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Post-resective liver failure is a frequent complication of liver surgery and it is due to portal hyperperfusion of the remnant liver and to arterial vasoconstriction, as buffer response of the hepatic artery. In this context, splenectomy allows a reduction of portal flow and increases the survival chance in preclinical models. SerpinB3 is over-expressed in the liver in oxidative stress conditions, as a mechanism of cell defense to provide survival by apoptosis inhibition and cell proliferation. In this study, the expression of SerpinB3 was assessed as predictor of liver damage in in vivo models of major hepatic resection with or without splenectomy. Wistar male rats were divided into 4 groups: group A received 30% hepatic resection, group B > 60% resection, group C > 60% resection with splenectomy and group D sham-operated. Before and after surgery liver function tests, echo Doppler ultrasound and gene expression were assessed. Transaminase values and ammonium were significantly higher in groups that underwent major hepatic resection. Echo Doppler ultrasound showed the highest portal flow and resistance of the hepatic artery in the group with > 60% hepatectomy without splenectomy, while the association of splenectomy determined no increase in portal flow and hepatic artery resistance. Only the group of rats without splenectomy showed higher shear-stress conditions, reflected by higher levels of HO-1, Nox1 and of Serpinb3, the latter associated with an increase of IL-6. In conclusion, splenectomy controls inflammation and oxidative damage, preventing the expression of Serpinb3. Therefore, SerpinB3 can be considered as a marker of post-resective shear stress.

Background: Generalized pustular psoriasis (GPP; MIM 614204) is a rare and severe pustular autoinflammatory skin disease in which acute generalized erythema and scaling develop with numerous sterile pustules. GPP shares skin manifestations, especially pustular skin reaction, with adult-onset immunodeficiency (AOID) with anti-interferon-γ autoantibodies, an autoimmune disease. Methods: Clinical examinations and whole-exome sequencing (WES) were performed on 32 patients with pustular psoriasis phenotypes and 21 patients with AOID with pustular skin reaction. Immunohistochemical and histopathological studies were performed. Results: WES identified three Thai patients presenting with similar pustular phenotypes—two with a diagnosis of AOID and the other with GPP. A heterozygous missense variant chr18:g.61325778C>A NM_006919.2: c.438G>T; NP_008850.1: p.Lys146Asn; rs193238900 in SERPINB3 was identified in two patients: one with GPP and the other with AOID. The other patient who had AOID carried a heterozygous missense variant chr18:g.61323147T>C NM_006919.2: c.917A>G; NP_008850.1: p.Asp306Gly in SERPINB3. Immunohistochemical studies showed overexpression of SERPINA1 and SERPINB3, a hallmark of psoriatic skin lesions. Conclusions: Genetic variants in SERPINB3 are associated with GPP and AOID with pustular skin reaction. The skin of patients with GPP and AOID carrying SERPINB3 mutations showed overexpression of SERPINB3 and SERPINA1. Clinically and genetically, GPP and AOID appear to share pathogenetic mechanisms.

Background & Aims Hypoxia and hypoxia-inducible factors (HIFs) are involved in chronic liver disease progression. We previously showed that hepatocyte HIF-2α activation contributed significantly to nonalcoholic fatty liver disease progression in experimental animals and human patients. In this study, using an appropriate genetic murine model, we mechanistically investigated the involvement of hepatocyte HIF-2α in experimental nonalcoholic steatohepatitis (NASH)-related carcinogenesis. Methods The role of HIF-2α was investigated by morphologic, cellular, and molecular biology approaches in the following: (1) mice carrying hepatocyte-specific deletion of HIF-2α (HIF-2α-/- mice) undergoing a NASH-related protocol of hepatocarcinogenesis; (2) HepG2 cells stably transfected to overexpress HIF-2α; and (3) liver specimens from NASH patients with hepatocellular carcinoma. Results Mice carrying hepatocyte-specific deletion of HIF-2α (hHIF-2α-/-) showed a significant decrease in the volume and number of liver tumors compared with wild-type littermates. These effects did not involve HIF-1α changes and were associated with a decrease of cell proliferation markers proliferating cell nuclear antigen and Ki67. In both human and rodent nonalcoholic fatty liver disease–related tumors, HIF-2α levels were strictly associated with hepatocyte production of SerpinB3, a mediator previously shown to stimulate liver cancer cell proliferation through the Hippo/Yes-associated protein (YAP)/c-Myc pathway. Consistently, we observed positive correlations between the transcripts of HIF-2α, YAP, and c-Myc in individual hepatocellular carcinoma tumor masses, while HIF-2α deletion down-modulated c-Myc and YAP expression without affecting extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and AKT-dependent signaling. In vitro data confirmed that HIF-2α overexpression induced HepG2 cell proliferation through YAP-mediated mechanisms. Conclusions These results indicate that the activation of HIF-2α in hepatocytes has a critical role in liver carcinogenesis during NASH progression, suggesting that HIF-2α–blocking agents may serve as novel putative therapeutic tools., Graphical abstract

