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Simple Summary: This review highlights SerpinB3's multifaceted roles in liver disease, from fibrosis, carcinogenesis and immune modulation to cell death protection. In different types of cancer its overexpression correlates with tumor aggressiveness; however, in acute oxidative stress conditions, SerpinB3 promotes cell survival. Novel therapeutic strategies targeting SerpinB3 through its upstream regulators are under development, while its therapeutic potential in acute medical conditions has also been proposed. SerpinB3, a member of the serine-protease inhibitor family, has emerged as a crucial player in various physiological and pathological processes. Initially identified as an oncogenic factor in squamous cell carcinomas, SerpinB3's intricate involvement extends from fibrosis progression and cancer to cell protection in acute oxidative stress conditions. This review explores the multifaceted roles of SerpinB3, focusing on its implications in fibrosis, metabolic syndrome, carcinogenesis and immune system impairment. Furthermore, its involvement in tissue protection from oxidative stress and wound healing underscores its potential as diagnostic and therapeutic tool. Recent studies have described the therapeutic potential of targeting SerpinB3 through its upstream regulators, offering novel strategies for cancer treatment development. Overall, this review underscores the importance of further research to fully elucidate the mechanisms of action of SerpinB3 and to exploit its therapeutic potential across various medical conditions. [ABSTRACT FROM AUTHOR]

LINKED CONTENT: This article is linked to Martini et al papers. To view these articles, visit https://doi.org/10.1111/apt.17804 and https://doi.org/10.1111/apt.17901 [ABSTRACT FROM AUTHOR]

Simple Summary: Hepatocellular Carcinoma (HCC) is a common and aggressive cancer with limited treatment options. Approximately half of all patients with HCC receive systemic therapy during their disease course, particularly in the advanced stages of the disease. Immunotherapy has been paradigm-shifting for the treatment of human cancers, with strong and durable antitumor activity in a subset of patients across a variety of malignancies including HCC. However, identifying new targets is crucial to improve the effectiveness of immunotherapy. This review discusses novel molecules, cell types, and mechanisms implicated in the immunosurveillance of HCC. Hepatocellular carcinoma (HCC) is a common and aggressive cancer with a high mortality rate. The incidence of HCC is increasing worldwide, and the lack of effective screening programs often results in delayed diagnosis, making it a challenging disease to manage. Immunotherapy has emerged as a promising treatment option for different kinds of cancers, with the potential to stimulate the immune system to target cancer cells. However, the current immunotherapeutic approaches for HCC have shown limited efficacy. Since HCC arises within a complex tumour microenvironment (TME) characterized by the presence of various immune and stromal cell types, the understanding of this interaction is crucial for the identification of effective therapy. In this review, we highlight recent advances in our understanding of the TME of HCC and the immune cells involved in anti-tumour responses, including the identification of new possible targets for immunotherapy. We illustrate a possible classification of HCC based on the tumour immune infiltration and give evidence about the role of SerpinB3, a serine protease inhibitor involved in the regulation of the immune response in different cancers. [ABSTRACT FROM AUTHOR]

Abstract: The liver is the main site of HBV replication, however extrahepatic organs, such as the lymphoid system, are an important reservoir of the virus. Viral DNA into different mononuclear cell subsets has been mainly detected in monocytes and B lymphocytes. The attachment site of the virus has been identified in the preS1 encoded protein of the virus envelope, the same involved in hepatocyte infection. The risk of HBV transmission by infected lymphocytes has been clearly documented in the setting of liver transplantation where de novo HBV infection has been found in up to about 80% of liver grafts from HBsAg negative but anti-HBc positive donors. In the hemodialysis setting the percentage of HBV DNA detection in mononuclear cells of HBsAg negative patients has been described in up to 54% of the cases. Vertical transmission studies indicate that HBV-infected mononuclear cells of the mother may result in viral infection of mononuclear cells of the newborns and possible HBV vaccine response failure. HBV can also infect bone marrow cells and in vitro studies demonstrate a block of hematopoiesis by HBV, supporting clinical observations of isolate cases of aplastic anemia associated to the infection. [Copyright &y& Elsevier]

