17,β-estradiol inhibits hepatitis C virus mainly by interference with the release phase of its life cycle

Rationale & Aim: Estrogen and estrogen-mediated signalling protect from hepatitis C virus through incompletely understood mechanisms. We aimed to ascertain which phase(s) of HCV life cycle is/are affected by estrogens. Methods: Huh7 cells infected with the JFH1 virus (genotype 2a) were exposed to dehydroepiandrosterone, testosterone, progesterone and 17β-estradiol (tested with/without its receptor antagonist fulvestrant). Dose-response curves were established to calculate IC50 values. To dissect how 17β-estradiol interferes with phases of HCV life cycle, its effects were measured on the HCV pseudo-particle system (viral entry), the sub-genomic replicon N17/JFH1 and the replicon cell line Huh7-J17 (viral replication). Finally, in a dual-step infection model, infectious supernatants, collected from infected cells exposed to hormones, were used to infect naïve cells. Results: Progesterone and testosterone showed no inhibitory effect on HCV; dehydroepiandrosterone was only mildly inhibitory. In contrast, 17β-estradiol inhibited infection by 64-67% (IC50 values 140 to 160 nM). Fulvestrant reverted the inhibition by 17β-estradiol in a dose-dependent manner. 17β-estradiol exerted only a slight inhibition (<20%) on HCV pseudo-particles, and had no effect on cells either transiently or stably (Huh7-J17 cells) expressing the N17/JFH1 replicon. In the dual-step infection model, a significant IC50 decline occurred between primary (134 nM) and secondary (100 nM) infections (p=0.02), with extracellular HCV RNA and infectivity being reduced to a higher degree in comparison to its intracellular counterpart. Conclusions: 17β-estradiol inhibits HCV acting through its intracellular receptors, mainly interfering with late phases (assembly/release) of the HCV life cycle

T-cell mediated responses against alpha-foetoprotein in hepatocellular carcinoma: Relationship with hepatitis C virus infection, tumour phenotype and patients’ survival

Background Alpha-foetoprotein (AFP) is a potential immunotherapeutic target in hepatocellular carcinoma (HCC). However, T-cell response (TR) to AFP is suppressed in HCC due to immune evasion. It is unknown whether HCV infection may pre-condition TR against AFP, or whether TR may influence the clinical course of HCC. Methods We prospectively enrolled 18 HCV+ treatment-naïve patients with cirrhosis (CC), 18 HCV+ HCC cases and 17 HCV- HCC cases. TR was quantified by ELISPOT using assays specific to interleukin (IL) 2, IL10 and granulocyte-monocyte colony stimulating factor (GM-CSF) on ex-vivo peripheral blood mononuclear cells (PBMC) stimulated in vitro with AFP peptides. Cytokine ratios were compared between groups and with clinicopathological features of HCC, including overall survival (OS). Results The proportion of AFP-specific responses was not different across the studied groups for any of the assayed cytokines. AFP-specific IL-2 responses were increased in larger (P = .02), multifocal tumours (P = .01) and correlated with advanced disease (P = .01). TRs did not correlate with other clinicopathological factors and did not predict for OS. Conclusion Tumour stage but not HCV infection is related to the emergence of anti-AFP TRs. These data enable formulation of a rationale for the further development of anti-AFP immunotherapy in HCC, facilitating optimal patient selection for future studies

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified

Oxidative Stress in Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a challenging disease caused by multiple factors, which may partly explain why it still remains an orphan of adequate therapies. This review highlights the interaction between oxidative stress (OS) and disturbed lipid metabolism. Several reactive oxygen species generators, including those produced in the gastrointestinal tract, contribute to the lipotoxic hepatic (and extrahepatic) damage by fatty acids and a great variety of their biologically active metabolites in a “multiple parallel-hit model”. This leads to inflammation and fibrogenesis and contributes to NAFLD progression. The alterations of the oxidant/antioxidant balance affect also metabolism-related organelles, leading to lipid peroxidation, mitochondrial dysfunction, and endoplasmic reticulum stress. This OS-induced damage is at least partially counteracted by the physiological antioxidant response. Therefore, modulation of this defense system emerges as an interesting target to prevent NAFLD development and progression. For instance, probiotics, prebiotics, diet, and fecal microbiota transplantation represent new therapeutic approaches targeting the gut microbiota dysbiosis. The OS and its counter-regulation are under the influence of individual genetic and epigenetic factors as well. In the near future, precision medicine taking into consideration genetic or environmental epigenetic risk factors, coupled with new OS biomarkers, will likely assist in noninvasive diagnosis and monitoring of NAFLD progression and in further personalizing treatments

Modulation of Oxidative Stress by 17 β-Estradiol and Genistein in Human Hepatic Cell Lines In Vitro

