatic glucose and lipid metabolism further increasing the risk of type 2 diabetes mellitus and of cardiovascular disease, independently of established risk factors. The incidence, prevalence, and severity of these complications are proportional to the histological severity of liver damage suggesting that NAFLD, but particularly nonalcoholic steatohepatitis, can also contribute to the low-grade inflammatory state through the systemic release of several markers of inflammation, oxidative stress, and of procoagulant factors. The clinical implication of these findings is that patients with NAFLD require a multidisciplinary evaluation, with a major focus on type 2 diabetes mellitus and cardiovascular disease complications and may benefit from more intensive surveillance and early treatment interventions to decrease the risk for cardiovascular and kidney complications., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: The PNPLA3/Adiponutrin rs738409 C/G single nucleotide polymorphism is associated with the severity of steatosis, steatohepatitis and fibrosis in patients with non-alcoholic fatty liver disease, as well as the severity of steatosis and fibrosis in patients with chronic hepatitis C (CHC)., Aim: To test in genotype 1(G1)-CHC patients, the putative association between the PNPLA3 variant and histological features of steatohepatitis, as well as their impact on the severity of fibrosis., Methods: Four hundred and thirty-four consecutively biopsied Caucasian G1-CHC patients were genotyped for PNPLA3 rs738409, its effect evaluated by using an additive model. Histological features of steatohepatitis in CHC were assessed using the Bedossa classification. Hepatic expression of PNPLA3 mRNA was evaluated in 63 patients., Results: The prevalence of steatohepatitis increased from 16.5% in patients with PNPLA3 CC, to 23.2% in CG and 29.2% in the GG genotype (P = 0.02). By multiple logistic regression, PNPLA3 genotype (OR 1.54, 95% CI 1.03-2.30, P = 0.03), together with age (OR 1.03, 95% CI 1.00-1.05, P = 0.02), BMI ≥ 30 (OR 2.06, 95% CI 1.04-4.10, P = 0.03) and homoeostasis model assessment (HOMA, OR 1.18, 95% CI 1.04-1.32, P = 0.006) were independently linked to steatohepatitis. When stratifying for obesity, PNPLA3 was associated with NASH in non-obese patients only (12.0% in CC vs. 18.3% in CG vs. 27.3% in GG, P = 0.01), including after correction for metabolic confounders (OR 2.06, 95% CI 1.26-3.36, P = 0.004). We showed an independent association between steatohepatitis (OR 2.05, 95% CI 1.05-4.02, P = 0.003) and severe fibrosis. Higher liver PNPLA3 mRNA was associated both with the severity of steatosis (adjusted P = 0.03) and steatohepatitis after adjusting for gender, age, BMI and HOMA (P = 0.002)., Conclusion: In patients with genotype 1 hepatitis C, the PNPLA3 G variant is associated with a higher risk of steatosis severity and steatohepatitis, particularly among non-obese subjects., (© 2015 John Wiley & Sons Ltd.)