Endothelial dysfunction plays a key role in the development of liver cirrhosis. Among the biomarkers of endothelial dysfunction, the soluble form of Vascular Adhesion Protein-1 (sVAP-1) is an unconventional and less known adhesion molecule endowed also with amine oxidase activity. The aim of this study was to explore and correlate the behavior of sVAP-1 with that of the soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) and with the severity of liver cirrhosis. A cross-sectional study was carried out by enrolling 28 controls, 59 cirrhotic patients without hepatocellular carcinoma, and 56 patients with hepatocellular carcinoma (HCC), mainly caused by alcohol abuse. The levels of adhesion molecules and of the pro-inflammatory cytokines (IL-6 and TNF-αα) were determined by immunoassay and the enzymatic activity of sVAP-1 by a fluorometric assay. In non-diabetic patients without HCC, a specific behavior of sVAP-1 was highlighted. Differently from sVCAM-1, sICAM-1, and cytokines, the sVAP-1 level was significantly increased only in the early stage of disease, and then, it decreased in the last stage (866 ± 390 ng/mL vs. 545 ± 316 ng/mL, in Child–Pugh class A vs. C, respectively, p < 0.05). Bivariate analysis correlates sVAP-1 to sVCAM-1, in the absence of HCC (Spearman's rho = 0.403, p < 0.01). Multiple linear regression analysis revealed that sVCAM-1 appears to be a predictor of sVAP-1 (β coefficient = 0.374, p = 0.021). In conclusion, in non-diabetic and non-HCC cirrhotic patients, sVAP-1 may be a potential prognostic biomarker that, together with sVCAM-1 and pro-inflammatory cytokines, may provide information on the progression of sinusoidal liver endothelium damage. [ABSTRACT FROM AUTHOR]

Summary: Background: SerpinB3 is a cysteine protease inhibitor involved in liver disease progression due to its proinflammatory and profibrogenic properties. The polymorphic variant SerpinB3‐PD (SB3‐PD), presents a substitution in its reactive centre loop, determining the gain of function. Aims: To disclose the clinical characteristics of a cohort of patients with cirrhosis in relation to the presence of SB3‐PD and to assess the effect of this genetic variant on fibrogenic and inflammatory cytokines in vitro. Methods: We assessed SB3 polymorphism in 90 patients with cirrhosis, prospectively followed up in our referral centre. We used HepG2 and HuH‐7 cells transfected to overexpress either wild‐type SB3 (SB3‐WT) or SB3‐PD to assess their endogenous effect, while LX2 and THP‐1 cells were treated with exogenous SB3‐WT or SB3‐PD proteins. Results: Patients carrying SB3‐PD had more severe portal hypertension and higher MELD scores, than patients carrying SB3‐WT. In multivariate analysis, SB3‐PD was an independent predictor of cirrhosis complications. Patients with SB3‐PD polymorphism presented with more severe liver fibrosis and inflammatory features. Hepatoma cells overexpressing SB3‐PD showed higher TGF‐β1 expression than controls. The addition of recombinant SB3‐PD induced an up‐regulation of TGF‐β1 in LX2 cells and a more prominent inflammatory profile in THP‐1 cells, compared to the effect of SB3‐WT protein. Conclusions: The polymorphic variant SB3‐PD is highly effective in determining activation of TGF‐β1 and inflammation in vitro. Patients with cirrhosis who carry SB3‐PD polymorphism may be more prone to develop severe liver disease progression. However, further validation studies are warranted to support the in vivo relevance of this polymorphism. [ABSTRACT FROM AUTHOR]