BACKGROUND/AIMS: estrogens and phytoestrogens exert hepatoprotection through mechanisms not clearly examined yet. Here, we investigated the protective effects exerted by 17\u3b2-estradiol and genistein against oxidative stress in hepatocytes and hepatic stellate cells (HSCs) and the involvement of specific receptors and the intracellular signalling. METHODS: Huh7.5 and LX-2, alone or in co-culture with Huh7.5, were treated with 17\u3b2-estradiol and genistein alone or in the presence of menadione and of estrogen receptors (ERs) and G protein-coupled-estrogenic-receptors (GPER) blockers. Cell viability, mitochondrial membrane potential and oxidant/antioxidant system were measured by specific kits. Western Blot was used for the analysis of Akt and p38-mitogen-activated-protein kinases (MAPK) activation and \u3b1-smooth-muscle actin expression. RESULTS: In Huh7.5, 17\u3b2-estradiol and genistein prevented the effects of peroxidation by modulating Akt and p38MAPK activation. Similar antioxidant and protective findings were obtained in LX-2 of co-culture experiments, only. ERs and GPER blockers were able to prevent the effects of 17\u3b2-estradiol and genistein. CONCLUSION: In Huh7.5 and LX-2, 17\u3b2-estradiol and genistein counteract the effects of peroxidation through the involvement of ERs and GPER and by an intracellular signalling related to Akt and p38MAPK. As concerning LX-2, paracrine factors released by Huh7.5 play a key role in protection against oxidative stress

Exposure to Plasma From Non-alcoholic Fatty Liver Disease Patients Affects Hepatocyte Viability, Generates Mitochondrial Dysfunction, and Modulates Pathways Involved in Fat Accumulation and Inflammation

Changes of lipidic storage, oxidative stress and mitochondrial dysfunction may be involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Although the knowledge of intracellular pathways has vastly expanded in recent years, the role and mechanisms of circulating triggering factor(s) are debated. Thus, we tested the hypothesis that factors circulating in the blood of NAFLD patients may influence processes underlying the disease. Huh7.5 cells/primary human hepatocytes were exposed to plasma from 12 NAFLD patients and 12 healthy subjects and specific assays were performed to examine viability, H2O2 and mitochondrial reactive oxygen species (ROS) release, mitochondrial membrane potential and triglycerides content. The involvement of NLRP3 inflammasome and of signaling related to peroxisome-proliferator-activating-ligand-receptor-\u3b3 (PPAR\u3b3), sterol-regulatory-element-binding-protein-1c (SREBP-1c), nuclear-factor-kappa-light-chain-enhancer of activated B cells (NF-kB), and NADPH oxidase 2 (NOX2) was evaluated by repeating the experiments in the presence of NLRP3 inflammasome blocker, MCC950, and through Western blot. The results obtained shown that plasma of NAFLD patients was able to reduce cell viability and mitochondrial membrane potential by about 48 and 24% (p < 0.05), and to increase H2O2, mitochondrial ROS, and triglycerides content by about 42, 19, and 16% (p < 0.05), respectively. An increased expression of SREBP-1c, PPAR\u3b3, NF-kB and NOX2 of about 51, 121, 63, and 46%, respectively, was observed (p < 0.05), as well. Those effects were reduced by the use of MCC950. Thus, in hepatocytes, exposure to plasma from NAFLD patients induces a NAFLD-like phenotype by interference with NLRP3-inflammasome pathways and the activation of intracellular signaling related to SREBP-1c, PPAR\u3b3, NF-kB and NOX2

Periostin Circulating Levels and Genetic Variants in Patients with Non-Alcoholic Fatty Liver Disease

Circulating periostin has been suggested as a possible biomarker in non-alcoholic fatty liver disease (NAFLD) in Asian studies. In the present study, we aimed to test its still controversial relevance in a Caucasian population. In patients with histologically-proven NAFLD (N. = 74; 10 with hepatocellular carcinoma, HCC) plasma periostin concentrations were analyzed. POSTN haplotype analysis was based on rs9603226, rs3829365, and rs1029728. Hepatitis C patients (N. = 81, 7 HCC) and healthy subjects (N. = 27) were used as controls. The median plasma periostin concentration was 11.6 ng/mL without differences amongst groups; it was not influenced by age, liver fibrosis or steatosis. However, possession of haplotype two (rs9603226 = G, rs3829365 = C, rs1028728 = A) was associated with lower circulating periostin compared to other haplotypes. Moreover, periostin was higher in HCC patients. At multivariate analysis, HCC remained the only predictor of high periostin. In conclusion, plasma periostin concentrations in Caucasians NAFLD patients are not influenced by the degree of liver disease, but are significantly higher in HCC. Genetically-determined differences may account for some of the variability. These data suggest extreme caution in predicting a possible future role of periostin antagonists as a rational therapeutic alternative for NAFLD, but show a potential periostin role in the management of NAFLD-associated HCC

