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Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Medicina, Endocrinologia, Metabolismo e Geriatria

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2019 - A round trip from nonalcoholic fatty liver disease to diabetes: molecular targets to the rescue? [Articolo su rivista]
Lonardo, Amedeo; Lugari, Simonetta; Ballestri, Stefano; Nascimbeni, Fabio; Baldelli, Enrica; Maurantonio, Mauro

Evidence suggests a close relationship between nonalcoholic fatty liver disease (NAFLD) and type two diabetes (T2D). On the grounds of prevalence of disease, both conditions account for a significant financial cost for health care systems and individuals. Aim of this review article is to explore the epidemiological basis and the putative molecular mechanisms underlying the association of NAFLD with T2D. Epidemiological studies have shown that NAFLD is associated to the development of incident T2D and either reversal or improvement of NAFLD will result into decreased risk of developing incident T2D. On the other side of the coin data have shown that T2D will worsen the course of NAFLD doubling the risk of disease progression (i.e. evolution from simple steatosis to advanced fibrosis, cirrhosis, hepatocellular carcinoma, liver transplant and death). Conversely, NAFLD will contribute to metabolic decompensation of T2D. The pathogenesis of T2D in NAFLD patients may be mediated by several hepatokines impairing metabolic control. Among these, Fetuin-B, which causes glucose intolerance and is increased in patients with T2D and NAFLD with fibrosis is one of the most promising. T2D may affect the progression of NAFLD by acting at different levels of the pathogenic cascade involving gut microbiota and expanded, inflamed, dysfunctional adipose tissue. In conclusion, T2D and NAFLD are mutually, closely and bi-directionally associated. An improved understanding of molecular pathogenesis underlying this bi-directional association may allow us to be able to prevent the development of T2D by halting the progression of NAFLD.

2018 - Do Nonalcoholic Fatty Liver Disease and Fetuin-A Play Different Roles in Symptomatic Coronary Artery Disease and Peripheral Arterial Disease? [Articolo su rivista]
Nascimbeni, Fabio; Romagnoli, Dante; Ballestri, Stefano; Baldelli, Enrica; Lugari, Simonetta; Sirotti, Valentina; Giampaoli, Valentina; Lonardo, Amedeo

Nonalcoholic fatty liver disease (NAFLD) is strongly associated with both atherosclerotic cardiovascular disease (CVD) and Fetuin-A. However, the association of Fetuin-A with atherosclerosis is more controversial. We hypothesized that the pathogenic interplay of NAFLD, Fetuin-A and atherosclerosis varies based on arterial site. Accordingly, we aimed to assess NAFLD prevalence, Fetuin-A values and their relationship with symptomatic atherosclerosis occurring in different localizations: coronary artery disease (CAD) vs. peripheral arterial disease (PAD).

2017 - Clinical features and natural history of cryptogenic cirrhosis compared to hepatitis C virus-related cirrhosis [Articolo su rivista]
Rinaldi, Luca; Nascimbeni, Fabio; Giordano, Mauro; Masetti, Chiara; Guerrera, Barbara; Amelia, Annalisa; Fascione, Maria Chiara; Ballestri, Stefano; Romagnoli, Dante; Zampino, Rosa; Nevola, Riccardo; Baldelli, Enrica; Iuliano, Natalina; Rosato, Valerio; Lonardo, Amedeo; Adinolfi, Luigi Elio

AIM To characterize natural history of cryptogenic cirrhosis (CC) and compare its clinical features and outcomes to those of hepatitis C virus (HCV)-related cirrhosis. METHODS A prospective cohort of 102 consecutive patients at their first diagnosis of CC were enrolled in this study. The clinical data and outcomes were compared to an age-and Child-Pugh class-matched cohort of 110 patients with HCV-related cirrhosis. Diagnosis of cirrhosis was based on compatible clinical and laboratory parameters, ultrasound/endoscopic parameters and, whenever possible, on histological grounds and transient elastography. All cases of cirrhosis without a definite etiology were enrolled in the CC group. The parameters assessed were: (1) severity of liver disease at the time of first diagnosis; (2) liver decompensation during follow-up; (3) hepatocellular carcinoma (HCC); (4) orthotopic liver transplantation; and (5) death. The independent associated factors were evaluated by multiple logistic regression analysis, and survival and its determinants by the Kaplan-Meier model, log-rank test and Cox regression. RESULTS At the first observation, median age was 66 and 65 years and male gender was 36% and 58% for CC and HCV cirrhosis, respectively. CC showed Child-Pugh class A/B/C of 47%/31%/22%, respectively. Compared to HCV cirrhosis, CC exhibited a significantly higher prevalence of metabolic syndrome (12% vs 54%, respectively), overweight/obesity, high BMI, impaired glucose tolerance, high blood pressure, dyslipidemia, hyperuricemia, cardiovascular diseases, extrahepatic cancer, and gallstones. Over a median period of 42 mo of follow-up, liver decompensation, HCC development and death for CC and HCV-related cirrhosis were 60.8%, and 54.4%, 16.7% and 17.2%, 39.2% and 30%, respectively. The median survival was 60 mo for CC. Independent predictors of death were age and Child-Pugh class at diagnosis. CC showed an approximately twofold higher incidence of HCC in Child-Pugh class A. CONCLUSION Undiagnosed nonalcoholic fatty liver disease has an etiologic role in CC that is associated with a poor prognosis, early HCC development, high risk of cardiovascular disease and extrahepatic cancer.

2017 - Multiclass HCV resistance to direct-acting antiviral failure in real-life patients advocates for tailored second-line therapies [Articolo su rivista]
Di Maio, Velia C.; Cento, Valeria; Lenci, Ilaria; Aragri, Marianna; Rossi, Piera; Barbaliscia, Silvia; Melis, Michela; Verucchi, Gabriella; Magni, Carlo F.; Teti, Elisabetta; Bertoli, Ada; Antonucci, Francescopaolo; Bellocchi, Maria C.; Micheli, Valeria; Masetti, Chiara; Landonio, Simona; Francioso, Simona; Santopaolo, Francesco; Pellicelli, Adriano M.; Calvaruso, Vincenza; Gianserra, Laura; Siciliano, Massimo; Romagnoli, Dante; Cozzolongo, Raffaele; Grieco, Antonio; Vecchiet, Jacopo; Morisco, Filomena; Merli, Manuela; Brancaccio, Giuseppina; Di Biagio, Antonio; Loggi, Elisabetta; Mastroianni, Claudio M.; Pace Palitti, Valeria; Tarquini, Pierluigi; Puoti, Massimo; Taliani, Gloria; Sarmati, Loredana; Picciotto, Antonino; Vullo, Vincenzo; Caporaso, Nicola; Paoloni, Maurizio; Pasquazzi, Caterina; Rizzardini, Giuliano; Parruti, Giustino; Craxì, Antonio; Babudieri, Sergio; Andreoni, Massimo; Angelico, Mario; Perno, Carlo F.; Ceccherini-Silberstein, Francesca; MARIANI COSTANTINI, Renato; Iapadre, N.; Grimaldi, A.; Cozzolongo, R.; Andreone, P.; Verucchi, G.; Menzaghi, B.; Quirino, T.; Pisani, Vincenzo; Torti, C.; Vecchiet, J.; Bruzzone, B.; De Maria, A.; Marenco, S.; Nicolini, L. A.; Viscoli, C.; Casinelli, K.; Delle Monache, M.; Lichtner, M.; Aghemo, A.; Boccaccio, V.; Bruno, S.; Cerrone, M.; Colombo, M.; D'Arminio Monforte, A.; Danieli, E.; Donato, Francesco; Gubertini, G.; Lleo, A.; Magni, C. F.; Mancon, A.; Monico, S.; Niero, F.; Russo, M. L.; Gnocchi, M.; Orro, A.; Milanesi, L.; Baldelli, E.; Bertolotti, M.; Borghi, V.; Mussini, C.; Brancaccio, Giulia; Gaeta, G. B.; Lembo, V.; Sangiovanni, V.; Di Marco, V.; Mazzola, A.; Petta, S.; D'Amico, maria ester; Cacciatore, P.; Consorte, A.; Pieri, A.; Polilli, E.; Sozio, F.; Antenucci, Francesca; Aragri, M.; Baiocchi, L.; Barbaliscia, S.; Biliotti, E.; Biolato, M.; Carioti, L.; Ceccherini-Silberstein, F.; Cerasari, G.; Cerva, C.; Ciotti, M.; D'Ambrosio, C.; D'Ettorre, G.; De Leonardis, F.; De Sanctis, A.; Di Maio, V. C.; Di Paolo, D.; Furlan, C.; Gallo, P.; Gasbarrini, A.; Giannelli, V.; Grieco, S.; Lambiase, L.; Lattanzi, B.; Lenci, I.; Lula, R.; Malagnino, V.; Manuelli, M.; Miglioresi, L.; Milana, M.; Moretti, A.; Nosotti, L.; Palazzo, D.; Pellicelli, A.; Romano, M.; Sarrecchia, C.; Sforza, Diego; Sorbo, M. C.; Spaziante, M.; Svicher, V.; Tisone, G.; Vespasiani-Gentilucci, U.; D'Adamo, G.; Mangia, A.; Maida, I.; Mura, M. S.; Falconi, L.; Di Giammartino, D.