Simple Summary: Cholangiocarcinoma is characterized by a very poor outcome and SerpinB3, a serine protease inhibitor, has recently been found to play a relevant role in malignant transformation in different cancers. The aim of this study was to analyze the expression of this biomarker in the serum and surgical specimens of cholangiocarcinoma in relation to clinical outcome. High levels of SerpinB3/4 were detected in tumoral tissue in 12.2% of CCA, which were characterized by a more advanced TNM stage and lower overall patient survival, independently of CCA subclass. In addition, patients who had detectable free or IgM-linked SerpinB3/4 in serum showed poorer survival. In conclusion, the present study provides evidence that SerpinB3/4, both in the serum and in tumoral tissue, could be considered a useful biomarker to identify the small subgroup of CCA patients with more aggressive tumor biology and dismal prognosis. Cholangiocarcinoma (CCA), the second most common primary liver tumor, is associated with a dismal outcome, and useful prognostic markers are not currently available in clinical practice. SerpinB3, a serine protease inhibitor, was recently found to play a relevant role in malignant transformation in different cancers. The aim of the present study was to determine the expression of SerpinB3/4 in tissue and serum samples of patients with CCA in relation to clinical outcomes. SerpinB3/4 was assessed in the tissue microarrays (TMAs) of 123 surgically resected CCAs. ELISA assays were carried out in 188 patients with CCA to detect the free and IgM-linked forms of SerpinB3/4. Overall survival was analyzed in relation to SerpinB3/4 expression, and Cox models were used to identify the variables associated with survival. High levels of SerpinB3/4 (TMA score 2+/3+) were detected in 15 tumors (12.2%), characterized by a more advanced TNM stage (III/IV: 64.3% vs. 31.3%; p = 0.031) and lower overall patient survival, independently of CCA subclass (intrahepatic CCA: median 1.1 (0.8—Not Estimable, NE) vs. 2.4 (1.8–3.4) years; p = 0.0007; extrahepatic CCA: median 0.8 (0.2—NE) vs. 2.2 (1.5–5.4) years; p = 0.011). Vascular invasion (p = 0.027) and SerpinB3/4 scores (p = 0.0016) were independently associated with mortality in multivariate analysis. Patients who had detectable free or IgM-linked SerpinB3/4 in their serum showed poorer survival (1 vs. 2.4 years, p = 0.015, for free SerpinB3/4, and 1 vs. 2.6 years, p = 0.0026, for SerpinB3/4–IgM). In conclusion, high levels of SerpinB3/4 in tissue and serum in CCA are associated with poor outcomes after surgery, regardless of tumor subclass. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) accounts for more than 75% of primary liver cancers, which are the second leading cause of cancer-related deaths. The GALAD (gender, age, AFP-L3, AFP, and des-carboxy-prothrombin) score is a diagnostic tool developed based on gender, age, alpha-fetoprotein, alpha-fetoprotein L3, and des-gamma-carboxy prothrombin, originally designed as a diagnostic tool for HCC in high-risk patients. Methods: We analyzed 212 patients with and without cirrhosis. The population study was divided into patients with liver cirrhosis without evidence of HCC at the time of serum sample collection for GALAD score determination and patients with liver cirrhosis and a confirmed diagnosis of HCC at the time of serum sample collection for GALAD score determination. Patients were followed up until death or liver transplantation. The association between variables and HCC mortality risk was performed, and the results were presented as hazard ratio (HR). The receiver operating characteristic (ROC) curve was used to assess the performance of the GALAD HCC diagnosis. The survival probability was explored using the non-parametric test, and the equality of survival amongst categories was assessed with the log-rank test. Results: Biomarkers were higher in the HCC group compared to cirrhosis. Kaplan–Meier survival probability analysis for individual GALAD categories revealed that a high GALAD level was associated with decreased survival during follow-up, and the difference between the curves was statistically significant (p = 0.01). Conclusions: Our findings suggest that the GALAD score has promise as a prognostic tool, with implications for improving patient management and treatment strategies for HCC. [ABSTRACT FROM AUTHOR]