Background & Aims: The accuracy of noninvasive tools for the diagnosis of severe fibrosis in patients with nonalcoholic fatty liver disease(NAFLD) in clinical practice is still limited. We aimed at assessing the diagnostic performance of combined noninvasive tools in two independent cohorts of Italian NAFLD patients., Methods: We analysed data from 321 Italian patients(179 Sicilian-training cohort, and 142 northern Italy-validation cohort) with an histological diagnosis of NAFLD. Severe fibrosis was defined as fibrosis ≥ F3 according to Kleiner classification. The APRI, AST/ALT, BARD, FIB-4, and NFS scores were calculated according to published algorithms. Liver stiffness measurement(LSM) was performed by FibroScan. Cut-off points of LSM, NFS and FIB-4 for rule-in or rule-out F3-F4 fibrosis were calculated by the reported formulas., Results: In the Sicilian cohort AUCs of LSM, NFS, FIB-4, LSM plus NFS, LSM plus FIB-4, and NFS plus FIB-4 were 0.857, 0.803, 0.790, 0.878, 0.888 and 0.807, respectively, while in the northern Italy cohort the corresponding AUCs were 0.848, 0.730, 0.703, 0.844, 0.850, and 0.733 respectively. In the training cohort, the combination of LSM plus NFS was the best performing strategy, providing false positive, false negative and uncertainty area rates of 0%,1.1% and 48% respectively. Similar results were obtained in the validation cohort with false positive, false negative and uncertainty area rates of 0%,7.3% and 40.8%., Conclusions: The combination of LSM with NFS, two complementary, easy-to-perform, and widely available tools, is able to accurately diagnose or exclude the presence of severe liver fibrosis, also reducing of about 50-60% the number of needed diagnostic liver biopsies., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background & Aims: Series studies have associated increased serum levels of ferritin with liver fibrosis in patients with nonalcoholic fatty liver disease. We aimed to determine the accuracy with which measurements of serum ferritin determine the presence and severity of liver fibrosis, and whether combining noninvasive scoring systems with serum ferritin analysis increases the accuracy of diagnosis of advanced liver fibrosis., Methods: We performed a retrospective analysis of data from 1014 patients with liver biopsy-confirmed nonalcoholic fatty liver disease. Three cut points of serum ferritin level, adjusted for sex, were established based on receiver operating characteristic curve analysis: 1.0-, 1.5-, and 2.0-fold the upper limit of normal. Three multiple logistic regression models were created to determine the association of these cutoff values with liver fibrosis, adjusting for age, sex, race, diabetes, body mass index, and level of alanine aminotransferase., Results: A greater proportion of patients with increased serum levels of ferritin had definitive nonalcoholic steatohepatitis and more-advanced fibrosis than patients without increased levels. In all models, serum level of ferritin was significantly associated with the presence and severity of liver fibrosis. However, for all 3 cutoff values, area under the receiver operating characteristic curve values were low (less than 0.60) for the presence of fibrosis or any stage of liver fibrosis; ferritin level identified patients with fibrosis with 16%-41% sensitivity and 70%-92% specificity. The accuracy with which noninvasive scoring systems identified patients with advanced fibrosis did not change with inclusion of serum ferritin values., Conclusions: Although serum levels of ferritin correlate with more-severe liver fibrosis, based on adjusted multiple logistic regression analysis, serum ferritin levels alone have a low level of diagnostic accuracy for the presence or severity of liver fibrosis in patients with nonalcoholic fatty liver disease., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Obesity is an established risk factor for many types of cancers, particularly for hepatocellular carcinoma (HCC), owing to its carcinogenic potential and the association with nonalcoholic fatty liver disease (NAFLD). HCC may develop in cirrhotic and noncirrhotic livers with NAFLD, particularly in the presence of multiple metabolic risk factors such as obesity and diabetes. This issue is alarming because the population potentially at higher risk is greatly increasing. This review summarizes current evidence linking obesity and liver cancer, and discusses recent advances on the mechanisms underlying this relationship., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 localizes to cellular structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10 and disrupts their integrity by inducing the degradation of PML. There are six PML isoforms with different C-terminal regions in ND10, of which PML isoform I (PML.I) is the most abundant. Depletion of all PML isoforms increases the plaque formation efficiency of ICP0-null mutant HSV-1, and reconstitution of expression of PML.I and PML.II partially reverses this improved replication. ICP0 also induces widespread degradation of SUMO-conjugated proteins during HSV-1 infection, and this activity is linked to its ability to counteract cellular intrinsic antiviral resistance. All PML isoforms are highly SUMO modified, and all such modified forms are sensitive to ICP0-mediated degradation. However, in contrast to the situation with the other isoforms, ICP0 also targets PML.I that is not modified by SUMO, and PML in general is degraded more rapidly than the bulk of other SUMO-modified proteins. We report here that ICP0 interacts with PML.I in both yeast two-hybrid and coimmunoprecipitation assays. This interaction is dependent on PML.I isoform-specific sequences and the N-terminal half of ICP0 and is required for SUMO-modification-independent degradation of PML.I by ICP0. Degradation of the other PML isoforms by ICP0 was less efficient in cells specifically depleted of PML.I. Therefore, ICP0 has two distinct mechanisms of targeting PML: one dependent on SUMO modification and the other via SUMO-independent interaction with PML.I. We conclude that the ICP0-PML.I interaction reflects a countermeasure to PML-related antiviral restriction.

The viral ubiquitin ligase ICP0 is required for efficient initiation of herpes simplex virus 1 (HSV-1) lytic infection and productive reactivation of viral genomes from latency. ICP0 has been shown to target a number of specific cellular proteins for proteasome-dependent degradation during lytic infection, including the promyelocytic leukemia protein (PML) and its small ubiquitin-like modified (SUMO) isoforms. We have shown previously that ICP0 can catalyze the formation of unanchored polyubiquitin chains and mediate the ubiquitination of specific substrate proteins in vitro in the presence of two E2 ubiquitin-conjugating enzymes, namely, UBE2D1 (UbcH5a) and UBE2E1 (UbcH6), in a RING finger-dependent manner. Using homology modeling in conjunction with site-directed mutagenesis, we identify specific residues required for the interaction between the RING finger domain of ICP0 and UBE2D1, and we report that point mutations at these residues compromise the ability of ICP0 to induce the colocalization of conjugated ubiquitin and the degradation of PML and its SUMO-modified isoforms. Furthermore, we show that RING finger mutants that are unable to interact with UBE2D1 fail not only to complement the plaque-forming defect of an ICP0-null mutant virus but also to mediate the derepression of quiescent HSV-1 genomes in cell culture. These data demonstrate that the ability of ICP0 to interact with cellular E2 ubiquitin-conjugating enzymes is fundamentally important for its biological functions during HSV-1 infection.

Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection.