Background & Aims: Despite the excellent efficacy of direct-acting antivirals (DAA) reported in clinical trials, virological failures can occur, often associated with the development of resistance-associated substitutions (RASs). This study aimed to characterize the presence of clinically relevant RASs to all classes in real-life DAA failures. Methods: Of the 200 virological failures that were analyzed in 197 DAA-treated patients, 89 with pegylated-interferon+ribavirin (PegIFN+RBV) and 111 without (HCV-1a/1b/1g/2/3/4=58/83/1/6/24/25; 56.8% treatment experienced; 65.5% cirrhotic) were observed. Sanger sequencing of NS3/NS5A/NS5B was performed by home-made protocols, at failure (N=200) and whenever possible at baseline (N=70). Results: The majority of the virological failures were relapsers (57.0%), 22.5% breakthroughs, 20.5% non-responders. RAS prevalence varied according to IFN/RBV use, DAA class, failure type and HCV genotype/subtype. It was 73.0% in IFN group vs 49.5% in IFN free, with the highest prevalence of NS5A-RASs (96.1%), compared to NS3-RASs (75.9% with IFN, 70.5% without) and NS5B-RASs (66.6% with IFN, 20.4% without, in sofosbuvir failures). In the IFN-free group, RASs were higher in breakthrough/non-responders than in relapsers (90.5% vs 40.0%, P<.001). Interestingly, 57.1% of DAA IFN-free non-responders had a misclassified genotype, and 3/4 sofosbuvir breakthroughs showed the major-RAS-S282T, while RAS-L159F was frequently found in sofosbuvir relapsers (18.2%). Notably, 9.0% of patients showed also extra target RASs, and 47.4% of patients treated with ≥2 DAA classes showed multiclass resistance, including 11/11 NS3+NS5A failures. Furthermore, 20.0% of patients had baseline-RASs, which were always confirmed at failure. Conclusions: In our failure setting, RAS prevalence was remarkably high in all genes, with a partial exception for NS5B, whose limited resistance is still higher than previously reported. This multiclass resistance advocates for HCV resistance testing at failure, in all three genes for the best second-line therapeutic tailoring.

2017 - NAFLD as a Sexual Dimorphic Disease: Role of Gender and Reproductive Status in the Development and Progression of Nonalcoholic Fatty Liver Disease and Inherent Cardiovascular Risk [Articolo su rivista]
Ballestri, Stefano; Nascimbeni, Fabio; Baldelli, Enrica; Marrazzo, Alessandra; Romagnoli, Dante; Lonardo, Amedeo

Nonalcoholic fatty liver disease (NAFLD) spans steatosis through nonalcoholic steatohepatis, cirrhosis, and hepatocellular carcinoma (HCC) associated with striking systemic features and excess cardiovascular and liver-related mortality. The pathogenesis of NAFLD is complex and multifactorial. Endocrine derangements are closely linked with dysmetabolic traits. For example, in animal and human studies, female sex is protected from dysmetabolism thanks to young individuals’ ability to partition fatty acids towards ketone body production rather than very low density lipoprotein (VLDL)-triacylglycerol, and to sex-specific browning of white adipose tissue. Ovarian senescence facilitates both the development of massive hepatic steatosis and the fibrotic progression of liver disease in an experimental overfed zebrafish model. Consistently, estrogen deficiency, by potentiating hepatic inflammatory changes, hastens the progression of disease in a dietary model of nonalcoholic steatohepatitis (NASH) developing in ovariectomized mice fed a high-fat diet. In humans, NAFLD more often affects men; and premenopausal women are equally protected from developing NAFLD as they are from cardiovascular disease. It would be expected that early menarche, definitely associated with estrogen activation, would produce protection against the risk of NAFLD. Nevertheless, it has been suggested that early menarche may confer an increased risk of NAFLD in adulthood, excess adiposity being the primary culprit of this association. Fertile age may be associated with more severe hepatocyte injury and inflammation, but also with a decreased risk of liver fibrosis compared to men and postmenopausal status. Later in life, ovarian senescence is strongly associated with severe steatosis and fibrosing NASH, which may occur in postmenopausal women. Estrogen deficiency is deemed to be responsible for these findings via the development of postmenopausal metabolic syndrome. Estrogen supplementation may at least theoretically protect from NAFLD development and progression, as suggested by some studies exploring the effect of hormonal replacement therapy on postmenopausal women, but the variable impact of different sex hormones in NAFLD (i.e., the pro-inflammatory effect of progesterone) should be carefully considered.

2017 - Ultrasonographic fatty liver indicator detects mild steatosis and correlates with metabolic/histological parameters in various liver diseases [Articolo su rivista]
Ballestri, Stefano; Nascimbeni, Fabio; Baldelli, Enrica; Marrazzo, Alessandra; Romagnoli, Dante; Targher, Giovanni; Lonardo, Amedeo

Background and aims Fatty liver is a common feature of different types of liver diseases. The sensitivity and specificity of ultrasonography for diagnosing fatty liver are variable. A semi-quantitative ultrasound score, i.e., the ultrasonographic fatty liver indicator (US-FLI), is closely associated with metabolic/histological variables in patients with nonalcoholic fatty liver disease (NAFLD). The main aims of this study were to assess the diagnostic performance of US-FLI in detecting varying degrees of histological steatosis, and to examine the association of US-FLI with metabolic/histological parameters in 352 biopsied patients with various chronic liver diseases (173 with hepatitis C [HCV], 23 with hepatitis B [HBV], 123 with NAFLD and 33 with other etiologies). Results US-FLI accurately detected mild steatosis (minimum amount 10% on histology) with a cut-off value ≥ 2 (sensitivity 90.1%, specificity 90%), moderate steatosis (≥ 30%) with a cut-off value ≥ 3 (sensitivity 86.4%, specificity 92.5%) and severe steatosis (> 66%) with a cut-off value ≥ 5 (sensitivity 88.5%, specificity 87%). US-FLI was correlated with steatosis percentage in each liver disease group as well as with lobular inflammation, ballooning, portal fibrosis, grading and staging in patients with NAFLD or HCV. US-FLI was also correlated with waist circumference, body mass index and insulin resistance both in the whole sample and in each liver disease group. Conclusions US-FLI accurately identifies histological severity and is correlated with metabolic parameters in patients with various steatogenic liver diseases. US-FLI is an easy and versatile tool for the screening of steatosis and the metabolic health of these patients.