In the last decades, studies on the inflammatory signaling pathways in multiple pathological contexts have revealed new targets for novel therapies. Among the family of G-protein-coupled Proteases Activated Receptors, PAR2 was identified as a driver of the inflammatory cascade in many pathologies, ranging from autoimmune disease to cancer metastasis. For this reason, many efforts have been focused on the development of potential antagonists of PAR2 activity. This work focuses on a small molecule, 1-Piperidine Propionic Acid (1-PPA), previously described to be active against inflammatory processes, but whose target is still unknown. Stabilization effects observed by cellular thermal shift assay coupled to in-silico investigations, including molecular docking and molecular dynamics simulations, suggested that 1-PPA binds PAR2 in an allosteric pocket of the receptor inactive conformation. Functional studies revealed the antagonist effects on MAPKs signaling and on platelet aggregation, processes mediated by PAR family members, including PAR2. Since the allosteric pocket binding 1-PPA is highly conserved in all the members of the PAR family, the evidence reported here suggests that 1-PPA could represent a promising new small molecule targeting PARs with antagonistic activity. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC), the most common form of liver cancer, is frequently diagnosed late due to the absence of symptoms during early disease, thus heavily affecting the overall survival of these patients. Soluble immunological factors persistently produced during cirrhosis have been recognized as promoters of chronic inflammation and neoplastic transformation. The aim of this pilot study was to evaluate the predictive value of the cytokine profiles for HCC development. A Luminex xMAP approach was used for the quantification of 45 proteins in plasma and ascitic fluids of 44 cirrhotic patients without or with HCC of different etiologies. The association with patient survival was also evaluated. Univariate analyses revealed that very low levels of interleukin 5 (IL-5) (<15.86 pg/mL) in ascites and IL-15 (<12.40 pg/mL) in plasma were able to predict HCC onset with an accuracy of 81.8% and a sensitivity of 95.2%. Univariate analyses also showed that HCC, hepatitis B virus/hepatitis C virus infections, low levels of IL-5 and granulocyte-macrophage colony-stimulating factor in ascitic fluids, and high levels of eotaxin-1, hepatocyte growth factor and stromal-cell-derived factor 1α in plasma samples were factors potentially associated with a poor prognosis and decreased survival. Our results suggest a potential protective role of some immune modulators that may act in the peritoneal cavity to counteract disease progression leading to HCC development. [ABSTRACT FROM AUTHOR]

Simple Summary: The Wnt-β-catenin signaling regulates liver homeostasis and repair in adulthood, while its abnormal regulation is involved in the development of several chronic diseases and tumors. SerpinB3 has been shown to induce β-catenin, and both molecules are overexpressed in tumors, particularly in those with poor prognoses. The aim of our study was to evaluate the ability of SerpinB3 to modulate the Wnt pathway in liver cancer and monocytic cells, the main type of inflammatory cells in the tumor microenvironment. We have demonstrated that SerpinB3 modulates the Wnt cascade upregulating the Wnt co-receptors, low-density lipoprotein receptor-related protein 5 (LRP-5) and LRP-6, as well as LRP-1, implicated in cell survival and invasiveness. These data were confirmed in experimental carcinogenesis. In conclusion, the upregulation of LRP family members by SerpinB3 determines Wnt signaling activation and increased cell survival and invasiveness. Abnormal activation of the Wnt-β-catenin signaling cascade is involved in tumor growth and dissemination. SerpinB3 has been shown to induce β-catenin, and both molecules are overexpressed in tumors, particularly in those with poor prognoses. The aim of this study was to evaluate the ability of SerpinB3 to modulate the Wnt pathway in liver cancer and in monocytic cells, the main type of inflammatory cells in the tumor microenvironment. The Wnt cascade, Wnt co-receptors, and low-density lipoprotein receptor-related protein (LRP) members were analyzed in different cell lines and human monocytes in the presence or absence of SerpinB3. The Wnt-β-catenin axis was also evaluated in liver tumors induced in mice with different extents of SeprinB3 expression. In monocytic cells, SerpinB3 induced a significant upregulation of Wnt-1/7, nuclear β-catenin, and c-Myc, which are associated with increased cell lifespan and proliferation. In liver tumors in mice, the expression of β-catenin was significantly correlated with the presence of SerpinB3. In hepatoma cells, Wnt co-receptors LRP-5/6 and LRP-1, implicated in cell survival and invasiveness, were upregulated by SerpinB3. The LRP pan-inhibitor RAP not only induced a decrease in LRP expression, but also a dose–dependent reduction in SerpinB3-induced invasiveness. In conclusion, SerpinB3 determines the activation of the Wnt canonical pathway and cell invasiveness through the upregulation of LRP family members. [ABSTRACT FROM AUTHOR]