Resume Objectif La pneumopathie interstitielle diffuse (PID) est un facteur majeur de pronostic chez les patients atteints de sclerodermie systemique (ScS). L’antigene de carcinome a cellules squameuses (SCCA1) est un inhibiteur des proteases a serine qui joue un role essentiel dans l’inflammation et la fibrose. Le SCCA1 est surexprime dans le tissu pulmonaire des patients presentant une fibrose pulmonaire idiopathique et peut etre detecte dans le serum comme un complexe immun circulant lie a l’IgM (SCCA-IgM). Nous avons souhaite explorer l’association entre le SCCA-IgM et les caracteristiques cliniques des patients atteints de ScS. Methodes Quatre-vingt-dix-sept patients consecutifs atteints de ScS (selon les criteres ACR/EULAR) ont ete recrutes dans l’etude. Les variables cliniques et serologiques et les atteintes d’organe ont ete consignees. L’atteinte pulmonaire a ete exploree par tomodensitometrie haute resolution (TDM-HR) et par des tests de la fonction respiratoire. Les taux seriques de SCCA-IgM ont ete mesures par un test ELISA valide (Hepa-IC, Xeptagen, Venise, Italie). Nous avons defini le seuil des valeurs seriques de SCCA-IgM > 200 UA/mL, calcule comme la moyenne + 3 ecarts types chez 100 sujets en bonne sante. Resultats Quarante-et-un patients (42,3 %) presentaient une PID. Les taux de SCCA-IgM etaient significativement plus eleves chez les patients avec PID que chez les patients sans PID : 218 (80–402) contre 87,5 (59–150) UA/mL, p Conclusion Le SCCA-IgM est associe a la pneumopathie interstitielle diffuse chez les patients sclerodermiques et pourrait etre utilise dans l’evaluation de la PID associee a la ScS.

Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of OSM was investigated in (1) selected cohorts of NAFLD/NASH HCC patients, (2) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM, (3) murine HCC xenografts, and (4) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM upregulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. © 2022 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease worldwide. In 20-30% of patients, NAFLD can progress into non-alcoholic steatohepatitis (NASH), eventually leading to fibrosis, cirrhosis and hepatocellular carcinoma development. SerpinB3 (SB3), a hypoxia-inducible factor-2α dependent cysteine protease inhibitor, is up-regulated in hepatocytes during progressive NAFLD and proposed to contribute to disease progression. In this study we investigated the pro-inflammatory role of SB3 by employing phorbol-myristate acetate-differentiated human THP-1 macrophages exposed in vitro to human recombinant SB3 (hrSB3) along with mice overexpressing SB3 in hepatocytes (TG/SB3) or knockout for SB3 (KO/SB3) in which NASH was induced by feeding methionine/choline deficient (MCD) or a choline-deficient, L-amino acid defined (CDAA) diets. In vivo experiments showed that the induction of NASH in TG/SB3 mice was characterized by an impressive increase of liver infiltrating macrophages that formed crown-like aggregates and by an up-regulation of hepatic transcript levels of pro-inflammatory cytokines. All these parameters and the extent of liver damage were significantly blunted in KO/SB3 mice. In vitro experiments confirmed that hrSB3 stimulated macrophage production of M1-cytokines such as TNFα and IL-1β and reactive oxygen species along with that of TGFβ and VEGF through the activation of the NF-kB transcription factor. The opposite changes in liver macrophage activation observed in TG/SB3 or KO/SB3 mice with NASH were associated with a parallel modulation in the expression of triggering receptor expressed on myeloid cells-2 (TREM2), CD9 and galectin-3 markers, recently detected in NASH-associated macrophages. From these results we propose that SB3, produced by activated/injured hepatocytes, may operate as a pro-inflammatory mediator in NASH contributing to the disease progression.

Squamous cell carcinoma antigen‐1 (SCCA1) overexpression is associated with poor prognosis and chemoresistance in several tumor types, however, the underlying mechanisms remain elusive. Here, we report SCCA1 in relation to the immune and peritumoral adipose tissue microenvironment in early and advanced esophageal adenocarcinoma (EAC). In our series of patients with EAC, free SCCA1 serum levels were associated with significantly worse overall survival, and SCCA1‐IgM serum levels showed a trend to a worse overall survival. Serum SCCA1 and intratumoral SCCA1 were inversely correlated with immune activation markers. In agreement with these findings, SCCA1 induced the expression of the immune checkpoint molecule programmed death ligand‐1 on monocytes and a direct correlation of these 2 molecules was observed in sequential tumor sections. Furthermore, SCCA1 mRNA expression within the tumor was inversely correlated with stem cell marker expression both within the tumor and in the peritumoral adipose tissue. In vitro, in EAC cell lines treated with different chemotherapeutic drugs, cell viability was significantly modified by SCCA1 presence, as cells overexpressing SCCA1 were significantly more resistant to cell death. In conclusion, poor prognosis in EAC overexpressing SCCA1 is due to reduced tumor chemosensitivity as well as intratumoral immunity impairment, likely induced by this molecule.