2016 - HCV NS3 sequencing as a reliable and clinically useful tool for the assessment of genotype and resistance mutations for clinical samples with different HCV-RNA levels [Articolo su rivista]
Di Maio, V. C.; Cento, V.; Di Paolo, D.; Aragri, M.; De Leonardis, F.; Tontodonati, M.; Micheli, V.; Bellocchi, M. C.; Antonucci, F. P.; Bertoli, A.; Lenci, I.; Milana, M.; Gianserra, L.; Melis, M.; Di Biagio, A.; Sarrecchia, C.; Sarmati, L.; Landonio, S.; Francioso, S.; Lambiase, L.; Nicolini, L. A.; Marenco, S.; Nosotti, L.; Giannelli, V.; Siciliano, M.; Romagnoli, Dante; Pellicelli, A.; Vecchiet, J.; Magni, C. F.; Babudieri, S.; Mura, M. S.; Taliani, G.; Mastroianni, C.; Vespasiani Gentilucci, U.; Romano, M.; Morisco, F.; Gasbarrini, A.; Vullo, V.; Bruno, S.; Baiguera, C.; Pasquazzi, C.; Tisone, G.; Picciotto, A.; Andreoni, M.; Parruti, G.; Rizzardini, G.; Angelico, M.; Perno, C. F.; Ceccherini Silberstein, F; Mariani, R.; Paoloni, M.; Iapadre, N.; Grimaldi, A.; Menzaghi, B.; Quirino, T.; Vecchiet, J.; Bruzzone, B.; De Maria, A.; Di Biagio, A.; Marenco, S.; Picciotto, A.; Viscoli, C.; Casinelli, K.; Delle Monache, M.; Lichtner, M.; Mastroianni, C.; Aghemo, A.; Bruno, S.; Cerrone, M.; Colombo, M.; D'Arminio Monforte, A.; Danieli, E.; Donato, F.; Gubertini, G.; Landonio, S.; Magni, C. F.; Mancon, A.; Micheli, V.; Monico, S.; Niero, F.; Puoti, M.; Rizzardini, G.; Russo, M. L.; Alfieri, R.; Gnocchi, M.; Orro, A.; Milanesi, L.; Baldelli, Enrica; Bertolotti, Marco; Borghi, Valentina; Mussini, C.; Romagnoli, D.; Brancaccio, G.; Caporaso, N.; Gaeta, G. B.; Lembo, V.; Morisco, F.; Calvaruso, V.; Craxí, A.; Di Marco, V.; Mazzola, A.; Petta, S.; D'Amico, E.; Cacciatore, P.; Consorte, A.; Pace Palitti, V.; Parruti, G.; Pieri, A.; Polilli, E.; Tontodonati, M.; Andreoni, M.; Angelico, M.; Antenucci, F.; Antonucci, F. P.; Aragri, M.; Armenia, D.; Baiocchi, L.; Bellocchi, M.; Biliotti, E.; Biolato, M.; Carioti, L.; Ceccherini Silberstein, F.; Cento, V.; Cerasari, G.; Cerva, C.; Ciotti, M.; D'Ambrosio, C.; D'Ettorre, G.; De Leonardis, F.; De Sanctis, A.; Di Maio, V. C.; Di Paolo, D.; Francioso, S.; Furlan, C.; Gallo, P.; Gasbarrini, A.; Giannelli, V.; Gianserra, L.; Grieco, A.; Grieco, S.; Lambiase, L.; Lattanzi, B.; Lenci, I.; Malagnino, V.; Manuelli, M.; Merli, M.; Miglioresi, L.; Milana, M.; Nosotti, L.; Palazzo, D.; Pasquazzi, C.; Pellicelli, A.; Perno, C. F.; Romano, M.; Santopaolo, F.; Santoro, M. M.; Sarmati, L.; Sarrecchia, C.; Sforza, D.; Siciliano, M.; Sorbo, M. C.; Spaziante, M.; Svicher, V.; Taliani, G.; Teti, E.; Tisone, G.; Vullo, V.; Mangia, A.; Babudieri, S.; Maida, I.; Melis, M.; Mura, M. S.; Falconi, L.; Di Giammartino, D.; Tarquini, P.

Objectives: This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen. Methods: NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays. Results: Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA > 3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤ 3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11). Conclusions: HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: A correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.

2016 - Nonalcoholic fatty liver disease is associated with an almost twofold increased risk of incident type 2 diabetes and metabolic syndrome. Evidence from a systematic review and meta-analysis [Articolo su rivista]
Ballestri, Stefano; Zona, Stefano; Targher, Giovanni; Romagnoli, Dante; Baldelli, Enrica; Nascimbeni, Fabio; Roverato, Alberto; Guaraldi, Giovanni; Lonardo, Amedeo

Background and Aim:: The magnitude of the risk of incident type 2 diabetes (T2D) and metabolic syndrome (MetS) among patients with nonalcoholic fatty liver disease (NAFLD) is poorly known. We gauged the risk of developing T2D and MetS in patients with NAFLD diagnosed by either serum liver enzymes (aminotransferases or gamma-glutamyltransferase [GGT]) or ultrasonography. Methods:: Pertinent prospective studies were identified through extensive electronic database research, and studies fulfilling enrolment criteria were included in the meta-analysis. Results: Overall, in a pooled population of 117020 patients (from 20 studies), who were followed-up for a median period of 5years (range: 3-14.7years), NAFLD was associated with an increased risk of incident T2D with a pooled relative risk of 1.97 (95% confidence interval [CI], 1.80-2.15) for alanine aminotransferase, 1.58 (95% CI, 1.43-1.74) for aspartate aminotransferase, 1.86 (95% CI, 1.71-2.03) for GGT (last vs first quartile or quintile), and 1.86 (95% CI, 1.76-1.95) for ultrasonography, respectively. Overall, in a pooled population of 81411 patients (from eight studies) who were followed-up for a median period of 4.5years (range: 3-11years), NAFLD was associated with an increased risk of incident MetS with a pooled relative risk of 1.80 (95% CI, 1.72-1.89) for alanine aminotransferase (last vs first quartile or quintile), 1.98 (95% CI, 1.89-2.07) for GGT, and 3.22 (95% CI, 3.05-3.41) for ultrasonography, respectively. Conclusions:: Nonalcoholic fatty liver disease, as diagnosed by either liver enzymes or ultrasonography, significantly increases the risk of incident T2D and MetS over a median 5-year follow-up.

2016 - Telomere length elongation after weight loss intervention in obese adults [Articolo su rivista]
Carulli, Lucia; Anzivino, Claudia; Baldelli, Enrica; Zenobii, Mf; Rocchi, Mb; Bertolotti, Marco

INTRODUCTION: Telomeres may be considered markers of biological aging, shorter telomere length is associated with some age-related diseases; in several studies short telomere length has also been associated to obesity in adults and adolescents. However the relationship between telomere complex functions and obesity is still not clear. Aim of the study was to assess telomere length (TL) in adults' obese subjects before and after weight loss obtained by placement of bioenteric intragastric balloon (BIB) for 6months. METHODS: We enrolled 42 obese subjects before and after BIB placement as weight loss intervention. Blood samples were collected in order to obtain DNA from leukocyte to measure TL by quantitative PCR. RESULTS: Data were analyzed only in 37 subjects with complete data; all presented important body weight loss (124.06±26.7 vs 105.40±23.14, p<0.001) and more interesting they presented a significant increase in TL (3.58±0.83 vs 5.61±3.29, p<0.001). Moreover we observed a significant positive correlation between TL elongation and weight loss (r=0.44, p=0.007) as well as an inverse correlation between TL at baseline and TL elongation (r=-0.35, p=0.03).The predictors of TL elongation were once again weight loss and short TL at baseline (respectively p=0.007 and p=0.003). CONCLUSIONS: Our study shows that weight loss is associated to telomere lengthening in a positive correlation: the greater weight loss the greater telomere lengthening; moreover telomere lengthening is more significant in those subjects with shortest telomeres at baseline.

2016 - The Role of Nuclear Receptors in the Pathophysiology, Natural Course, and Drug Treatment of NAFLD in Humans [Articolo su rivista]
Ballestri, Stefano; Nascimbeni, Fabio; Romagnoli, Dante; Baldelli, Enrica; Lonardo, Amedeo

Nonalcoholic fatty liver disease (NAFLD) describes steatosis, nonalcoholic steatohepatitis with or without fibrosis, and hepatocellular carcinoma, namely the entire alcohol-like spectrum of liver disease though observed in the nonalcoholic, dysmetabolic, individual free of competing causes of liver disease. NAFLD, which is a major public health issue, exhibits intrahepatic triglyceride storage giving rise to lipotoxicity. Nuclear receptors (NRs) are transcriptional factors which, activated by ligands, are master regulators of metabolism and also have intricate connections with circadian control accounting for cyclical patterns in the metabolic fate of nutrients. Several transcription factors, such as peroxisome proliferator-activated receptors, liver X receptors, farnesoid X receptors, and their molecular cascades, finely regulate energetic fluxes and metabolic pathways. Dysregulation of such pathways is heavily implicated in those metabolic derangements characterizing insulin resistance and metabolic syndrome and in the histogenesis of progressive NAFLD forms. We review the role of selected NRs in NAFLD pathogenesis. Secondly, we analyze the role of NRs in the natural history of human NAFLD. Next, we discuss the results observed in humans following administration of drug agonists or antagonists of the NRs pathogenically involved in NAFLD. Finally, general principles of treatment and lines of research in human NAFLD are briefly examined.

2016 - Type 2 diabetes in non-alcoholic fatty liver disease and hepatitis c virus infection—liver: The “Musketeer” in the spotlight [Articolo su rivista]
Ballestri, Stefano; Nascimbeni, Fabio; Romagnoli, Dante; Baldelli, Enrica; Targher, Giovanni; Lonardo, Amedeo

The pathogenesis of type 2 diabetes (T2D) involves chronic hyperinsulinemia due to systemic and hepatic insulin resistance (IR), which if uncorrected, will lead to progressive pancreatic beta cell failure in predisposed individuals. Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of fatty (simple steatosis and steatohepatitis) and non-fatty liver changes (NASH-cirrhosis with or without hepatocellular carcinoma (HCC)) that are commonly observed among individuals with multiple metabolic derangements, notably including visceral obesity, IR and T2D. Hepatitis C virus (HCV) infection is also often associated with both hepatic steatosis and features of a specific HCV-associated dysmetabolic syndrome. In recent years, the key role of the steatotic liver in the development of IR and T2D has been increasingly recognized. Thus, in this comprehensive review we summarize the rapidly expanding body of evidence that links T2D with NAFLD and HCV infection. For each of these two liver diseases with systemic manifestations, we discuss the epidemiological burden, the pathophysiologic mechanisms and the clinical implications. To date, substantial evidence suggests that NAFLD and HCV play a key role in T2D development and that the interaction of T2D with liver disease may result in a “vicious circle”, eventually leading to an increased risk of all-cause mortality and liver-related and cardiovascular complications. Preliminary evidence also suggests that improvement of NAFLD is associated with a decreased incidence of T2D. Similarly, the prevention of T2D following HCV eradication in the era of direct-acting antiviral agents is a biologically plausible result. However, additional studies are required for further clarification of mechanisms involved.