A novel bioconjugate for hepatocellular carcinoma (HCC) targeting was obtained by pullulan re-programming, which involves the backbone oxidation and conjugation of targeting peptide and doxorubicin (Doxo) through a releasable linker. Preliminary in vivo studies showed that the oxidation of 40 glucopyranose units (GPU) out of 100 remarkably reduced the pullulan unspecific liver tropism. This oxidized polymer was functionalized with PreS1 to selectively target the HCC and with rhodamine (Rhod) as label to perform in vitro cell up-take investigations. PreS1 and Rhod were conjugated to the aldehydes present along the oxidized pullulan backbone through a 3.4 and 2 kDa PEG spacer, respectively, and by reductive amination. The resulting PreS1-Pull-Rhod contained a mean of 8 PreS1 per oxidized pullulan chain. Cell culture studies were performed by using HepG2/SERPINB3 cells that overexpress the serpine B3 receptor and control HepG2/EMPTY cells that do not overexpress the receptor. A comparative study by cytofluorimetry and confocal microscopy performed using PreS1-Pull-Rhod and Pull-Rhod (control polymer) showed that PreS1 conveys to the conjugate high cell selectivity. Afterwards, the oxidized pullulan was exploited to generate a targeted drug delivery system by conjugation of Doxo to the polymer backbone through a hydrazone pH-sensitive bond and NH 2 -PEG 3.4 kDa -PreS1. The PreS1-Pull-Doxo conjugate showed a two-fold increase of anticancer activity with respect to the control Pull-Doxo towards HepG2/SERPINB3 cells. [ABSTRACT FROM AUTHOR]

In liver cirrhosis, portal hypertension is a consequence of enhanced intrahepatic vascular resistance and portal blood flow. Significant vasodilation in the arterial splanchnic district is crucial for an increase in portal flow. In this pathological condition, increased levels of circulating endogenous vasodilators, including nitric oxide, prostacyclin, carbon monoxide, epoxyeicosatrienoic acids, glucagon, endogenous cannabinoids, and adrenomedullin, and a decreased vascular response to vasoconstrictors are the main mechanisms underlying splanchnic vasodilation. In this review, the molecular pathways leading to splanchnic vasodilation will be discussed in detail. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Foot ulcerations are a disabling complication of diabetes and no treatment is currently available based on disease mechanisms. The protein serpin B3 (SB3) was identified as a positive biomarker of successful diabetic wound healing; therefore, its exogenous administration may promote healing. The topical administration of SB3 is challenging due to its protein nature. Physical entrapment in wet sol–gel silica can stabilize the protein's conformation and permit its sustained delivery. However, irreversible syneresis and poor viscoelastic properties hamper wet sol–gel silica application as a semisolid vehicle. To overcome these limits, a sol–gel silica/hydroxypropylmethylcellulose (HPMC) hydrogel blend was developed. SB3 entrapped in 8% SiO2 wet sol–gel silica preserved its structure, was stabilized against denaturation, and was slowly released for at least three days. Blending a silica gel with an HPMC–glycerol (metolose-G) hydrogel permitted spreadability without affecting the protein's release kinetics. When administered in vivo, SB3 in silica/metolose-G—but not in solution or in metolose-G alone—accelerated wound healing in SB3 knockout and diabetic mouse models. The results confirmed that SB3 is a new pharmacological option for the treatment of chronic ulcers, especially when formulated in a slow-releasing vehicle. Silica–metolose-G represents a novel type of semisolid dosage form which could also be applied for the formulation of other bioactive proteins. [ABSTRACT FROM AUTHOR]

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. 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. 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). 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. • Cardiac complications are a leading cause of mortality after OLT • Pre-operative cardiological risk stratification is challenging and not standardized • Standard chest CT is routinely performed before OLT to exclude contraindications • CAC score on standard chest CT may be used as an early cardiac risk stratification tool [ABSTRACT FROM AUTHOR]