Background & Aims: Increased visceral adiposity is considered the hallmark of the metabolic syndrome, whose hepatic manifestation is nonalcoholic fatty liver disease (NAFLD), although a subset of patients does not have visceral obesity. Our study aimed to compare metabolic alterations and liver damage in patients with NAFLD with and without visceral obesity., Methods: Four hundred and thirty one consecutive patients with liver biopsy-confirmed NAFLD were divided in three groups according to waist circumference, the simplest surrogate marker of visceral obesity. One hundred and thirty three patients (31%) had a waist circumference ≤94 (males) and ≤80 cm (females) (group A), 157 (36%) between 94 and 102, and 80 and 88 (B), and the remaining 141 (33%) had values higher than 102 and 88 cm (C)., Results: Significant trends for older age, higher prevalence of female gender, lower HDL, higher triglycerides, altered glucose metabolism, hypertension, and metabolic syndrome were observed with increasing visceral adiposity. In contrast, non-alcoholic steatohepatitis (NASH) detected in 55% and 72% of patients with normal and increased waist circumference, respectively, and the presence of fibrosis ≥2 were not associated with visceral adiposity. Alanine aminotransferase (ALT), ferritin, HOMA-IR >4, and severe steatosis were independently associated with NASH, whereas ferritin and impaired glucose tolerance were associated with fibrosis ≥2., Conclusions: Patients with normal waist circumference, despite milder metabolic alterations, may have NASH and are at risk of developing fibrosis, suggesting that once NAFLD is present, visceral obesity is not a major determinant of liver damage severity., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background/aim: To test the short-term clinical usefulness of venesection associated with lifestyle counselling as against counselling alone on insulin resistance and liver enzymes in subjects with non-alcoholic fatty liver disease (NAFLD), using a propensity score approach., Methods: We carried out a 6- to 8-month observational analysis of 198 NAFLD patients in three Italian referral centres (79 venesection and 119 counselling alone). Insulin resistance was measured by the homeostasis model assessment (HOMA) method. Logistic regression was used to identify factors associated with normal HOMA and normal alanine aminotransferase (ALT) at the end of observation. The results were adjusted for the propensity score to be enrolled in the venesection programme, based on clinical and laboratory data, including common HFE polymorphisms and liver biopsy (available in 161 cases)., Results: After adjustment for propensity and changes in BMI, venesection was significantly associated with normal HOMA [all cases: odds ratio (OR) 3.00; 95% confidence interval (CI) 1.51-5.97; cases with histology: OR 2.29; 95% CI 1.08-4.87] and ALT within normal limits (all cases: OR 2.56; 95% CI 1.29-5.10; cases with histology: OR 2.81; 95% CI 1.20-5.24). The results were confirmed in an analysis of 57 pairs matched for propensity, where venesection similarly increased the probability of normal HOMA (OR 3.27; 95% CI 1.16-7.84) and normal ALT (OR 5.60; 95% CI 2.09-15.00). Similar data were obtained in the subset of cases with normal basal ferritin (<350 ng/ml)., Conclusion: Iron depletion by venesection favours the normalization of insulin resistance and raised liver enzymes in non-haemochromatosis patients with NAFLD.

The metabolic syndrome encompasses metabolic and cardiovascular risk factors which predict diabetes and cardiovascular disease (CVD) better than any of its individual components. Nonalcoholic fatty liver disease (NAFLD) comprises a disease spectrum which includes variable degrees of simple steatosis (nonalcoholic fatty liver, NAFL), nonalcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is the hepatic manifestation of the metabolic syndrome, with insulin resistance as the main pathogenetic mechanism. Recent data indicate that hyperinsulinemia is probably the consequence rather than cause of NAFLD and NAFLD can be considered an independent predictor of cardiovascular disease. Serum free fatty acids derived from lipolysis of visceral adipose tissue are the main source of hepatic triglycerides in NAFLD, although hepatic de novo lipogenesis and dietary fat supply contribute to the pathogenesis of NAFLD. Approximately 10-25% NAFLD patients develop NASH, the evolutive form of hepatic steatosis. Presumably in a genetically predisposed environment, this increased lipid overload overwhelms the oxidative capacity and reactive oxygen species are generated, leading to lipid peroxidation, cytokine induction, chemoattraction of inflammatory cells, hepatic stellate cell activation and finally fibrogenesis with extracellular matrix deposition. No currently available therapies for NAFLD and NASH exist. Recently nuclear receptors have emerged as key regulators of lipid and carbohydrate metabolism for which specific pharmacological ligands are available, making them attractive therapeutic targets for NAFLD and NASH., (Copyright 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Inherited factors play a major role in the predisposition to nonalcoholic fatty liver disease (NAFLD), and the rs738409 C-->G polymorphism of PNPLA3/adiponutrin, encoding for the isoleucine-to-methionine substitution at residue 148 (I148M) protein variant, has recently been recognized as a major determinant of liver fat content. However, the effect of the rs738409 polymorphism on the severity of liver fibrosis in patients with NAFLD is still unknown. In this study, we considered 253 Italian patients, 179 healthy controls, and 71 family trios with an affected child with NAFLD. Analyses were replicated in 321 patients from the United Kingdom. The rs738409 polymorphism was determined by TaqMan assays. Liver histology was scored according to Kleiner et al. Hepatic expression of genes regulating liver damage was assessed by real-time polymerase chain reaction in 52 patients. The rs738409 GG genotype was more prevalent in patients than in controls (14% versus 3%, adjusted odds ratio [OR] = 3.29, 95% confidence interval [CI] = 1.8-6.9), and in the family study, the G allele was overtransmitted to affected children (P = 0.001). In Italian and United Kingdom patients, adiponutrin genotype influenced alanine aminotransferase levels and the severity of steatosis. Adiponutrin genotype was associated with the expression of genes involved in the steatosis-related liver damage, including the proapoptotic molecule Fas ligand. In the whole series combined, adiponutrin genotype was associated with steatosis grade >1 (OR = 1.35, 95% CI = 1.04-1.76), nonalcoholic steatohepatitis (OR = 1.5, 95% CI = 1.12-2.04), and fibrosis stage >1 (OR = 1.5, 95% CI = 1.09-2.12), independent of age, body mass index, and diabetes. Adiponutrin genotype demonstrated a dose effect with heterozygote risk intermediate between CC and GG homozygotes., Conclusion: In patients with NAFLD, adiponutrin rs738409 C-->G genotype, encoding for I148M, is associated with the severity of steatosis and fibrosis and the presence of nonalcoholic steatohepatitis.