2014 - Age-associated alterations in cholesterol homeostasis: evidence from a cross-sectional study in a Northern Italy population. [Articolo su rivista]
Bertolotti, Marco; Mussi, Chiara; Pellegrini, E; Magni, A; Del Puppo, M; Ognibene, Silvia; Carulli, Lucia; Anzivino, C; Baldelli, Enrica; Loria, Paola; Carulli, N.

BACKGROUND: The modifications of cholesterol metabolism associated with aging are ill-defined. The objective of this study was to define age-associated alterations of the different metabolic pathways controlling cholesterol homeostasis by analyzing circulating sterols. METHODS: We analyzed serum samples collected from 201 adult (75 male, 126 female) subjects within the epidemiological MICOL study (Multicentrica Italiana Colelitiasi). The age range was 38-79 years; 103 had evidence of gallstones. The concentrations of the different sterols, recognized as markers of the main pathways of cholesterol homeostasis, were analyzed by gas chromatography-mass spectrometry, including lathosterol (synthesis), campesterol and sitosterol (absorption), and 7α-hydroxy-4-cholesten-3-one (degradation to bile acids). RESULTS: A significant direct correlation was detected between age and cholesterol levels (r =0.34, P<0.01). The lathosterol/cholesterol ratio was lower in older age quartiles (P<0.05 by analysis of variance), with an inverse correlation between the lathosterol/cholesterol ratio and age (r=-0.32, P<0.01). Such correlation was particularly evident in females. The campesterol/cholesterol and sitosterol/cholesterol ratios were inversely correlated with aging in control, but not in gallstone patients. The levels of 7α-hydroxy-4-cholesten-3-one were not correlated with age. CONCLUSION: These data show a reduction of cholesterol synthesis with aging which is associated with increased circulating cholesterol levels. The finding might be related to a reduced metabolic need for cholesterol in advancing age, leading to a downregulation of the main mechanisms of cholesterol intake in the liver. A different age-related behavior was observed in gallstone-free versus gallstone patients regarding cholesterol absorption. The possible implications in terms of the pharmacological management of hypercholesterolemia in the elderly remain to be defined.

2014 - In Vivo Degradation of Cholesterol to Bile Acids Is Reduced in Patients Receiving Parenteral Nutrition. [Articolo su rivista]
Carulli, Lucia; Del Puppo, M; Anzivino, Claudia; Zambianchi, L; Gabbi, C; Baldelli, Enrica; Odoardi, Mr; Loria, Paola; Carulli, Nicola; Bertolotti, Marco

Background. Artificial nutrition is frequently associated with hepatobiliary complications, probably due to the inherent derangement of the gastrointestinal tract physiology. Alterations of hepatic lipid metabolism are likely to be involved. The aim of the present study was to investigate the effect of artificial nutrition on bile acid production, a key event in cholesterol homeostasis, in humans. Patients and Methods. Eleven patients receiving artificial nutrition, either parenteral nutrition (PN; n = 6) or enteral nutrition (EN; n = 5) with no previous history of liver disease, underwent analysis of cholesterol 7α-hydroxylation rates in vivo, a measure of bile acid formation, by isotope release analysis after intravenous injection of [7α-(3)H]cholesterol. The results were compared with those obtained in a population of 16 age-matched control subjects. Results. Hydroxylation rates were lower in patients with artificial nutrition (PN: 94 ± 13 mg/d; EN: 230 ± 39 mg/d, mean ± SEM) when compared with controls (385 ± 47 mg/d) (P < .01, 1-way analysis of variance). In a patient receiving EN, hydroxylation rates increased 3.5-fold after treatment with the cholecystokinin analogue ceruletide (20 µg bid for 2 weeks intramuscularly). Serum lathosterol-to-cholesterol ratio, a marker of cholesterol synthesis, was also significantly reduced in artificial nutrition, whereas serum levels of fibroblast growth factor 19 (FGF19) were increased. Conclusion. In vivo 7α-hydroxylation is suppressed in artificial nutrition, particularly in PN. The finding associates with reduced cholesterol production, possibly as a metabolic consequence. The data suggest a regulatory role of gastrointestinal hormones and FGF19 on bile acid production and might suggest a pathophysiological basis for some common complications of artificial nutrition, such as gallstone disease and cholestasis.

2014 - Nonalcoholic fatty liver disease and aging: epidemiology to management [Articolo su rivista]
Bertolotti, Marco; Lonardo, Amedeo; Mussi, Chiara; Baldelli, Enrica; Pellegrini, Elisa; Ballestri, Stefano; Romagnoli, Dante; Loria, Paola

Nonalcoholic fatty liver disease (NAFLD) is common in the elderly, in whom it carries a more substantial burden of hepatic (nonalcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma) and extra-hepatic manifestations and complications (cardiovascular disease, extrahepatic neoplasms) than in younger age groups. Therefore, proper identification and management of this condition is a major task for clinical geriatricians and geriatric hepatologists. In this paper, the epidemiology and pathophysiology of this condition are reviewed, and a full discussion of the link between NAFLD and the aspects that are peculiar to elderly individuals is provided; these aspects include frailty, multimorbidity, polypharmacy and dementia. The proper treatment strategy will have to consider the peculiarities of geriatric patients, so a multidisciplinary approach is mandatory. Non-pharmacological treatment (diet and physical exercise) has to be tailored individually considering the physical limitations of most elderly people and the need for an adequate caloric supply. Similarly, the choice of drug treatment must carefully balance the benefits and risks in terms of adverse events and pharmacological interactions in the common context of both multiple health conditions and polypharmacy. In conclusion, further epidemiological and pathophysiological insight is warranted. More accurate understanding of the molecular mechanisms of geriatric NAFLD will help in identifying the most appropriate diagnostic and therapeutic approach for individual elderly patients.

2014 - Pathogenesis and significance of hepatitis C virus steatosis: An update on survival strategy of a successful pathogen [Articolo su rivista]
Lonardo, Amedeo; Adinolfi, Luigi Elio; Restivo, Luciano; Ballestri, Stefano; Romagnoli, Dante; Baldelli, Enrica; Nascimbeni, Fabio; Loria, Paola

Hepatitis C virus (HCV) is a successful pathogen on the grounds that it exploits its host's metabolism to build up viral particles; moreover it favours its own survival by inducing chronic disease and the development of specific anatomic changes in the infected organ. Steatosis, therefore, is associated with HCV infection by necessity rather than by chance alone. Approximately 6% of HCV patients have steatohepatitis. Interestingly, HCV steatosis occurs in the setting of multiple metabolic abnormalities (hyperuricemia, reversible hypocholesterolemia, insulin resistance, arterial hypertension and expansion of visceral adipose tissue) collectively referred to as "hepatitis C-associated dysmetabolic syndrome" (HCADS). General, nonalcoholic fatty liver disease (NAFLD)-like, mechanisms of steatogenesis (including increased availability of lipogenic substrates and de novo lipogenesis; decreased oxidation of fatty substrates and export of fatty substrates) are shared by all HCV genotypes. However, genotype 3 seemingly amplifies such steatogenic molecular mechanisms reported to occur in NAFLD via more profound changes in microsomal triglyceride transfer protein; peroxisome proliferator-activated receptor alpha; sterol regulatory element-binding proteins and phosphatase and tensin homologue. HCV steatosis has a remarkable clinical impact in as much as it is an acknowledged risk factor for accelerated fibrogenesis; for impaired treatment response to interferon and ribavirin; and development of hepatocellular carcinoma. Recent data, moreover, suggest that HCV-steatosis contributes to premature atherogenesis via both direct and indirect mechanisms. In conclusion, HCV steatosis fulfills all expected requirements necessary to perpetuate the HCV life cycle. A better understanding of the physiology of HCADS will likely result in a more successful handling of disease with improved antiviral success rates. © 2014 Baishideng Publishing Group Inc. All rights reserved.