Background and aims: Cholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem-like compartment is available. Methods: Enrichment of CCA stem-like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT-PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC-like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3-transfected MON (MONSB3+) cells in immune-deficient NOD-SCID/IL2Rgnull (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients. Results: SB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c-MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (β-catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3-overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up-regulation of the proto-oncogene β-catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients. Conclusion: These findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

Aims Cardiac complications are a leading cause of mortality after orthotopic liver transplantation (LT) and pre-operative risk stratification is challenging. We evaluated whether coronary artery calcium (CAC) score calculated on a standard (non-thin layer, non-ECG gated) chest computed tomography (CT) predicted cardiac outcome after LT. Methods We included a consecutive series of LT recipients who underwent pre-operative cardiac evaluation including stress-testing or cardiac catheterization in high-risk patients. Patients with a history of coronary artery disease or coronary revascularization were excluded. The CAC score was calculated from the chest CT routinely performed before LT. CAC values were not available at the time of pre-transplant cardiac evaluation and did not affect LT eligibility. The primary end-point included peri-operative arrhythmic cardiac arrest and sustained ventricular arrhythmias; heart failure, myocardial infarction and cardiac death within 1-year after LT. Results The study population consisted of 301 patients (median age 56 years, 76% males). At chest CT, 49% had CAC = 0; 27% had CAC = 1–99, 15% had CAC = 100–399 and 9% CAC > 400. The primary end-point incidence increased from 7% in patients with CAC = 0 to 27% in patients with CAC > 400 (p = 0.007). At multivariable analysis including traditional risk factors, CAC remained an independent predictor of cardiac events (p = 0.01). Conclusions CAC score calculated on a standard chest CT stratified the risk of cardiac events in patients who underwent LT after negative pre-transplant cardiac evaluation. These findings suggest that evaluation of CAC from a standard chest CT performed for other reasons can be used as an early cardiac risk stratification tool before LT.

Simple Summary Cell proliferation and escape from apoptosis are important pathological features of hepatocellular carcinoma, one of the tumors with the highest mortality rate worldwide. The aim of the study was to evaluate the expression of the pro-apoptotic p66shc and the anti-apoptotic SerpinB3 molecules in relation to clinical outcome in patients with hepatocellular carcinoma and to evaluate their effect on cell fate and tumor growth. In patients with hepatocellular carcinoma the best survival was observed in the subgroup with p66shc levels below median values and SerpinB3 levels above median values. Mice p66shc knockout showed high levels of SerpinB3, while in hepatoma cells overexpressing SerpinB3, p66shc expression was trivial. Hepatoma cells overexpressing SerpinB3 were more prone to die after oxidizing treatments. These cells injected in nude mice developed tumors five times smaller than those from controls. Tumors originating from hepatoma cells overexpressing SerpinB3 showed typical features of necroptosis. In conclusion, in patients affected by hepatocellular carcinoma, the pattern characterized by p66shc downregulation and elevated SerpinB3 levels was associated with markedly better survival. This pattern favored necroptosis in experimental high-stress conditions. Abstract Cell proliferation and escape from apoptosis are important pathological features of hepatocellular carcinoma (HCC), one of the tumors with the highest mortality rate worldwide. The aim of the study was to evaluate the expression of the pro-apoptotic p66shc and the anti-apoptotic SerpinB3 in HCCs in relation to clinical outcome, cell fate and tumor growth. p66shc and SerpinB3 were evaluated in 67 HCC specimens and the results were correlated with overall survival. Proliferation and cell death markers were analyzed in hepatoma cells overexpressing SerpinB3, under different stress conditions. p66shc−/− mice and xenograft models were also used to assess the effects of p66shc and SerpinB3 on tumor growth. In patients with HCC, the best survival was observed in the subgroup with p66shc levels below median values and SerpinB3 levels above median values. Mice p66shc−/− showed high levels of SerpinB3, while in HepG2 cells overexpressing SerpinB3, p66shc expression was trivial. HepG2 overexpressing SerpinB3 cells were more prone to die after oxidizing treatments, such as diamide or high concentration H2O2. These cells injected in nude mice developed tumors five times smaller than those from control HepG2 cells. Tumors originating from HepG2 overexpressing SerpinB3 cells showed decreased activated Caspase-8, with concomitant increase of RIP3K and decreased levels of cleaved RIP3K, typical features of necroptosis. In conclusion, in patients affected by HCC, the pattern characterized by p66shc downregulation and elevated SerpinB3 levels was associated with markedly better survival. This pattern favored necroptosis in experimental high-stress conditions.