Background & Aims: Mutations in the hemochromatosis gene (HFE) (C282Y and H63D) lead to parenchymal iron accumulation, hemochromatosis, and liver damage. We investigated whether these factors also contribute to the progression of fibrosis in patients with nonalcoholic fatty liver disease (NAFLD)., Methods: We studied clinical, histologic (liver biopsy samples for hepatocellular iron accumulation), serologic (iron and enzyme levels), and genetic (HFE genotype) data from 587 patients from Italy with NAFLD and 184 control subjects., Results: Iron accumulation predominantly in hepatocyes was associated with a 1.7-fold higher risk of a fibrosis stage greater than 1 (95% confidence interval [CI]: 1.2-2.3), compared with the absence of siderosis (after adjustment for age, body mass index, glucose tolerance status, and alanine aminotransferase level). Nonparenchymal/mixed siderosis was not associated with moderate/severe fibrosis (odds ratio, 0.72; 95% CI: 0.50-1.01). Hepatocellular siderosis was more prevalent in patients with HFE mutations than in those without; approximately one third of patients with HFE mutations had parenchymal iron accumulation (range, 29.8%-35.7%, depending on HFE genotype). Predominantly hepatocellular iron accumulation occurred in 52.7% of cases of patients with HFE mutations. There was no significant association between either the presence of HFE mutations or specific HFE genotypes and the severity of liver fibrosis., Conclusions: Iron deposition predominantly in hepatocyes is associated with more severe liver damage in patients with NAFLD. However, HFE mutations cannot be used to identify patients with hepatocellular iron accumulation., (Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Chronic hepatitis C (CHC) has been associated with type 2 diabetes and insulin resistance, but the extent of impairment in insulin action, the target pathways involved, and the role of the virus per se have not been defined. In this study, we performed a euglycemic hyperinsulinemic clamp (1 mU x minute(-1) x kg(-1)) coupled with infusion of tracers ([6,6-(2)H(2)]glucose, [(2)H(5)]glycerol) and indirect calorimetry in 14 patients with biopsy-proven CHC, who were selected not to have any features of the metabolic syndrome, and in seven healthy controls. We also measured liver expression of inflammatory cytokines/mediators and tested their association with the metabolic parameters. Compared to controls, in patients with CHC: (1) total glucose disposal (TGD) during the clamp was 25% lower (P = 0.003) due to impaired glucose oxidation (P = 0.0002), (2) basal endogenous glucose production (EGP) was 20% higher (P = 0.011) and its suppression during the clamp was markedly reduced (P = 0.007), and (3) glycerol appearance was not different in the basal state or during the clamp, but lipid oxidation was less suppressed by insulin (P = 0.004). Lipid oxidation was higher in patients with CHC who had more steatosis and was directly related to EGP, TGD, and glucose oxidation. The decreased insulin-stimulated suppression of EGP was associated with increased hepatic suppressor of cytokine signaling 3 (SOCS3; P < 0.05) and interleukin-18 (P < 0.05) expression., Conclusion: Hepatitis C infection per se is associated with peripheral and hepatic insulin resistance. Substrate competition by increased lipid oxidation and possibly enhanced hepatic expression of inflammatory cytokines/mediators could be involved in the defective glucose regulation.