2013 - ABCB4 and ABCB11 mutations in intrahepatic cholestasis of pregnancy in an Italian population [Articolo su rivista]
Anzivino, Claudia; Odoardi, Maria Rosaria; Meschiari, Erica; Baldelli, Enrica; Facchinetti, Fabio; Neri, Isabella; Ruggiero, Giuseppe; Zampino, Rosa; Bertolotti, Marco; Loria, Paola; Carulli, Lucia

Background: Genetic alterations in the ATP-binding cassette subfamily B member 4 (ABCB4) and ATPbinding cassette subfamily B member 11 (ABCB11) have been associated to the onset of intrahepatic cholestasis of pregnancy (ICP) in predisposed women. Aims: To identify new and/or frequent ABCB4 and ABCB11 genes variants in a cohort of Italian patients with ICP and to evaluate the possible pathogenetic role for the novel mutations identified. Methods: DNA of 33 unrelated Italian women with obstetric cholestasis were screened for mutations in the entire coding sequence of ABCB4 and ABCB11 genes. Polymerase chain reaction and automated sequencing was performed on the 27 coding exons of both genes. Results: Genotyping revealed 11 mutations, 5 of whom were novel variants: 2 localized on ABCB4 (p.I587DfsX603, p.I738LfsX744) and 3 on ABCB11 (p.V284D, p.Q558H, p.P731S). The most severe phenotypes were associated with the variants p.I587DfsX603, p.I738LfsX744 and p.V284D. Moreover, the already described mutation p.N510S found in ABCB4 seems to be strictly involved in the onset of ICP in that particular patient. Conclusions: Our data support the hypothesis of a significant involvement of ABCB4 mutations in the onset of ICP, but also confirm an important role for ABCB11 mutations in increasing the susceptibility to cholestasis of pregnancy.

2013 - Classical and innovative insulin sensitizing drugs for the prevention and treatment of NAFLD [Articolo su rivista]
Carulli, Lucia; Mauro, Maurantonio; Lionel, Hebbard; Baldelli, Enrica; Loria, Paola; George, Jacob

Background. Nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disorder worldwide, comprises a spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. NASH is associated with an increased risk of hepatocellular carcinoma (HCC) and cardiometabolic disease. Insulin resistance (IR) is the underlying pathogenic mechanism for NAFLD, the presence of which in turn, is a strong predictor for the development of metabolic disorders. Hence, therapy of NAFLD with insulin-sensitizing drugs (ISDs) should ideally improve the key hepatic histological changes (steatosis, inflammation and fibrosis), but should also reduce cardiometabolic and cancer risk. Objectives. In this review, the rationale for the use of ISDs and the evidence for their efficacy are detailed. In particular, the mechanism of action, potential for use, limitations and untoward effects of metformin and thiazolidinediones are systematically reviewed. Further, we discuss novel ISDs that may have potential clinical utility in NAFLD. Results and Conclusion. Despite the theoretical prediction that ISDs might have beneficial effects on disease outcomes, evidence that ISDs are able to alter the natural history of NAFLD are presently not available. The exploration of novel strategies exploiting “non-conventional” ISDs is encouraged

2013 - Relationship of serum fetuin-a levels with coronary atherosclerotic burden and NAFLD in patients undergoing elective coronary angiography. [Articolo su rivista]
Ballestri, S; Meschiari, Erica; Baldelli, Enrica; Musumeci, Francesca Elena; Romagnoli, D; Trenti, T; Zennaro, Rg; Lonardo, A; Loria, Paola

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) patients are prone to coronary artery disease (CAD). Fetuin-A inhibits arterial calcification, induces insulin resistance, and is increased in NAFLD. Data on fetuin-A levels in CAD are conflicting. We tried to ascertain whether NAFLD and CAD are associated and if fetuin-A predicts CAD and/or NAFLD. METHODS: CAD was diagnosed by ≥50% stenosis in coronary arteries and NAFLD by ultrasound imaging in the absence of any other liver disease. Seventy patients who underwent elective coronarography at our hospital were recruited in this cross-sectional study. Twenty-four patients had no CAD (9 with and 15 without NAFLD) and 46 had CAD (20 with and 26 without NAFLD). Standard anthropometric indices and metabolic parameters were recorded. Fetuin-A was determined by enzyme-linked immunosorbent assay (ELISA). Visceral fat thickness and visceral/subcutaneous fat ratio were assessed by ultrasonography. RESULTS: NAFLD was not associated with CAD, probably owing to the limited series. Fetuin-A was significantly lower, whereas visceral fat thickness and visceral/subcutaneous fat ratio were higher in patients with CAD versus those without CAD. Younger age and higher body mass index (BMI), waist circumference, triglycerides, fasting glucose, homeostasis model assessment, spleen area, subcutaneous fat thickness, and prevalence of metabolic derangements were associated with NAFLD. At multivariate analysis, elevated fetuin-A levels were an independent negative predictor of CAD [odds ratio (OR)=0.995, P=0.049]. Fetuin-A was an independent predictor of NAFLD (OR=1.005, P=0.036) in the model including BMI. CONCLUSIONS: This prospective cross-sectional study demonstrates high fetuin-A levels to be independently associated with NAFLD and a lower risk of coronarographically diagnosed CAD.

2012 - Increased appearance rate of 27-hydroxycholesterol in vivo in hypercholesterolemia: A possible compensatory mechanism. [Articolo su rivista]
Bertolotti, Marco; Del Puppo, M; Corna, F; Anzivino, Claudia; Gabbi, Chiara; Baldelli, Enrica; Carulli, Lucia; Loria, Paola; Galli Kienle, M; Carulli, Nicola

BACKGROUND AND AIMS: The first step in the alternative pathway of bile acid biosynthesis is the 27-hydroxylation of cholesterol, which takes place both in liver and extrahepatic tissues. This pathway is believed to play a role in peripheral cholesterol degradation. Aim of this study was to investigate theimpact of hyperlipidemia on 27-hydroxycholesterol appearance rate, and to assess the effects induced by treatment with statins. METHODS AND RESULTS: Seven patients with familial hypercholesterolemia and eight patients with familial combined hyperlipidemia underwent determination of 27-hydroxylation rates in vivo by i.v. infusion of deuterated 27-hydroxycholesterol. Isotope enrichment was assayed by gas chromatography-mass spectrometry, allowing to calculate 27-hydroxycholesterol appearance rates. Six normocholesterolemic subjects wereregarded as controls. In some hypercholesterolemic patients the infusions were repeated during treatment with atorvastatin or rosuvastatin. Hydroxylation rates were higher in hypercholesterolemic patients (8.7 ± 2.5 mg/h; controls,3.4 ± 2.0 mg/h; combined hyperlipidemia, 4.4 ± 1.6 mg/h; mean ± SD, P < 0.01 vs both). After statin treatment, both plasma cholesterol levels and hydroxylation rates dropped by nearly 50%. No difference was detectable between the two statins. A linear correlation was shown between plasma cholesterol and 27-hydroxylation rates. CONCLUSION: Hypercholesterolemia associates withincreased 27-hydroxycholesterol appearance rates, which decrease during hypocholesterolemic treatment. The correlation with cholesterol levels supports the view that 27-hydroxylation may act as a compensatory mechanism in a conditionof larger plasma cholesterol pool. A regulatory role for hepatic and extrahepatic nuclear receptors seems reasonable. These data prompt novel pharmacological approaches for the management of hypercholesterolemia and the prevention of atherosclerosis.

2010 - Homocysteine potentiates seizures and cell loss induced by pilocarpine treatment [Articolo su rivista]
Baldelli, Enrica; Leo, Giuseppina; Andreoli, Nicola; K., Fuxe; Biagini, Giuseppe; Agnati, Luigi Francesco

Patients affected by recurrent seizures frequently present increased homocysteine plasma levels in consequence of treatment with antiepileptic drugs. Homocysteine is proconvulsant and can affect the response to antiepileptic drugs. In addition, high homocysteine plasma levels represent a risk factor for cardiovascular and neurodegenerative diseases. To better define the role of increased homocysteine in epilepsy, we analyzed the effects of homocysteine pretreatment in the pilocarpine model of status epilepticus (SE), which is used to mimic temporal lobe epilepsy (TLE) in rodents. Precisely, we investigated whether a moderate hyperhomocysteinemia, unable to cause seizures, could sensitize rats to pilocarpine and cooperate in inducing brain lesions. We found that a subthreshold dose of pilocarpine (200 mg/kg) is sufficient to induce SE in the majority (approximately 90%) of rats pretreated with homocysteine for 2 weeks, whereas only 40% of saline-treated controls developed SE following the same pilocarpine dose. Furthermore, homocysteine pretreatment led to a significant increase in neuronal cell loss evaluated by counting toluidine blue-stained or Fluoro-Jade-positive cells in hippocampal and parahippocampal regions. Pilocarpine augmented amyloid beta expression in both animal groups. However, pretreatment with homocysteine favored the intraneuronal fibrillar conformation of amyloid beta, thus promoting neurodegeneration. These findings indicate that increased homocysteine levels enhance seizure activity and neurodegeneration in pilocarpine-treated rats and suggest that similar detrimental effects may occur in patients affected by TLE.