We have investigated the specificity of cell-mediated cytotoxicity for autologous liver cells in 10 patients with acute hepatitis B in relation to the expression of hepatitis B virus (HBV) antigens and IgG on the surface of hepatocytes. Hepatitis B core antigen (HBcAg) was expressed on hepatocytes from six patients and hepatitis B surface antigen (HBsAg) from four, though always in association with HBcAg. Peripheral blood mononuclear cells (PBMC) were significantly cytotoxic in eight of the patients, and fractionation experiments revealed that the cytotoxic effect was equally mediated by T and non-T cells. Monoclonal antibody blocking experiments showed that HBcAg was the major target antigen recognized by T cells, although some inhibition of cytotoxicity was observed after pretreatment of target cells with monoclonal anti-HBs in patients with liver cell surface HBsAg. In contrast, non-T cells were not consistently inhibited by either monoclonal anti- HBc or anti-HBs. These findings further suggest that the HBV nucleoprotein serves as a target for recognition by cytotoxic T cells. [ABSTRACT FROM AUTHOR]

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%, p <0.001). 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. • An AKI-AKD-CKD continuum has been hypothesized, whereby initial kidney injury can lead to persistent injury and eventually CKD. • AKD is a condition that is not yet well characterized in patients with cirrhosis. • According to this concept, AKD may occur as an AKI-AKD or without a previous episode of AKI (NAKI-AKD) in patients with cirrhosis. • According to our data, AKD has a very negative impact on survival and other complications of cirrhosis. • AKD is a potentially reversible condition that can recur mostly as a consequence of repeated AKI. [ABSTRACT FROM AUTHOR]

• Le complexe antigène de carcinome à cellules squameuses-IgM (SCCA-IgM) est associé à la pneumopathie interstitielle diffuse (PID) dans la sclérodermie systémique (ScS). • Le SCCA-IgM semble pouvoir améliorer la performance diagnostique de la capacité pulmonaire totale (CPT) en permettant d'identifier les patients à très faible risque de PID associée à la ScS. • Le SCCA-IgM pourrait s'avérer particulièrement utile dans l'évaluation précoce de la PID associée à la ScS. La pneumopathie interstitielle diffuse (PID) est un facteur majeur de pronostic chez les patients atteints de sclérodermie systémique (ScS). L'antigène de carcinome à cellules squameuses (SCCA1) est un inhibiteur des protéases à sérine qui joue un rôle essentiel dans l'inflammation et la fibrose. Le SCCA1 est surexprimé dans le tissu pulmonaire des patients présentant une fibrose pulmonaire idiopathique et peut être détecté dans le sérum comme un complexe immun circulant lié à l'IgM (SCCA-IgM). Nous avons souhaité explorer l'association entre le SCCA-IgM et les caractéristiques cliniques des patients atteints de ScS. Quatre-vingt-dix-sept patients consécutifs atteints de ScS (selon les critères ACR/EULAR) ont été recrutés dans l'étude. Les variables cliniques et sérologiques et les atteintes d'organe ont été consignées. L'atteinte pulmonaire a été explorée par tomodensitométrie haute résolution (TDM-HR) et par des tests de la fonction respiratoire. Les taux sériques de SCCA-IgM ont été mesurés par un test ELISA validé (Hepa-IC, Xeptagen, Venise, Italie). Nous avons défini le seuil des valeurs sériques de SCCA-IgM > 200 UA/mL, calculé comme la moyenne + 3 écarts types chez 100 sujets en bonne santé. Quarante-et-un patients (42,3 %) présentaient une PID. Les taux de SCCA-IgM étaient significativement plus élevés chez les patients avec PID que chez les patients sans PID : 218 (80–402) contre 87,5 (59–150) UA/mL, p <0.01. Chez les patients positifs pour le SCCA-IgM, la PID était plus fréquente (69,7 % contre 28,1 %, p < 0,0001) et la capacité pulmonaire totale (CPT) plus faible (p <0.05) que chez les patients négatifs. Aucune différence n'a été notée pour les autres caractéristiques cliniques et sérologiques. L'analyse multivariée a associé le SCCA-IgM au diagnostic de PID (OR 10,6 ; IC 2,9–38,4 ; p = 0,001). Le SCCA-IgM est associé à la pneumopathie interstitielle diffuse chez les patients sclérodermiques et pourrait être utilisé dans l'évaluation de la PID associée à la ScS. [ABSTRACT FROM AUTHOR]