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[Background] The combination of alpha-fetoprotein (AFP) and squamous cell carcinoma antigen immunocomplex (SCCA-IgM) have been proposed for its use in the screening of hepatocellular carcinoma (HCC). Current screening programs for all cirrhotic patients are controversial and a personalized screening is an unmet need in the precision medicine era., [Aim] To determine the role of the combination of SCCA-IgM and AFP in predicting mid- and long-term appearance of HCC., [Methods] Two-hundred and three cirrhotic patients (Child A 74.9%, B 21.2%, C 3.9%) were followed-up prospectively every six months to screen HCC by ultrasound and AFP according to European Association for the Study of the Liver guidelines. The estimation cohort was recruited in Italy (30.5%; 62/203) and validation cohort from Spain (69.5%; 141/203). Patients underwent to evaluate SCCA-IgM by enzyme-linked immunosorbent assay (Hepa-IC, Xeptagen, Italy) and AFP levels at baseline. Patients were followed-up for 60 mo, being censored at the time of the appearance of HCC., [Results] There were 10.8% and 23.1% of HCC development at two- and five-years follow-up. Patients with HCC showed higher levels of SCCA-IgM than those without it (425.72 ± 568.33 AU/mL vs 195.93 ± 188.40 AU/mL, P = 0.009) during the five-year follow-up. In multivariate analysis, after adjusting by age, sex, aspartate transaminase and Child-Pugh, the following factors were independently associated with HCC: SCCA-IgM [Hazard ratio (HR) = 1.001, 95%CI: 1.000-1.002; P = 0.003], AFP (HR = 1.028, 95%CI: 1.009-1.046; P = 0.003) and creatinine (HR = 1.564 95%CI: 1.151-2.124; P = 0.004). The log-rank test of the combination resulted in 7.488 (P = 0.024) in estimation cohort and 11.061 (P = 0.004) in the validation cohort, and a 100% of correctly classified rate identifying a low-risk group in both cohorts in the two-year follow-up., [Conclusion] We have constructed a predictive model based on the combination of SCCA-IgM and AFP that provides a new HCC screening method, which could be followed by tailored HCC surveillance for individual patients, especially for those cirrhotic patients belonging to the subgroup identified as low-risk of HCC development.

This paper deals with the combination of a proline-based moiety with biologically active gold centers in the oxidation states +1 and +3. In particular, six Au(i)/(iii)-proline dithiocarbamato (DTC) complexes with general formulae [Au(DTC)2] and [AuIIIX2(DTC)] (X = Cl, Br) are reported here. After the synthesis of the ligand and the complexes, all derivatives were characterized via several techniques and tested for their stability in DMSO/water media. This study was focused on the demonstration of a peculiar behavior of Au(iii)-DTC species in solution. Finally, the complexes were screened for their antiproliferative activity against 2 human cancer cell lines, namely HepG2 and HepG2/SB3, taken as models of hepatocellular carcinoma. The latter, chosen for its aggressiveness due to the upregulation of the anti-apoptotic protein SerpinB3, was selectively inhibited in terms of growth by some Au(iii)-DTC complexes.

Aims In hepatocellular carcinoma (HCC), the regulatory protease Dipeptidyl-peptidase IV (DPPIV/CD26), that possesses pro-apoptotic properties, has been found abnormally regulated. The protease inhibitor SerpinB3, exerting anti-apoptotic activity, has also been described to be upregulated, especially in HCCs with poor prognosis. The aim of this study was to investigate the possible relationship between these two molecules in HCC patients and in experimental models. Materials and methods DPPIV/CD26 and SerpinB3 expression was measured in liver specimens of 67 patients with HCC. HepG2 and Huh7 cells, stably transfected to overexpress SerpinB3, and respective control cells were used to assess biological and metabolic modifications of DPPIV/CD26 activity induced by this serpin. Key findings DPPIV/CD26 and SerpinB3 were localized in the same tumoral areas and both molecules were correlated with the grade of tumor differentiation, with the highest values detected in GI tumors. Cell lines over-expressing SerpinB3 displayed upregulation of DPPIV/CD26, likely as a feedback mechanism, due to the DPPIV/CD26 protease activity inhibition by SerpinB3, as confirmed by the similar behavior induced by the inhibitor Sitagliptin. Moreover, they exhibited lower glycogen storage and higher lipid accumulation, typical effects of DPPIV/CD26. Significance A close connection between SerpinB3 and DPPPIV has been identified, but further studies are required to better understand the mechanism by which these proteins communicate and exert metabolic effects in HCC.

Tenofovir and entecavir are nowadays the first-line treatment in hepatitis B virus (HBV)-related cirrhosis. Both drugs were shown to be effective in HBV suppression and well tolerated. The effects of tenofovir on bone mineral density (BMD), however, were shown to worsen the rate of osteoporosis, which is already a common feature in cirrhosis. In contrast, entecavir seems to have no effect on mineral metabolism. The aim of our study was to compare the effects of nucleos(t)ide analogs on bone density in HBV-related cirrhosis.Fourty-eight patients were treated with tenofovir and 22 patients were treated with entecavir, and were followed prospectively from 2008 to 2013. To evaluate BMD, laboratory examinations, dual-X-ray absorptiometry, and Fracture Risk Assessment Tool were assessed.During the study, no difference was found between the two groups in the plasmatic concentration of calcium, phosphate, vitamin D, parathyroid hormone, or creatinine. Dual-X-ray absorptiometry showed no difference in the T-score and Fracture Risk Assessment Tool showed no significant difference in the 10-year risk of osteoporotic fractures in the two groups. On univariate and multivariate analyses, the only predictors of osteoporosis development were the prognostic scores of liver disease and BMI.Both tenofovir and entecavir are effective in treating HBV in cirrhotic patients. The known effects of tenofovir on BMD do not worsen osteoporotic fractures risk compared with entecavir in these patients.