2009 - Farmacoresistenza e sclerosi ippocampale in un modello di epilessia del lobo temporale: evidenze a favore di una relazione con la lesione dell’area CA3Pharmacoresistance and hippocampal sclerosis in a model of temporal lobe epilepsy: evidence for a relationship with lesion of the CA3 region [Relazione in Atti di Convegno]
Longo, Daniela; Baldelli, Enrica; Manca, Lidia; G., Gatti; E., Perucca; M., Avoli; Biagini, Giuseppe

Temporal lobe epilepsy (TLE) with hippocampal sclerosis is the most common type of pharmacoresistantepilepsy in adults.We investigated whether hippocampal damage could influence the response to antiepilepticdrugs in the pilocarpine model of TLE. Sprague-Dawley rats were injected with intraperitoneal (i.p.) pilocarpine(380 mg/kg), and the provoked status epilepticus (SE) was quelled after 30 or 120 minutes with i.p.diazepam (20 mg/kg). After 3 weeks, when all the animals developed spontaneous recurrent seizures, weimplanted osmotic minipumps to assure a constant release of carbamazepine (CBZ, 4 mg/kg/h) or vehicle(epileptic controls). After one week, during which we monitored seizure frequency, we sacrificed the animalsto assess the hippocampal damage.We found a highly predictable ischemic-hemorrhagic lesion in the CA3stratum lacunosum-molecolare of rats exposed to 120 min SE. Although ablating the perforant path terminalfield, this lesion was significantly (p < 0.05) less pronounced in animals with a SE of 30 minutes.All the rats wereresistant to CBZ.Moreover, rats exposed to 120 minutes of SE showed a 6-fold increase in frequency of spontaneousseizures during CBZ administration. These data suggest that the loss of direct inputs from the entorhinalcortex to CA3 can worsen the response to CBZ treatment in a model of TLE.

2009 - Glia-derived steroids modulate epileptogenesis in a model of temporal lobe epilepsy [Abstract in Rivista]
Longo, Daniela; Baldelli, Enrica; G., Bertazzoni; M., Avoli; Biagini, Giuseppe

Purpose: The conversion of cholesterol into pregnenolone by cytochromeP450 cholesterol side-chain cleavage enzyme (P450 scc) is therate-limiting step in the steroid synthesis. Glial cells and neurons bothexpress P450 scc and synthesize neurosteroids, which are positive modulatorsof GABAergic transmission. Astrocytes become activated followingstatus epilepticus (SE), but it is presently unclear whether thisactivation leads to enhanced neurosteroidogenesis.Method: We studied the time course of P450 scc immunoreactivitychanges after pilocarpine-induced SE in adult (8 week-old) and young (3weeks-old) rats. To evaluate the role of P450 scc upregulation, we usedthe 5a-reductase inhibitor finasteride (100/kg s.c. for 3 weeks) to suppressthe synthesis of neurosteroids.Results: We demonstrated that P450 scc is upregulated in the CA3 hippocampalregion. Moreover the extent of P450 scc induction was directlyrelated to the onset of spontaneous recurrent seizures in adult (8-weekold)rats. In 3-week-old rats compared with adults, higher P450 scc levelsand a longer latent period were found. Interestingly, adult epileptic rats,treated with finasteride, compared with a group of vehicle-treated rats,presented anticipated generalized seizures (p<0.01). In young rats, finasterideanticipates seizures in approximately 50% of the animals.Conclusion: These findings suggest that neurosteroids can modulateepileptogenesis in the pilocarpine model of temporal lobe epilepsy.

2009 - Neurosteroids and epileptogenesis in the pilocarpine model: evidence for a relationship between P450scc induction and length of the latent period [Articolo su rivista]
Biagini, Giuseppe; Longo, Daniela; Baldelli, Enrica; Zoli, Michele; M. A., Rogawski; G., Bertazzoni; M., Avoli

Purpose: Cytochrome P450 cholesterol side-chaincleavage enzyme (P450scc) catalyzes the initialstep in the biosynthesis of neurosteroids withinthe brain. We sought to determine which cellsexpress P450cc and whether neurosteroids play arole in the regulation of epileptogenesis followingpilocarpine-induced status epilepticus (SE).Methods: Rats experienced uninterrupted SE orSE terminated with diazepam at 60, 120, and180 min. P450scc induction in CA3 hippocampuswas determined by double immunolabeling withP450scc antiserum and monoclonal antibodiesagainst GFAP (astrocytes), RIP (oligodendrocytes),or heme oxygenase-1 (microglia).Results: SE was associated with P450scc inductionin many astrocytes and a small number of microgliaand oligodendrocytes in the hippocampal CA3strata radiatum and lacunosum-moleculare. Theextent of P450scc induction increased withincreasing SE duration. Paradoxically, increasedP450scc induction in rats experiencing SE for180 min or more was associated with the delayedonset of spontaneous recurrent seizures. Treatmentwith the 5a-reductase inhibitor finasteride(100 mg/kg/day for 25 days), which inhibits thesynthesis of c-aminobutyric acid (GABA)A receptormodulating neurosteroids such as allopregnanolone,was associated with a significantreduction in time to the onset of spontaneous seizuresin rats exposed to 180-min but not 90-minSE.Discussion: P450scc is induced by SE in a diversepopulation of hippocampal glia. Induction ofP450scc is associated with the delayed onsetof spontaneous seizures. Conversely, inhibitionof neurosteroid synthesis accelerated the onset ofspontaneous seizures, but only in animals exhibitingsignificant increases in P450scc. These findingssuggest that induction of neurosteroid synthesis inreactive glial cells is associated with delayed onsetof spontaneously recurrent seizures.

2008 - Characterizationof a focal vascular lesionaffecting entorhinalcortex-CA3connections after statusepilepticus [Abstract in Rivista]
Biagini, Giuseppe; Longo, Daniela; Baldelli, Enrica; Contri, Miranda; Nichelli, Paolo Frigio; U., Guerrini; L., Sironi; P., Gelosa; G., Bertazzoni; M., Avoli

Pilocarpine mimics temporal lobeepilepsy by inducing SE associatedwith damage in hippocampal andextrahippocampal areas. We havecharacterized a vascular lesion thatdestroys the perforant path (PP) inCA3 after SE. This lesion (white arrows,Figure 1) was evident withmagnetic resonance imaging 1 day aftera SE lasting for 120 min and its appearancewas delayed by limiting SEto 60 or 30 min. Rats exposed to SEfor 30 min developed the lesion unlesssubsequently treated with diazepam(20 mg/kg s.c. for 3 days), whichprotected 50% of the animals. Antibodiesfor astrocytes (GFAP), dendrites(MAP2) and PPnerve terminals(mGluR2/3), showed areas of immunoreactivityloss in which we localizedincreased staining for laminin, inthe basement membrane of vessels.This lesion was uncommon in young(3-week-old) rats. The latent periodfor seizure appearance was similar inadult rats exposed to SE only or to SEfollowed by neuroprotection with diazepam;however, the frequency ofspontaneous seizures was significantlylower (p < 0.01) in the neuroprotectedgroup. Moreover, spontaneousseizures were delayed in youngrats (p < 0.01) exposed to 60 min SEcompared to adults experiencing asimilar SE. To investigate the role ofdamaged CA3 in seizure activity, were-induced SE in adult and young epilepticrats. Using FosB/FosB markers,we found induction of FosB/FosB immunopositivity in CA3neurons of young but not in adult rats.These experiments reveal that SE canproduce a focal lesion in the PP, whichaffects the role of the hippocampus inepileptic rats.

2008 - Glia-derived neurosteroids modulate epileptogenesis in a model of temporal lobe epilepsy [Abstract in Rivista]
Longo, Daniela; Baldelli, Enrica; G., Bertazzoni; M., Avoli M; Biagini, Giuseppe

The conversion of cholesterol intopregnenolone by the enzyme cholesterolside-chain cleavage cytochromeP450 (P450scc) is the rate-limitingstep in the steroid synthesis. Glialcells and neurons both expressP450scc and synthesize neurosteroidsthat act as positive modulators ofGABAergic transmission. Astrocytesbecome activated following statusepilepticus (SE), but it is presently unclearwhether this activation leads toenhanced neurosteroid synthesis. Westudied the time course of P450sccimmunoreactivity changes afterpilocarpine-induced SE and its relationshipwith epileptogenesis. Wefound that P450scc is upregulated inthe CA3 hippocampal region, afterSE. Induction of P450scc was mainlyobserved in astrocytes identified by aGFAP antibody, although hemeoxygenase-1-positive microglial cells,RIP-positive oligodendrocytes andNeuN-neurons were also stained forP450scc (Fig. 1). The extent ofP450scc induction was directly relatedwith the onset of spontaneouslyrecurrent seizures in adult (8-weekold)rats. In addition, in young (3-week-old) rats the induction ofP450scc and the latent period durationwere much larger than in adultrats. To further evaluate the role ofP450scc upregulation, we used the5-reductase inhibitor finasteride(100 mg/kg/day for approximately 3weeks) to suppress the synthesis ofGABA-modulating neurosteroidssuch as allopregnanolone. Interestingly,adult epileptic rats, comparedwith a group of vehicle-treated ratsthat experienced a similar SE, presentedwith significantly (p<0.01) anticipatedgeneralized seizures whentreated with finasteride. In young rats,we observed early generalized seizuresin approximately 50% of the animals.These findings suggest thatneurosteroids can modulate epileptogenesisin the pilocarpine modelof temporal lobe epilepsy.