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 (25 th ; 75 th 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). Log 10 AFP and log 10 PIVKA-II time-changes differed in HCC + and HCC - patients. In HCC + patients, both log 10 AFP and log 10 PIVKA-II showed an increasing trend over time. In HCC - patients, log 10 PIVKA-II variations were minimal as compared to log 10 AFP variations. The percent increase of log 10 AFP at 6 months vs. baseline was 11% (95%CI 5 to 17%) and 5% (95%CI 1 to 8%) for log 10 PIVKA-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. [ABSTRACT FROM AUTHOR]

Objective: Interstitial lung disease (ILD) is the major determinant of prognosis in patients with systemic sclerosis (SSc). Squamous Cell Carcinoma Antigen (SCCA1) is a serin protease inhibitor which plays a pivotal role in inflammation and fibrosis. SCCA1 is overexpressed in pulmonary tissue of patients with idiopathic pulmonary fibrosis and can be detectable in serum as circulating immune complex bound to IgM (SCCA-IgM). We aimed to investigate the association between SCCA-IgM and clinical features of patients with SSc.Methods: Ninety-seven patients with SSc (ACR/EULAR criteria) were consecutively enrolled in the study. Clinical and serological variables and organ involvement were recorded. Pulmonary involvement was investigated by high-resolution CT (HRCT) and respiratory function tests. SCCA-IgM serum levels were measured by a validated ELISA assay (Hepa-IC, Xeptagen, Venice, Italy). We set the cut-off value for serum levels of SCCA-IgM >200 AU/ml, calculated as mean+3 standard deviations in 100 healthy subjects.Results: Forty-one (42.3%) patients were affected with ILD. SCCA-IgM values were significantly higher in patients with ILD than in those without: 218 (80-402) vs. 87.5 (59-150) AU/mL, P=0.003. Patients with positive SCCA-IgM had more frequently ILD (69.7% vs. 28.1%, P≤0.0001) and a lower total lung capacity (TLC) (P=0.024) compared with negative ones. No differences were found in any other clinical and serological features. At multivariate analysis, SCCA-IgM was found to be associated with ILD diagnosis (OR 10.6, IC 2.9-38.4, P=0.001).Conclusion: SCCA-IgM is associated with interstitial lung disease in scleroderma patients and might be used in the assessment of SSc-ILD. [ABSTRACT FROM AUTHOR]

Complications of chronic liver diseases – particularly hepatocellular carcinoma (HCC) – are a major cause of mortality worldwide. Several studies have shown that high or increasing levels of serum Squamous Cell Carcinoma Antigen-Immunoglobulin M complex (SCCA-IgM) are associated with development of HCC in patients with advanced liver disease and worse survival in patients with liver cancer. The aim of the present study was to assess, in patients with advanced liver disease, differences in long-term clinical outcomes in relation to baseline levels of serum SCCA-IgM. Ninety one consecutive outpatients with liver cirrhosis of different etiologies, without hepatocellular carcinoma at presentation, were enrolled from April 2007 to October 2012 in a prospective study. For a median time of 127 months, patients were bi-annually re-evaluated. SCCA-IgM complex levels were determined with a validated enzyme-linked immunosorbent assay. The results provided evidence that serum SCCA-IgM is a predictor of overall survival. The best cut-off to discriminate both HCC-free and overall survival rates was 120 AU/mL. Patients with baseline values higher than this threshold showed a substantial increase in both HCC incidence rate and all-cause mortality rate. In conclusion, a single measurement of serum SCCA-IgM helps to identify those patients with liver cirrhosis with increased risks of HCC development and mortality. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Viral hepatitis infection is a major global issue and a leading cause of liver disease and associated deaths. Over time, patients infected with hepatitis B (HBV) or C virus (HCV) develop cirrhosis and, eventually, hepatocellular carcinoma (HCC). For this reason, they need to be constantly monitored. Current Asian guidelines recommend the determination of serum alpha-fetoprotein (AFP) together with liver ultrasounds every six months to detect HCC nodules. However, both methods have several limitations, and other biomarkers have been studied for monitoring cirrhosis, including SCCA-IgM, an immune-complex formed by Squamous Cell Carcinoma Antigen and IgM. To date, SCCA-IgM has been validated as a novel biomarker for liver diseases only in European populations. The aim of our study was to analyze SCCA-IgM as a biomarker to monitor cirrhosis evolution in an Asian cohort of patients and to compare its performance to that of AFP. We analyzed the concentration of AFP and SCCA-IgM in serum samples obtained from a group of Asian adult patients with cirrhosis or HCC and a control group of patients admitted for gastrointestinal disorders. In untreated patients and similarly to AFP, SCCA-IgM levels were significantly higher in patients with cirrhosis compared to those with HCC. In addition, SCCA-IgM, but not AFP serological levels, were significantly lower in HCC patients who were treated with surgical resection compared to those who received a different therapy. [ABSTRACT FROM AUTHOR]