Unlabelled: It is uncertain whether patients with nonalcoholic fatty liver disease (NAFLD) and normal alanine aminotransferase (ALT) have a milder disease and should undergo liver biopsy. We reviewed the histological data of 458 Italian patients with NAFLD in whom liver biopsy was indicated by altered liver enzymes (395 cases, 86%), or persistently elevated ferritin or long-lasting severe steatosis (63 cases). Factors associated with nonalcoholic steatohepatitis (NASH) and fibrosis >/= 2 were identified by multivariate analysis. Patients with normal ALT were significantly older, had lower body mass index, fasting triglycerides, insulin resistance according to homeostasis model assessment (HOMA-IR), ALT, and gamma-glutamyltransferase, but a higher prevalence of hypertension. NASH was diagnosed in 59% and 74% of the patients with normal and increased ALT, respectively (P = 0.01). In the overall series of patients, NASH was independently predicted by ALT (odds ratio [OR], 1.11; 95% confidence interval [CI], 1.04-1.19 per 10-IU/mL increase) and diabetes (OR, 1.5; 95% CI, 1.1-2.0). The same variables were selected in patients with increased ALT, whereas in those with normal ALT, HOMA-IR and ALT were independent predictors. Severe fibrosis was independently predicted by serum ferritin (OR, 1.04; 95% CI, 1.001-1.08 per 50-ng/mL increase), ALT (OR, 1.07; 95% CI, 1.02-1.14), and diabetes (OR, 1.8; 95% CI, 1.4-2.3) in the overall series, serum ferritin and diabetes in those with increased ALT, and only HOMA-IR (OR, 1.97; 95% CI, 1.2-3.7) in patients with normal ALT., Conclusion: Normal ALT is not a valuable criterion to exclude patients from liver biopsy. Alterations in glucose metabolism and insulin resistance in subjects with normal ALT should also be considered in the selection of NAFLD cases for histological assessment of disease severity and progression.

The risk of chronic liver disease and liver-related mortality is increased in patients with type 2 diabetes mellitus. Several cohort studies have suggested a metabolic pathway from nonalcoholic fatty liver, nonalcoholic steatohepatitis, cryptogenic cirrhosis, and eventually hepatocellular carcinoma. Although cardiovascular risk remains the major cause for excess mortality in type 2 diabetes mellitus, the risk of progressive liver disease should no longer be underscored.

Objectives: Hyperferritinemia is frequently observed in nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome characterized by hepatic insulin resistance and considered high cardiovascular risk. Iron depletion by phlebotomy has been reported to decrease insulin resistance in NAFLD in small, uncontrolled studies. Aims of this study were to define the relationship between ferritin and iron stores in patients with NAFLD, the effect of iron depletion on insulin resistance, and whether basal ferritin levels influence treatment outcome., Methods: Subjects were included if ferritin and/or ALT were persistently elevated after 4 months of standard therapy. Sixty-four phlebotomized subjects were matched 1:1 for age, sex, ferritin, obesity, and ALT levels with patients who underwent lifestyle modifications only. Insulin resistance was evaluated by insulin levels, determined by RIA and the HOMA-R index, at baseline and after 8 months., Results: Baseline ferritin levels were associated with body iron stores (P<0.0001). Iron depletion produced a significantly larger decrease in insulin resistance (P=0.0016 for insulin, P=0.0042 for HOMA-R) compared with nutritional counseling alone, independent of changes in BMI, baseline HOMA-R, and the presence of the metabolic syndrome. Iron depletion was more effective in reducing HOMA-R in patients in the top two tertiles of ferritin concentrations (P<0.05 vs controls), and in carriers of the mutations in the HFE gene of hereditary hemochromatosis (P<0.05 vs noncarriers)., Conclusions: Given that phlebotomy reduces insulin resistance, which is associated with liver tissue damage, future studies should evaluate the effect of iron depletion on liver histology and cardiovascular end points.

Hepatic steatosis has been associated with fibrosis, but it is unknown whether the latter is independent of the etiology of fat infiltration. We analyzed the relationship between clinical characteristics, insulin resistance (HOMA-R) and histological parameters in 132 patients with "viral" steatosis caused by genotype 3 chronic hepatitis C (CHC-3) and 132 patients with "metabolic" steatosis caused by nonalcoholic fatty liver disease (NAFLD), matched by age, BMI, and degree of liver fat accumulation. Tests of liver function were comparable in the two study populations. The prevalence of features of insulin resistance was higher in NAFLD, as was HOMA-R (P = .008). Logistic regression analysis confirmed that steatosis was associated with a high viral load and low serum cholesterol in CHC-3, and with high aminotransferase, glucose, ferritin and hypertriglyceridemia in NAFLD. At univariate analysis, advanced fibrosis was associated with steatosis in NAFLD, but not in CHC-3. Other parameters related to fibrosis severity were HOMA-R and a low platelet count in CHC-3, and high aminotransferases, HOMA-R, ferritin and low HDL-cholesterol in NAFLD. On multivariate analysis, only low platelet count (OR = 0.78; 95% CI, 0.67-0.92) and HOMA-R (OR = 2.98; 1.13-7.89) were independent predictors of advanced fibrosis in CHC-3. In NAFLD, severe fibrosis was predicted by fat grading (OR = 3.03; 1.41-6.53), ferritin (OR = 1.13; 1.03-1.25) and HOMA-R (OR = 1.16; 1.02-1.31). In conclusion, insulin resistance is an independent predictor of advanced fibrosis in both NAFLD and CHC-3, but the extent of steatosis contributes to advanced disease only in NAFLD. Virus-induced hepatic steatosis as seen in CHC-3 does not contribute significantly to liver fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is consistently associated with features of the metabolic syndrome, a condition carrying a high risk of cardiovascular events. We measured the vasodilatory response of the brachial artery in response to ischemia (a test of endothelial function) (FMV) as well as cardiovascular risk profile in 52 NAFLD cases and 28 age- and sex-matched controls. The 10-year risk of coronary events was calculated according to the Framingham equation and the scores derived from the PROCAM study and NCEP-ATPIII proposals. FMV was 6.33% +/- 5.93% in NAFLD versus 12.22% +/- 5.05% in controls (P < .0001), and higher in pure fatty liver (9.93%) compared with nonalcoholic steatohepatitis (4.94%) (P = .010). No differences were observed in flow-independent vasodilation (response to sublingual nitroglycerin). Percent FMV was negatively associated with insulin resistance (homeostasis model assessment) in the whole population (r = -0.243; P = .030). In logistic regression analysis, NAFLD was associated with a percent FMV in the lower tertile (OR, 6.7; 95% CI, 1.26-36.1), after adjustment for age, sex, body mass index, and insulin resistance. Among NAFLD patients, low FMV was associated with nonalcoholic steatohepatitis (adjusted OR, 6.8; 95% CI, 1.2-40.2). The 10-year probability of cardiovascular events was moderately increased in NAFLD, and particularly in nonalcoholic steatohepatitis. In conclusion, our study provides evidence of endothelial dysfunction and increased risk of cardiovascular events in NAFLD. The risk of advanced liver disease is well recognized in NAFLD patients, but the large majority of cases might experience cardiovascular disease in the long term, indirectly limiting the burden of liver failure.