2008 - Network hyperexcitability within the deep layers of pilocarpine-treated entorhinal cortex. [Articolo su rivista]
P., De Guzman; Y., Inaba; Baldelli, Enrica; M., De Curtis; Biagini, Giuseppe; M., Avoli

In this study we report that in the presence of normal buffer, epileptiform dischargesoccur spontaneously (duration=2.60±0.49 s) or can be induced by electrical stimuli(duration=2.50±0.62 s) in the entorhinal cortex (EC) of brain slices obtained frompilocarpine-treated rats but not in those from age-matched, nonepileptic control (NEC)animals. These network-driven epileptiform events consist of field oscillatory sequences atfrequencies greater than 200 Hz that most often initiate in the lateral EC and propagateto the medial EC with 4–63 ms delays. The NMDA receptor antagonist CPP depressesthe rate of occurrence (P <0.01) of these spontaneous epileptiform discharges but failsin blocking them. Paradoxically, stimulus-induced epileptiform responses are enhanced induration during CPP application.However, concomitant application ofNMDAand non-NMDAglutamatergic antagonists abolishes spontaneous and stimulus-induced epileptiform events.Intracellular recordings from lateral EC layer V cells indicate a lower frequency of spontaneoushyperpolarizing postsynaptic potentials in pilocarpine-treated tissue than in NEC (P <0.002)both under control conditions and with glutamatergic receptor blockade; the reversal potentialof pharmacologically isolated GABAA receptor-mediated inhibitory postsynaptic potentialshas similar values in the two types of tissue. Finally, immunohistochemical analysis showsthat parvalbumin-positive interneurons are selectively reduced in number in EC deep layers.Collectively, these results indicate that reduced inhibition within the pilocarpine-treated EClayer V may promote network epileptic hyperexcitability.

2008 - Nuclear receptors and obstructive cholestasis in Humans: increased heaptic expression of short heterodimer partner : a protective response? [Abstract in Rivista]
Bertolotti, Marco; Gabbi, Chiara; Anzivino, C; Baldelli, Enrica; Carulli, Lucia; Carulli, Nicola

alteration in hepatic expression of nuclear receptors durinhg obstructive cholestasis, appear to be mediated by increaased expression of SHPa nd appear to be finalized to stimulate extrusion of biliary lipid out of teh liver and to inhibit further uptake of organic anions into the liver cell itself.

2008 - On the key role played by altered protein conformation in Parkinson’s disease [Articolo su rivista]
Agnati, Luigi Francesco; Baldelli, Enrica; Andreoli, Nicola; A. S., Woods; Vellani, Vittorio; D., Marcellino; D., Guidolin; K., Fuxe

On the basis of the previously proposed hierarchic organisation of the central nervous system (CNS) and of its syntropic behaviour, a view of neurodegenerative diseases focusing on the assemblage of abnormal multimeric proteins (pathologic protein mosaics (PMs)) is proposed. Thus, the main focus of the present paper is on Parkinson’s disease (PD) as a neurodegenerative disease, which has as crucial feature protein conformational alterations and formation of pathological PMs. Two interconnected cellular dysfunctions are discussed as main pathogenic factors of PD syndromes, namely mitochondrial deficits (i.e. energy failure, especially critical for Substantia Nigra DA neurons) and conformational protein alterations (due to genetic or environmental causes). Conformational protein alterations can trigger pathological phenomena via the loss and/or the gain of new functions. In particular, altered proteins can lead to the formation of abnormal PMs, which can, inter alia, cause distortion of cellular structures, toxic functions and/or formation of improper membrane ion channels. In view of the fact that disordered proteins can easily acquire unwanted conformation, the “disorder index” (DI) for proteins involved in PD has been evaluated. It has been found that both α-synuclein and tau-protein have high DI. This datum is in agreement with the observation that these two proteins synergistically promote polymerisation of each other into amyloid fibrils, favouring the formation of Lewy bodies.

2008 - Proepileptic influence of a focal vascular lesion affecting entorhinal cortex-CA3 connections after status epilepticus. [Articolo su rivista]
Biagini, Giuseppe; Baldelli, Enrica; Longo, Daniela; Contri, Miranda; U., Guerrini; L., Sironi; P., Gelosa; Zini, Isabella; M., Avoli

In limbic seizures, neuronal excitation is conveyed from the entorhinalcortex directly to CA1 and subicular regions. This phenomenonis associated with a reduced ability of CA3 to respond toentorhinal cortex inputs. Here, we describe a lesion that destroys theperforant path in CA3 after status epilepticus (SE) induced bypilocarpine injection in 8-week-old rats. Using magnetic resonanceimaging, immunohistochemical, and ultrastructural analyses, wedetermined that this lesion develops after 30 minutes of SE and ischaracterized by microhemorrhages and ischemia. After a longerperiod of SE, the lesion invariably involves the upper blade ofthe dentate gyrus. Adult rats treated with subcutaneous diazepam(20 mg kgj1 for 3 days) did not develop the dentate gyrus lesionand had less frequent spontaneous recurrent seizures (p G 0.01).Young (3-week-old) rats rarely (20%) developed the CA3 lesion,and their spontaneous seizures were delayed (p G 0.01). To investigatethe role of the damaged CA3 in seizure activity, wereinduced SE in adult and young epileptic rats. Using FosB/$FosBmarkers, we found induction of FosB/$FosB immunopositivity inCA3 neurons of young but not in adult rats. These experimentsindicate that SE can produce a focal lesion in the perforant path thatmay affect the roles of the hippocampus in epileptic rats.

2006 - Endogenous neurosteroids modulate epileptogenesis in a model of temporal lobe epilepsy [Articolo su rivista]
Biagini, Giuseppe; Baldelli, Enrica; Longo, Daniela; L., Pradelli; Zini, Isabella; M. A., Rogawski; M., Avoli

Neurosteroids modulate seizure susceptibility, but their role in the regulation of epileptogenesis is unknown. Status epilepticus (SE) induces temporal lobe epileptogenesis following a latent period in which glial cells are activated. Here, we found that P450scc, the rate-limiting enzyme in steroid synthesis.. is upregulated in hippocampal glia during the latent period after pilocarpine-induced SE in rats. More prolonged SE was associated with greater P450scc expression and longer latencies to the development of seizures, suggesting that enhanced steroid synthesis retards epileptogenesis. The 5 alpha-reductase inhibitor finasteride, which blocks neurosteroid synthesis, reduced the latent period, indicating that glia-derived neurosteroids may be antiepileptogenic. (c) 2006 Elsevier Inc. All rights reserved.

2006 - Neurosteroidi ed epilettogenesi: evidenze a favore di una relazione tra induzione dell’enzima P450scc e durata della fase di latenza nel modello della pilocarpina. [Articolo su rivista]
Baldelli, Enrica; Longo, Daniela; Zini, Isabella; Zoli, Michele; M., Avoli; Biagini, Giuseppe

Neurosteroids are GABAA receptor agonists able to modulate seizure susceptibility. They are mainly synthesizedin astrocytes, a type of glial cells that are activated by neuronal damage. Interestingly, status epilepticus (SE)induces neuronal damage and a chronic epileptic condition that becomes evident following a “latent” period,in which glial cells are highly activated. Investigating glial cell activation in the pilocarpine model, wefound that P450scc, the rate-limiting enzyme in neurosteroid synthesis, is upregulated in hippocampal astrocytesduring the latent period. The induction of P450scc immunoreactivity was stronger with prolonged statusepilepticus and was associated with longer latencies to the development of seizures, suggesting thatenhanced neurosteroid synthesis retards epileptogenesis. Accordingly, the 5α-reductase inhibitor finasteride,which blocks neurosteroid synthesis, reduced the latent period, thus indicating that glia-derived neurosteroidsmay be antiepileptogenic.