BACKGROUND The role of serum biomarkers in the surveillance of hepatocellular carcinoma (HCC) is controversial. OBJECTIVE We assessed the diagnostic performances of alpha-fetoprotein (AFP) and protein-induced by vitamin-K-absence/antagonist-II (PIVKA-II) in 388 cirrhotic patients with chronic liver disease (CLD). METHODS Biomarkers were quantified by automated chemiluminescent-enzyme-immunoassays (Fujirebio, Tokyo, Japan) at HCC diagnosis in 258 patients (204 males; median age 66.9 years) and in 130 cirrhotics without HCC (104 males; median-age 60.6 years). CLD etiology in HCC/non-HCC was CHB in 48/35, CHC in 126/56 and Non-Viral in 84/39. RESULTS Overall AUROC values for AFP and PIVKA-II were 0.698 (95%CI = 0.642-0.753, P< 0.001) and 0.780 (95%CI = 0.730-0.831, P< 0.001). AFP/PIVKA-II AUROC (95%CI) were: 0.822 (0.728-0.915)/0.833 (0.739-0.926) in CHB, 0.648 (0.560-0.736)/0.732 (0.650-0.814) in CHC; 0.640 (0.540-0.740)/0.806 (0.722-0.889) in Non-Viral-CLD. AFP/PIVKA-II diagnostic accuracy was 40.5-59.8%/62.7-73.5% and combining both markers 78.2% for CHB, 77% for Non-Viral-CLD and 75% for CHC. AFP correlated with ALT in HCC patients with CHC (ρ= 0.463/P< 0.001) and Non-Viral CLD (ρ= 0.359/P= 0.047), but not in CHB (treated with antivirals). PIVKA-II correlated with tumour size independently of CLD-etiology (P< 0.001) and AFP in CHB patients only (P= 0.007). CONCLUSION The diagnostic performance of AFP and PIVKA-II is significantly influenced by the etiology and activity of CLD; their combination provides a better diagnostic accuracy.

A recent study on our metal-dithiocarbamato complexes pointed out the antiproliferative properties and the druglikeness of some new patented derivatives. In this work, the best compounds have been encapsulated in micellar nanocarriers, being also carbohydrate-functionalized on their hydrophilic surface to investigate the possibility of a cancer-selective delivery. In particular, the nonionic block copolymer Pluronic® F127 (PF127) has been chemically modified with sugars and the derivatives characterized by means of NMR spectroscopy and FT-IR spectrophotometry. Then, the two selected complexes (β-[Ru2(PipeDTC)5]Cl (PipeDTC = piperidine dithiocarbamate) and [Cu(ProOMeDTC)2] (ProOMeDTC = L-proline methyl ester dithiocarbamate)), have been loaded into the hydrophobic core of PF127 micelles and cancer-targeting counterparts. These nanoformulations have been studied for their dimensions (DLS, TEM) and stability, and tested for their cytotoxicity against aggressive human cancer cell lines. The in vitro results were paralleled with mechanistic studies through Confocal Laser Scanning Microscopy and xCELLigence analysis.

In 2012, the KDIGO group proposed new definitions for acute kidney injury (AKI), acute kidney disease (AKD) and chronic kidney disease (CKD). According to the definition adapted by the International Club of Ascites, AKI has been extensively investigated in patients with cirrhosis. On the contrary, there are currently no data on the epidemiology and clinical outcomes associated with AKD. The aim of the study was to assess the prevalence and the impact of AKD on the clinical course and survival of patients with cirrhosis.A total of 272 consecutive patients with cirrhosis attending our outpatient clinic were included in the study. Clinical and laboratory data were collected at inclusion. Patients were followed-up until death, liver transplant or the end of follow-up.During follow-up, 80 patients developed AKD (29.4%). Forty-two (52.5%) recovered from the first episode of AKD and 26 maintained a normal renal function up to the end of follow-up. Sixteen patients developed a second episode of AKD. Globally, 36 patients (45.0%) died with AKD. Finally, AKD progressed to CKD in 11 patients (13.8%). The 5-year survival rate was significantly lower in patients who developed AKD than in those who did not (34.8% vs. 88.8%, p0.001). The 5-year rates of complications of cirrhosis and of hospitalizations were also higher in patients with AKD than in those without AKD.AKD is frequent in patients with cirrhosis. It can be reversible, but it may recur and progress to CKD. AKD has a very negative impact on morbidity and mortality in patients with cirrhosis.Renal impairment has a very negative impact on patients with cirrhosis. Renal impairment seems to be characterized by a very dynamic course, which is defined according to renal function and length of the impairment as acute kidney injury, acute kidney disease and chronic kidney disease. The role of acute kidney disease is currently unknown. Our study shows for the first time that acute kidney disease is frequent in patients with cirrhosis and has a very negative impact on survival.