Plasma levels of adiponectin are decreased in patients with nonalcoholic fatty liver disease (NAFLD), but the relationship among plasma adiponectin, insulin sensitivity, and histological features is unclear. In 174 NAFLD patients and 42 controls, we examined plasma adiponectin concentrations in relation to 1) lipid profile, indices of insulin resistance, and features of the metabolic syndrome (n = 174); 2) hepatic insulin resistance (clamp technique with tracer infusion) (10 patients); and 3) histological features at liver biopsy (n = 116). When the data from all subjects were combined, plasma adiponectin levels were positively associated with increased age, female gender, and plasma high-density lipoprotein levels, and negatively associated with waist circumference, body mass index, triglycerides, indices of insulin resistance, and aminotransferase levels, and also predicted the presence of the metabolic syndrome. In step-wise regression, increased age, female gender, waist circumference, triglyceride levels, and homeostasis model assessment independently associated with adiponectin (adjusted R(2), 0.329). In NAFLD, adiponectin was only associated with increased age, female gender, and triglycerides (adjusted R(2), 0.245). When the measured histological parameters were included in the model, plasma adiponectin levels were also inversely proportional to the percentage of hepatic fat content (adjusted R(2), 0.221), whereas necroinflammation and fibrosis did not fit in the model. Adiponectin was negatively correlated with insulin-suppressed endogenous glucose production during the clamp (P = 0.011). The results demonstrate that decreased levels of circulating adiponectin in NAFLD are related to hepatic insulin sensitivity and to the amount of hepatic fat content. Hypoadiponectinemia in NAFLD is part of a metabolic disturbance characterized by ectopic fat accumulation in the central compartment.

Objectives: Metformin proved useful in the treatment of nonalcoholic fatty liver disease (NAFLD), but its superiority over nutritional treatment and antioxidants has never been demonstrated. We aimed to compare the usefulness of metformin versus prescriptive diet or vitamin E., Methods: In an open label, randomized trial, nondiabetic NAFLD patients were given metformin (2 g/day; n = 55) for 12 months. The control cases were given either vitamin E (800 IU/day; n = 28) or were treated by a prescriptive, weight-reducing diet (n = 27). Outcome measures were liver enzymes, insulin resistance (homeostasis model assessment), parameters of the metabolic syndrome, and histology., Results: Aminotransferase levels improved in all groups, in association with weight loss. The effects in the metformin arm were larger (p < 0.0001), and alanine aminotransferase normalized in 56% of cases (odds ratio (OR) versus. controls, 3.11; 95% confidence interval (CI), 1.56-6.20; p= 0.0013). In multivariate analysis, metformin treatment was associated with higher rates of aminotransferase normalization, after correction for age, gender, basal aminotransferases, and change in body mass index (OR, 5.98; 95% CI, 2.05-17.45). Differences were maintained when the two control groups were separately analyzed. The distribution of positive criteria for the metabolic syndrome was reduced only in the metformin arm (p= 0.001, signed rank test). A control biopsy in 17 metformin-treated cases (14 nonresponders) showed a significant decrease in liver fat (p= 0.0004), necroinflammation, and fibrosis (p= 0.012 for both). No side effects were observed during metformin treatment., Conclusions: Metformin treatment is better than a prescriptive diet or vitamin E in the therapy of NAFLD patients receiving nutritional counseling. Limited histological data support an association between improved aminotransferases and biopsy findings, which require confirmation in a double-blind trial with appropriate statistical power based on liver histology.