2006 - Neurosteroids modulate epileptogenesis in a model of temporal lobe epilepsy [Abstract in Rivista]
Longo, Daniela; Baldelli, Enrica; L., Pradelli; Zini, Isabella; M. A., Rogawski; M., Avoli; Biagini, Giuseppe

Neurosteroids modulate seizure susceptibility, but their role in the regulation of epileptogenesis is unknown. Status epilepticus inducestemporal lobe epileptogenesis following a latent period in which glial cells are activated. Here, we found that P450scc, the rate-limiting enzyme insteroid synthesis, is upregulated in hippocampal glia during the latent period after pilocarpine-induced SE in rats. More prolonged SE wasassociated with greater P450scc expression and longer latencies to the development of seizures, suggesting that enhanced steroid synthesis retardsepileptogenesis. The 5alpha-reductase inhibitor finasteride, which blocks neurosteroid synthesis, reduced the latent period, indicating that glia-derivedneurosteroids may be antiepileptogenic.

2006 - Subiculum network excitability is increased in a rodent model of temporal lobe epilepsy [Articolo su rivista]
P., de Guzman; Y., Inaba; Biagini, Giuseppe; Baldelli, Enrica; C., Mollinari; D., Merlo; M., Avoli

In this study, we used in vitro electrophysiology along with immunohistochemistry and molecular techniques to study the subiculum-a limbic structure that gates the information flow from and to the hippocampus-in pilocarpine-treated epileptic rats. Comparative data were obtained from age-matched nonepileptic controls (NEC). Subicular neurons in hippocampal-entorhinal cortex (EC) slices of epileptic rats were: (i) hyperexcitable when activated by CA1 or EC inputs; and (ii) generated spontaneous postsynaptic potentials at higher frequencies than NEC cells. Analysis of pharmacologically isolated, GABA(A) receptor-mediated inhibitory postsynaptic potentials revealed more positive reversal potentials in epileptic tissue (-67.8 +/- 6.3 mV, n = 16 vs. -74.8 +/- 3.6 mV in NEC, n = 13; P < 0.001) combined with a reduction in peak conductance (17.6 +/- 11.3 nS vs. 41.1 +/- 26.7 nS in NEC; P < 0.003). These electrophysiological data correlated in the epileptic subiculum with (i) reduced levels of mRNA expression and immunoreactivity of the neuron-specific potassium-chloride cotransporter 2; (ii) decreased number of parvalbumin-positive cells; and (iii) increased synaptophysin (a putative marker of sprouting) immunoreactivity. These findings identify an increase in network excitability within the subiculum of pilocarpine-treated, epileptic rats and point at a reduction in inhibition as an underlying mechanism. (c) 2006 Wiley-Liss, Inc.

2005 - Impaired activation of CA3 pyramidal neurons in the epileptic hippocampus [Articolo su rivista]
Biagini, Giuseppe; G., D'Arcangelo; Baldelli, Enrica; M., D'Antuono; V., Tancredi; M., Avoli

We employed in vitro and ex vivo imaging tools to characterize the function of limbic neuron networks in pilocarpine-treated and age-matched, nonepileptic control (NEC) rats. Pilocarpine-treated animals represent an established model of mesial temporal lobe epilepsy. Intrinsic optical signal (IOS) analysis of hippocampal-entorhinal cortex (EC) slices obtained from epileptic rats 3 wk after pilocarpine-induced status epilepticus (SE) revealed hyperexcitability in many limbic areas, but not in CA3 and medial EC layer III. By visualizing immunopositivity for FosB/Delta FosB-related proteins-which accumulate in the nuclei of neurons activated by seizures-we found that: (1) 24 h after SE, FosB/Delta FosB immunoreactivity was absent in medial EC layer III, but abundant in dentate gyrus, hippocampus proper (including CA3) and subiculum; (2) FosB/Delta FosB levels progressively diminished 3 and 7 d after SE, whereas remaining elevated (p < 0.01) in subiculum; (3) FosB/Delta FosB levels sharply increased 2 wk after SE (and remained elevated up to 3 wk) in dentate gyrus and in most of the other areas but not in CA3. A conspicuous neuronal damage was noticed in medial EC layer III, whereas hippocampus was more preserved. IOS analysis of the stimulus-induced responses in slices 3 wk after SE demonstrated that IOSs in CA3 were lower (p < 0.05) than in NEC slices following dentate gyrus stimulation, but not when stimuli were delivered in CA3. These findings indicate that CA3 networks are hypoactive in comparison with other epileptic limbic areas. We propose that this feature may affect the ability of hippocampal outputs to control epileptiform synchronization in EC.

2004 - Hypoactivity of CA3 pyramidal neurons in the pilocarpine model of mesial temporal lobe epilepsy [Articolo su rivista]
Baldelli, E.; Zini, I.; Biagini, G.; D'Arcangelo, G.; Tancredi, V.; D'Antuono, M.; Avoli, M.

Reduced CA3-driven interictal activity in hippocampal-entorhinal cortex (EC) slices obtained from pilocarpine-treated rodents is accompanied by the persistence of ictal discharges originating from EC. Therefore, loss of CA3 pyramidal cells could be critical for unveiling limbic seizures in mesial temporal lobe epilepsy (MTLE). We analyzed here this issue by means of in vitro and ex vivo imaging along with histopathology in pilocarpine-treated and age-matched, non-epileptic control (NEC) rats. By visualizing immunopositivity for FosB/ΔFosB-related proteins - which accumulate in the nuclei of seizing neurons - we found that activated neurons can be found in many hippocampal and parahippocampal regions but not in CA3. Intrinsic optical signal (IOS) analysis of the stimulus-induced responses in slices obtained 3 weeks after SE revealed that IOSs in CA3 were lower (p &lt; 0.05) than in NEC slices following dentate gyrus stimulation, while comparable responses were elicited by stimuli delivered in the CA3 pyramidal layer. We conclude that in epileptic rats, and perhaps in MTLE patients, mechanisms other than neuronal loss hamper CA3 network synchronization and thus the ability of hippocampal outputs to control EC ictogenesis.

2004 - Ipoattività dei neuroni piramidali dell’area CA3 in un modello animale di epilessia del lobo temporale. [Articolo su rivista]
Baldelli, Enrica; Zini, Isabella; Biagini, Giuseppe; G., D'Arcangelo; V., Tancredi; M., D'Antuono; M., Avoli

Rational and Aims - Reduced CA3-driven interictal activity has been reported in hippocampal-entorhinal cortex (EC)-slices obtained from pilocarpine-treated rodents during 4-aminopyridine (4AP) treatment. In addition, ictal discharges initiating in EC persist throughout the experiment in these slices, while they disappear within the first 2 hours of 4AP superfusion in non-epileptic control (NEC) tissue. This evidence has led us to hypothesize that CA3 cell damage, which is known to occur in several models of mesial temporal lobe epilepsy (MTLE), may release EC ictogenesis. However, this in vitro data could also be related to the artificial condition set to record seizure activity in the slice. We addressed this issue by determining in vivo the extent of hippocampal network activation by using a marker sensitive to repetitive seizure activity: ΔFosB. Methods – We induced status epilepticus (SE) in Sprague-Dawley male rat by injecting pilocarpine i.p. (380 mg/kg). The animals where then sacrificed at 1, 3, 7, 14, 21 days and processed for ΔFosB immunodetection and other histopathological studies; alternatively, at 21 days they were used for in vitro analysis of the hippocampal-entorhinal cortex slice by intrinsic optical signal detection after electrical stimulation.Results – Immediately after SE, ΔFosB immunoreactivity was widespread in all the hippocampal regions, while no positive cell nuclei were found in NEC. In the following days, a general decrease in ΔFosB levels was observed in all the positive areas, reaching the lowest levels 7 days after. However, the subiculum presented a smoother decrease and ΔFosB levels were significantly higher than in the other areas. A sharp increase in ΔFosB immunoreactivity occurred in the dentate gyrus 14 days after SE, when positive cell nuclei reappeared also in other regions. Twenty-one days after SE, ΔFosB levels were increased in all the hippocampal regions apart CA3 and the medial entorhinal cortex. The histopathological analysis demonstrated a large damage in layer III of the medial entorhinal cortex, while CA was generally preserved. By counting pyramidal neurons in CA3, only a 10% decrease in cell number was found (p<0.05 vs NEC). Hence, we postulated that CA3 could be hypoactive in pilocarpine-treated spontaneously epileptic animals. This hypothesis was confirmed by analyzing the intensity of intrinsic optical signals detected in CA3 after maximal stimulation of the dentate gyrus. Again, by directly stimulating the CA3 pyramidal layer we were able to record IOSs as intense as in NEC, proving that this region is spared from damage.Conclusions - These findings indicate that in epileptic rats, and perhaps in MTLE patients, mechanisms other than neuronal loss hamper CA3 network synchronization and thus the ability of hippocampal output ability to control ictogenesis in the EC.