BACKGROUND: The time-related variability of HCC biomarkers has not been investigated so far. OBJECTIVE: To assess the changes of alpha-fetoprotein (AFP) and protein induced by vitamin-K absence/antagonist-II (PIVKA-II) in patients with HCC (HCC+) as compared to patients without HCC (HCC-). METHODS: AFP and PIVKA-II were measured by a single laboratory using an automated chemiluminescent-enzyme-immunoassay (Fujirebio Inc., Tokyo, Japan) in 1163 sera of 418 cirrhotics (31.1% HBV, 58.6% HCV, 10.3% non-viral etiology) undergoing ultrasound HCC surveillance. The mean (range) number of effective time-points available for analysis was 2.8 (2.0 to 3.0); 124 patients with HCC were matched with 294 who remained HCC free for at least 12 months after the last specimen. AFP and PIVKA-II changes were estimated over time by means of a random-effect generalized least squares (RE-GLS) regression model under the missingness at random assumption. RESULTS: Patients with and without HCC had comparable chronic liver disease etiology and staging. AFP/PIVKA-II median (25th; 75th percentile) values at the latest time-point were 4.2 (2.6; 8.6) ng/mL/32 (25; 42) mAU/mL in HCC- and 8.4 (4.4; 32.1) ng/mL/66 (32; 192) mAU/mL in HCC+ (p< 0.001). Log10AFP and log10PIVKA-II time-changes differed in HCC+ and HCC- patients. In HCC+ patients, both log10AFP and log10PIVKA-II showed an increasing trend over time. In HCC- patients, log10PIVKA-II variations were minimal as compared to log10AFP variations. The percent increase of log10AFP at 6 months vs. baseline was 11% (95%CI 5 to 17%) and 5% (95%CI 1 to 8%) for log10PIVKA-II in HCC+vs. HCC- patients. CONCLUSIONS: The present retrospective study of the biological variability of AFP and PIVKA-II suggests that their time-related changes may serve as potential predictors of HCC. This topic needs to be addressed by longitudinal studies.

Introduction and objectives: SerpinB3 is a cysteine protease inhibitor involved in several biological activities. It is progressively expressed in chronic liver disease, but not in normal liver. The role in vascular reactivity of this serpin, belonging to the same family of Angiotensin II, is still unknown. Our aim was to evaluate the in vivo and in vitro effects of SerpinB3 on systemic and splanchnic hemodynamics. Material and methods: Different hemodynamic parameters were evaluated by ultrasonography in two colonies of mice (transgenic for human SerpinB3 and C57BL/6J controls) at baseline and after chronic carbon tetrachloride (CCl4) treatment. In vitro SerpinB3 effect on mesenteric microvessels of 5 Wistar-Kyoto rats was analyzed measuring its direct action on: (a) preconstricted arteries, (b) dose–response curves to phenylephrine, before and after inhibition of angiotensin II type 1 receptors with irbesartan. Hearts of SerpinB3 transgenic mice and of the corresponding controls were also analyzed by morphometric assessment. Results: In SerpinB3 transgenic mice, cardiac output (51.6 ± 21.5 vs 30.1 ± 10.8 ml/min, p = 0.003), hepatic artery pulsatility index (0.85 ± 0.13 vs 0.65 ± 0.11, p

Background: Infection can trigger autoimmunity but the nature of the pathogen could enhance or inhibit the onset of the disease. In this study we tested the tolerogenic effect of the surface Hepatitis B Virus capside protein preS1 in a lupus mouse model. Objectives: To evaluate the effect of administration of Hepatitis B virus preS1 subunit to NZB/WF1 mice Methods: Mice (10 each group) were injected with a total volume of 200μl of 32.5 μg PreS1 in 200μl PBS (group 1), 200μl PBS (group 2), 3 times at 11th, 14th and 17th weeks of age. Urine samples were collected weekly to evaluate proteinuria by reactive strips. Blood samples were collected before every injection, at 22th and 28th weeks, and at death to evaluate levels of anti-C1q and anti-dsDNA by home-made ELISA tests. Tissues were harvested for histological analyses. Proteinuria free and survival rates were analyzed by Kaplan-Meier method using Mantel-Cox test for comparisons. Neurocognitive functions were evaluated with Staircase, Y-Maze and Forced Swim Test. Results: Anti-dsDNA and Anti-C1q levels (OD±SD) were significantly higher in PBS than in preS1 treated mice (Figure 1). Mean proteinuria levels (mg/dl±SD) were higher in PBS than in preS1 treated mice. Proteinuria free survival rate ( Severe segmental and focal glomerulonephritis in both groups with large proliferation of mesangial and endothelial cells and Russell bodies were found by histological analysis. Mice treated with preS1 had more chronic inflammation in the kidneys and extramedullary hematopoiesis in the spleen and liver. Notably, in salivary glands, severe acute inflammation was observed in group 1 and chronic inflammation in group 2. Conclusion: HBV capsid protein preS1 causes a delay in the onset of lupus-like glomerulonephrits and ameliorates behavioral disorders in NZW/BF1 mice. This peptide seems to induce a tolerizing rather than a pathogenic effect in these autoimmune prone mice models. References: [1] F. McKinneyet al; T-cell exhaustion, co-stimulation and clinical outcome in autoimmunity and infection. Nature523, 612–616 (2015). [2] T-cell exhaustion, co-stimulation and clinical outcome in autoimmunity and infection. Nature523, 612–616 (2015). [3] D. R. Gettset al, Virus infection, antiviral immunity, and autoimmunity. Immunol. Rev. 255, 197–209 (2013) Disclosure of Interests: None declared