Aims/hypothesis: Non-alcoholic fatty liver disease (NAFLD) has been associated with the metabolic syndrome. However, it is not clear whether insulin resistance is an independent feature of NAFLD, and it remains to be determined which of the in vivo actions of insulin are impaired in this condition., Methods: We performed a two-step (1.5 and 6 pmol min(-1) kg(-1)) euglycaemic insulin clamp coupled with tracer infusion ([6,6-2H2]glucose and [2H5]glycerol) and indirect calorimetry in 12 non-obese, normolipidaemic, normotensive, non-diabetic patients with biopsy-proven NAFLD and six control subjects., Results: In NAFLD patients, endogenous glucose production (basal and during the clamp) was normal; however, peripheral glucose disposal was markedly decreased (by 30% and 45% at the low and high insulin doses, respectively, p<0.0001) at higher plasma insulin levels (p=0.05), due to impaired glucose oxidation (p=0.003) and glycogen synthesis (p<0.001). Compared with control subjects, glycerol appearance and lipid oxidation were significantly increased in NAFLD patients in the basal state, and were suppressed by insulin to a lesser extent (p<0.05-0.001). The lag phase of the in vitro copper-catalysed peroxidation of LDL particles was significantly shorter in the patients than in the control subjects (48+/-12 vs 63+/-13 min, p<0.04). Lipid oxidation was significantly related to endogenous glucose production, glucose disposal, the degree of hepatic steatosis, and LDL oxidisability., Conclusions/interpretation: Insulin resistance appears to be an intrinsic defect in NAFLD, with the metabolic pattern observed indicating that adipose tissue is an important site.

The role of insulin resistance in non-alcoholic fatty liver disease is suggested by laboratory data (hyperinsulinemia and decreased sensitivity to endogenous and exogenous insulin). The clinical association with features of the metabolic syndrome, particularly in the most aggressive stages of the disease, further confirms a causative role. Fat accumulation in the liver may stem either from genetic defects, primarily responsible for insulin resistance, or excessive calorie intake and visceral obesity, and is mediated by adipocytokines (leptin, adiponectin, tumour necrosis factor-alpha). Progression of fatty liver to steatohepatitis may be the result of an imbalance between pro-inflammatory and anti-inflammatory cytokines, triggering the formation of reactive oxygen species and intrahepatic lipid peroxidation. This process may also be promoted or accelerated by pro-oxidant xenobiotics or environmental factors. Insulin resistance provides a target for specific treatment of non-alcoholic fatty liver, and insulin-sensitising agents (metformin or thiazolidinediones) as well as lifestyle changes to reduce visceral adiposity are the most promising therapeutic options. Future trials need to be performed in order to test the long-term effectiveness of these treatments on the basis of clinically relevant histological outcomes.

The clinical significance of liver disease is frequently underestimated in patients with metabolic disorders. In patients followed up in a metabolic unit for diabetes, obesity or hyperlipidemia (n=147), we studied the prevalence and the severity of liver disease, and its relationship with the metabolic syndrome (MS). Cases cared for in a liver unit (n=179) were used as controls. Patients in the metabolic series were older and had a higher prevalence of coronary heart disease. Criteria for the metabolic syndrome were fulfilled in 64% and 22% of cases, respectively (P<0.0001). Liver biopsy was obtained in 44 and 66% of cases. Metabolic patients had a more severe steatosis score (P<0.0001), whereas the scores of fibrosis and necroinflammation were less severe (P=0.0059 and 0.0007, respectively). Histological criteria for non-alcoholic steatohepatitis (NASH) were present in 82% of metabolic cases and 68% cases in the liver series (P=0.057). Liver disease in patients routinely cared for in metabolic units is similar to that observed in patients cared for in liver units, and potentially may progress to terminal liver failure. Liver biopsy is recommended for diagnostic and prognostic purposes, as well as for testing treatment effects in controlled trials.

The mechanism(s) determining the progression from fatty liver to steatohepatitis is currently unknown. Our goal was to define the relative impact of iron overload, genetic mutations of HFE, and insulin resistance on the severity of liver fibrosis in a population of subjects with nonalcoholic fatty liver disease (NAFLD) who had low prevalence of obesity and no overt symptoms of diabetes. In a cohort of 263 prospectively enrolled patients with NAFLD, 7.4% of patients had signs of peripheral iron overload and 9% had signs of hepatic iron overload, but 21.1% had hyperferritinemia. The prevalence of C282Y and H63D HFE mutations was similar to the general population and mutations were not associated with iron overload. Although subjects were on average only moderately overweight, insulin sensitivity, measured both in the fasting state and in response to oral glucose, was lower. Univariate analysis demonstrated that the presence of severe fibrosis was independently associated with older age, female sex, overweight, aspartate/alanine aminotransferase ratio, serum ferritin level, fasting glucose and insulin levels, decreased insulin sensitivity, and with histologic features (degree of necroinflammation and steatosis). After adjustment for body mass index (BMI), age, sex, and degree of steatosis, ferritin levels (odds ratio [OR] = 1.77; 95% CI = 1.21- 2.58; P =.0032) and the oral glucose insulin sensitivity (OR = 0.53; CI = 0.33-0.87; P =.0113) were independent predictors of severe fibrosis. In conclusion, the current study indicates that insulin resistance is a major, independent risk factor for advanced fibrosis in patients with NAFLD. Increased ferritin levels are markers of severe histologic damage, but not of iron overload. Iron burden and HFE mutations do not contribute significantly to hepatic fibrosis in the majority of patients with NAFLD.