Gas6/TAM system: potential prognostic biomarker for Multiple Sclerosis

Introduction: The protein growth arrest specific 6 (Gas6) and its tyrosine kinase receptors Tyro-3, Axl, Mer (TAMs) are ubiquitous proteins involved in regulation of inflammation and apoptotic body clearance. Gas6 and TAMs have been associated with neuronal remyelination and stimulation of oligodendrocyte survival. However, few data are available on their role in multiple sclerosis (MS). Objectives/Aims: Objectives/Aims: In this study we evaluated if soluble levels of these molecules, determined at MS diagnosis in cerebrospinal fluid (CSF) and serum, correlated with progression with short-term disease severity. Methods: Methods: We conducted a retrospective cohort study enrolling 64 patients with different forms of MS, the Radiological Isolated Syndrome (RIS), the Clinical Isolated Syndrome (CIS) and Relapsing-Remitting (RR). At diagnosis, we collected serum, CSF, and clinical-radiological data: lesion load, spinal cord, and gadolinium-enhancing (Gad+) lesions, and expanded disability status score (EDSS). During the last clinical follow-up EDSS, MS severity score (MSSS) and Age-Related MS severity (ARMSS) were assessed. Gas6 and TAMs were determined by ELISA kit (R&D Systems), while neurofilaments (NFLs) levels, for neuronal damage assessment, by SimplePlexTM fluorescence-based immunoassay. Statistical analyses were conducted with STATA software to determine Mann–Whitney, Kruskal-Wallis test and Spearman’s rank correlation coefficient significance. Results: Results: At diagnosis, RIS and CIS showed higher values of sMer and sTyro-3, compared to RRMS (p = 0.007 and p = 0.018). Serum sAxl was higher in patients untreated or first-line disease modifying treatments (DMTs) versus patients with high-efficacy DMTs (p = 0.04). Moreover, serum Axl was associated with EDSS ≤ 3 at diagnosis (p = 0.037) and EDSS progression in patients with EDSS ≤ 3 (p = 0.017). Similarly, high levels of Gas6 in CSF were associated with EDSS ≤ 3 at diagnosis (p = 0.04), and high levels of Gas6 in serum to a lower MSSS (r2 = -0.32 and p = 0.01). Results significances were confirmed by multivariate analyses. In our cohort, serum and CSF NFLs levels were confirmed as markers of disability in EDSS (p = 0.005 and p = 0.002) and MSSS (r2 = 0.27 and p =0.03; r2 = 0.39 and p = 0.001). Conclusion: Conclusion: Taken together, our results suggest that Gas6 and its receptors, particularly Axl, might have a neuroprotective role and prognostic potential in MS. Disclosures: Disclosures: Nothing to disclos

Role of Osteopontin as a Potential Biomarker of Pulmonary Arterial Hypertension in Patients with Systemic Sclerosis and Other Connective Tissue Diseases (CTDs)

Pulmonary arterial hypertension (PAH) is a severe complication of connective tissue diseases (CTD). Its early diagnosis is essential to start effective treatment. In the present paper, we aimed to evaluate the role of plasma osteopontin (OPN) as a candidate biomarker of PAH in a cohort of CTD patients. OPN is a pleiotropic protein involved in inflammation and fibrogenesis and, therefore, potentially promising in this specific clinical context. We performed a cross-sectional observational study on a cohort of 113 CTD patients (females N = 101, 89.4%) affected by systemic sclerosis N = 88 (77.9%), mixed connective tissue disease N = 10 (8.8%), overlap syndrome N = 10 (8.8%) or undifferentiated connective tissue disease N = 5 (4.4%). CTD-PAH patients showed significantly higher OPN plasma values than patients with CTD alone (241.0 (188.8-387.2) vs. 200.7 (133.5-281.6) ng/mL; p = 0.03). Although OPN levels were directly correlated with age and inversely with glomerular filtration rate, they remained associated with PAH at multivariate analysis. In conclusion, OPN was significantly associated with PAH among patients with CTD, suggesting it may have a role as a non-invasive disease biomarker of PAH

Improvement of insulin sensitivity in diabetic and non diabetic patients with chronic hepatitis C treated with direct antiviral agents

The increased incidence of type 2 diabetes mellitus among hepatitis C virus (HCV) infected patients is likely due to viral-induced insulin resistance (IR). Indeed, control of diabetes in these patients benefits of successful antiviral treatment; whether the same applies to subtler alterations of glucose metabolism is unknown. We aimed to fill this gap