Cirrhosis represents the end stage of chronic liver disease, characterized by high mortality and morbidity. The prevalence of liver disease is difficult to assess, given its clinical latency up to the late stage.We aimed to assess the prevalence of unrecognized chronic liver disease and cirrhosis using surrogate indicators from medical records of family physicians.Medical records of 139,104 subjects, collected from 99 family physicians of the Veneto region, were used. Persistently high transaminases were used as indicators of occult chronic liver disease; thrombocytopenia, unrelated to haematological malignancies, was used as indicator of occult cirrhosis. Diagnosis of chronic liver disease and cirrhosis was assessed using ICD9-CM-1997 codes.Alteration of transaminases was found in 32.7% of the subjects, and among them only one-third had an already diagnosed liver disease. Patients with diagnosis of cirrhosis were 0.3%, while thrombocytopenia, indicator of occult cirrhosis, was detected in 1.3% of the remaining population. Patients with overt and occult cirrhosis showed a higher metabolic profile, with significantly higher prevalence of arterial hypertension, obesity and diabetes than the general population.A large proportion of patients with chronic liver disease is still undiagnosed. Surrogate biochemical indicators might be useful for disease recognition.

// Matteo Fassan 1 , Stefano Realdon 2 , Luca Vianello 1 , Santina Quarta 3 , Alberto Ruol 4 , Carlo Castoro 5 , Marco Scarpa 5 , Giovanni Zaninotto 6 , Vincenza Guzzardo 1 , Vanna Chiarion Sileni 7 , Patrizia Pontisso 3 , Massimo Rugge 1, 8 1 Department of Medicine (DIMED), Surgical Pathology Unit, University of Padua, Padua, Italy 2 Gastroenterology Unit, Istituto Oncologico Veneto, IOV-IRCCS, Padua, Italy 3 Department of Medicine (DIMED), 5th Medical Clinic, University of Padua, Padua, Italy 4 Department of Surgical, Oncological and Gastroenterological Sciences (DiSCOG), 3rd Surgical Clinic, University of Padua, Padua, Italy 5 Esophageal and Digestive Tract Surgical Unit, Veneto Institute of Oncology, IOV-IRCCS, Padua, Italy 6 Imperial College London, Department of Surgery and Cancer, Division of Surgery, London, UK 7 Melanoma & Esophageal Oncology Unit, Istituto Oncologico Veneto, IOV-IRCCS, Padua, Italy 8 Veneto Tumour Registry, Veneto Region, Padua, Italy Correspondence to: Patrizia Pontisso, email: patrizia@unipd.it Keywords: SCCA, esophageal adenocarcinoma, Barrett’s carcinogenesis, preneoplastic lesions Received: July 29, 2016 Accepted: November 22, 2016 Published: December 22, 2016 ABSTRACT Squamous Cell Carcinoma Antigen (SCCA) is consistently overexpressed in many different solid tumors, and has been associated with both tumor aggressiveness and chemoresistance. No data, however, is currently available on SCCA expression during esophageal Barrett’s carcinogenesis, nor on SCCA expression’s role on esophageal adenocarcinoma chemoresistance. The SCCA immunohistochemical expression was assessed in a series of 100 biopsy samples covering the whole histological spectrum of Barrett’s oncogenesis. Squamous native mucosa was characterized by a moderate to strong cytoplasmic and nuclear SCCA expression in suprabasal, medium, and superficial layers. On the other hand, almost half of the considered lesions did not express SCCA; the other half featured weak to moderate SCCA expression. The relationship between SCCA protein expression and tumor response to neoadjuvant chemotherapy was assessed in 90 esophageal adenocarcinoma specimens (40 biopsy and 50 surgery specimens), stratified according to Mandard tumor regression grade. As observed in other settings, the presence of SCCA expression clustered in the group of tumors characterized by a lower responsiveness to neoadjuvant treatments. The present results suggest an involvement of SCCA in a subset of Barrett-related tumors, and prompt to consider the SCCA-protein expression as response-predictive marker of neoadjuvant therapy in esophageal adenocarcinomas.