Several physiological and pathophysiological conditions, including changes in body fat, food intake, and insulin resistance, are known to be associated with variations in plasma ghrelin concentrations. We tested the hypothesis that insulin resistance exerts a primary role by measuring ghrelin in 86 patients with nonalcoholic fatty liver disease (NAFLD), a condition in which insulin resistance is relatively independent of obesity. Compared with 40 matched healthy subjects, patients with NAFLD had similar glucose levels and higher plasma insulin and insulin resistance [homeostasis model assessment (HOMA)-R index] by over 60%. Ghrelin was reduced (mean +/- SD, 226 +/- 72 pmol/liter in NAFLD vs. 303 +/- 123 in controls; P < 0.0001). In relation to quartiles of body mass index, ghrelin progressively decreased in controls (P = 0.003), but not in patients (P = 0.926). In relation to quartiles of HOMA-R, ghrelin decreased in both groups, and significantly correlated with HOMA-R. After adjustment for age and sex, HOMA-R was the sole factor significantly associated with low ghrelin in the whole group (odds ratio, 5.79; 95% confidence interval, 2.62-12.81; P < 0.0001) and specifically in NAFLD (2.96; 1.12-7.79; P = 0.028). The study suggests that insulin resistance is a major factor controlling ghrelin levels in subjects with and without NAFLD.

This is a report of a combined experience of a nephrologist, who had been previously working for many years in a public hospital, and a business planning manager coming from a financial administration department of a multinational company. The Istituto Clinico Humanitas is a multi-specialist hospital accreditated with the National Health System, containing 473 beds available: 398 inpatient, 28 intensive care and 47 day hospital beds. Human resources include approximately 250 medical doctors, 500 nurses and ancillary workers. In 2002 the hospital treated about 17,500 inpatients, 18,450 day hospital patients, nearly 18,600 surgical interventions and 470,000 outpatients examinations and tests were performed. The detailed clinical activity of the general medicine and the nephrology units in 2002, the role of the scientific direction and that of the Humanitas Foundation have been presented. The work experience, in particular the advantages and disavantages of public versus private environment, represents another important topic of this paper.

Nonalcoholic fatty liver disease (NAFLD) has been associated with the insulin-resistance syndrome, at present defined as the metabolic syndrome, whose limits were recently set. We assessed the prevalence of the metabolic syndrome in 304 consecutive NAFLD patients without overt diabetes, on the basis of 3 or more criteria out of 5 defined by the U.S. National Institutes of Health (waist circumference, glucose, high-density lipoprotein [HDL]-cholesterol, triglycerides, and arterial pressure). The prevalence of the metabolic syndrome increased with increasing body mass index, from 18% in normal-weight subjects to 67% in obesity. Insulin resistance (Homeostasis Model Assessment method) was significantly associated with the metabolic syndrome (odds ratio [OR], 2.5; 95% CI, 1.5-4.2; P <.001). Liver biopsy was available in 163 cases (54%). A total of 120 patients (73.6%) were classified as having nonalcoholic steatohepatitis (NASH); 88% of them had a metabolic syndrome (vs. 53% of patients with pure fatty liver; P <.0001). Logistic regression analysis confirmed that the presence of metabolic syndrome carried a high risk of NASH among NAFLD subjects (OR, 3.2; 95% CI, 1.2-8.9; P =.026) after correction for sex, age, and body mass. In particular, the syndrome was associated with a high risk of severe fibrosis (OR, 3.5; 95% CI, 1.1-11.2; P =.032). In conclusion, the presence of multiple metabolic disorders is associated with a potentially progressive, severe liver disease. The increasing prevalence of obesity, coupled with diabetes, dyslipidemia, hypertension, and ultimately the metabolic syndrome puts a very large population at risk of forthcoming liver failure in the next decades.

Background & Aims: Nonalcoholic steatohepatitis (NASH) may progress to cirrhosis; whether NASH plays also a role in the development of hepatocellular carcinoma (HCC) is unknown., Methods: Among 641 cirrhosis-associated HCCs, we retrospectively identified 44 patients with cryptogenic cirrhosis (CC). Of these, 23 were actively followed up and were compared in a case-control study with viral- and alcohol-associated HCC. Family and personal history of diabetes, hypertension, coronary heart disease, dyslipidemia, obesity, and biochemical data were compared between groups. Iron status and presence of mutations in the HFE gene of familiar hemochromatosis were also determined., Results: Family history was not different in relation to etiology. The prevalence of obesity and diabetes was significantly higher in patients with CC. Although liver function was similar, CC patients had higher glucose, cholesterol, and triglyceride plasma levels, increased parameters of insulin resistance, and lower aminotransferase levels. Iron status and prevalence of mutations in the HFE gene did not differ. Logistic regression analysis identified in sequence hypertriglyceridemia, diabetes, and normal aminotransferases as independent factors associated with HCC arising in CC., Conclusions: Features suggestive of NASH are more frequently observed in HCC arising in patients with CC than in age- and sex-matched HCC patients of well-defined viral or alcoholic etiology. HCC may represent a late complication of NASH-related cirrhosis.