After the first successful pregnancy in a liver transplant recipient in 1978, much evidence has accumulated on the course, outcomes and management strategies of pregnancy following liver transplantation. Generally, liver transplantation restores sexual function and fertility as early as a few months after transplant. Considering that one third of all liver transplant recipients are women, that approximately one-third of them are of reproductive age (18–49 years), and that 15% of female liver transplant recipients are paediatric patients who have a >70% probability of reaching reproductive age, the issue of pregnancy after liver transplantation is rather relevant, and obstetricians, paediatricians, and transplant hepatologists ever more frequently encounter such patients. Pregnancy outcomes for both the mother and infant in liver transplant recipients are generally good, but there is an increased incidence of preterm delivery, hypertension/preeclampsia, foetal growth restriction, and gestational diabetes, which, by definition, render pregnancy in liver transplant recipients a high-risk one. In contrast, the risk of congenital anomalies and the live birth rate are comparable to those of the general population. Currently there are still no robust guidelines on the management of pregnancies after liver transplantation. The aim of this position paper is to review the available evidence on pregnancy in liver transplant recipients and to provide national Italian recommendations for clinicians caring for these patients.

The only first-line treatment approved for advanced hepatocellular carcinoma (HCC) is sorafenib. Since many patients experience drug resistance, the discovery of more effective therapeutic strategies represents an unmet clinical need. MicroRNA (MiR)-122 is downregulated in most HCCs, while oncogenic SerpinB3 is upregulated. Here, we assessed the relationship between miR-122 and SerpinB3 and their influence on cell phenotype and sorafenib resistance in HCC. A bioinformatics analysis identified SerpinB3 among hypothetical miR-122 targets. In SerpinB3-overexpressing HepG2 cells, miR-122 transfection decreased SerpinB3 mRNA and protein levels, whereas miR-122 inhibition increased SerpinB3 expression. Luciferase assay demonstrated the interaction between miR-122 and SerpinB3 mRNA. In an HCC rat model, high miR-122 levels were associated with negative SerpinB3 expression, while low miR-122 levels correlated with SerpinB3 positivity. A negative correlation between miR-122 and SerpinB3 or stem cell markers was found in HCC patients. Anti-miR-122 transfection increased cell viability in sorafenib-treated Huh-7 cells, while miR-122 overexpression increased sorafenib sensitivity in treated cells, but not in those overexpressing SerpinB3. In conclusion, we demonstrated that miR-122 targets SerpinB3, and its low levels are associated with SerpinB3 positivity and a stem-like phenotype in HCC. MiR-122 replacement therapy in combination with sorafenib deserves attention as a possible therapeutic strategy in SerpinB3-negative HCCs.

Background: SerpinB3 (SB3) is a hypoxia and hypoxia-inducible factor (HIF)-2&alpha, dependent cysteine-protease inhibitor up-regulated in hepatocellular carcinoma (HCC), released by cancer cells and able to stimulate proliferation and epithelial-to-mesenchymal-transition. Methods: In the study we employed transgenic and knock out SerpinB3 mice, liver cancer cell line, human HCC specimens, and mice receiving diethyl-nitrosamine (DEN) administration plus choline-deficient L-amino acid refined (CDAA) diet (DEN/CDAA protocol). Results: We provide detailed and mechanistic evidence that SB3 can act as a paracrine mediator able to affect the behavior of surrounding cells by differentially up-regulating, in normoxic conditions, HIF-1&alpha, and HIF-2&alpha, SB3 acts by (i) up-regulating HIF-1&alpha, transcription, facilitating cell survival in a harsh microenvironment and promoting angiogenesis, (ii) increasing HIF-2&alpha, stabilization via direct/selective NEDDylation, promoting proliferation of liver cancer cells, and favoring HCC progression. Moreover (iii) the highest levels of NEDD8-E1 activating enzyme (NAE1) mRNA were detected in a subclass of HCC patients expressing the highest levels of HIF-2&alpha, transcripts, (iv) mice undergoing DEN/CDAA carcinogenic protocol showed a positive correlation between SB3 and HIF-2&alpha, transcripts with the highest levels of NAE1 mRNA detected in nodules expressing the highest levels of HIF-2&alpha, transcripts. Conclusions: These data outline either HIF-2&alpha, and NEDDylation as two novel putative therapeutic targets to interfere with the procarcinogenic role of SerpinB3 in the development of HCC.

Early detection is the most effective mean of improving prognosis for many fatal diseases such as cancer. In this context, the Surface Enhanced Resonance Raman Scattering (SERRS) technique is being proposed as alternative to fluorescent methods in detection of biomarkers, because SERRS nanostructures are bright as fluorescent tags but more stable and clearly detectable using the narrow Raman “fingerprints” of a suitable reporter. Here we show that biocompatible SERRS active gold nanostructures, functionalized with an engineered PreS1 peptide (AuNP@PEG-PreS1), detect the presence of the SerpinB3 antigen overexpressed on liver tumor cells, a biomarker of the onset of liver cell carcinomatous transformation. A proper engineering of the targeting unit, linked to the nanostructure by a polymer chain, affords a sensitivity and specificity larger than 80%, at subnanomolar concentrations. Taking into account the high sensitivity of SERRS and that SB3 overexpression is an early event in liver cell carcinomatous transformation, AuNP@PEG-PreS1 nanostructures could be used in routine diagnostic activities, to improve the accuracy of HCC detection in particular in patients with chronic liver diseases.