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-Kabsence/ 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. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma, the most frequent solid tumor of the liver, has a very poor prognosis, being the second most common cause of death from cancer worldwide. The incidence and mortality of this liver tumor are increasing in most areas of the world as a consequence of aging and the emerging of new risk factors such as the metabolic syndrome, beside the recognized role of hepatitis B and C viral infections and alcohol abuse. Despite the increasing knowledge on the molecular mechanisms underlying hepatic carcinogenesis, effective therapeutic strategies are still an unmet clinical need. Efforts have been made to develop selective drugs as well as effective targeted drug delivery systems. The development of novel drug carriers for therapeutic molecules can indeed offer a valuable strategy to ameliorate the efficacy of HCC treatment. In this review, we discuss recent drug delivery strategies for HCC treatment based on the exploitation of targeted nanoparticles (NPs). Indeed, a few of these platforms have achieved an advanced stage of preclinical development. Here, we review the most promising drug nanovehicles based on both synthetic and natural polymers, including polysaccharides that have emerged for their biocompatibility and biodegradability. To maximize site-selectivity and therapeutic efficacy, drug delivery systems should be functionalized with ligands which can specifically recognize and bind targets expressed by HCC, namely cell membrane associated antigens, receptors or biotransporters. Cell surface and intracellular molecular targets are exploited either to selectively deliver drug-loaded nanovehicles or to design novel selective therapeutics. In conclusion, the combination of novel and safe drug delivery strategies based on site-specific targeted drug nanovehicles with therapeutic molecular targets may significantly improve the pharmacological efficacy for the treatment of HCC. [ABSTRACT FROM AUTHOR]

Introduction Liver cirrhosis is characterized by structural and hemodynamic changes that affect mainly the liver, the kidney and the vascular system. Cytochrome P-450 (CYP) is a variegated family of enzymes that, among many other activities, metabolize arachidonic acid to the vasoactive epoxyeicosatrienoic acids (EETs). Aim To investigate in an animal model of cirrhosis the m-RNA expression of CYPs in liver, kidney and aorta and to evaluate the effect of epoxygenase inhibition by N -(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH). Methods In aorta, liver and kidney from 3 control, 3 cirrhotic and 6 cirrhotic rats treated with MS-PPOH, quantitative real-time PCR reactions were performed and the m-RNA expression of CYP2J3, CYP2J4, CYP2J10, CYP2C11, CYP2C12 and CYP2C23 was calculated. Results In cirrhotic rats, the gene expression of hepatic CYP2C11 and CYP2J10 was increased, of aortic CYP2J4 was increased, of aortic CYP2C12 was reduced and of renal CYP2C11 was increased. In cirrhotic rats, MS-PPOH reduced CYP2J10 hepatic and CYP2C11 renal gene expression to levels similar to the ones of control rats. Conclusions Changes in CYPs gene expression may contribute to the hemodynamic alterations typical of cirrhosis. The altered gene expression of CYPs can, in some cases, be reversed by epoxygenase inhibition. [ABSTRACT FROM AUTHOR]

Aim The identification and surveillance of patients with liver dysfunctions and the discovering of new disease biomarkers are needed in the clinical practice. The aim of this study was to investigate on Survivin-immunoglobulin ( Ig) M immune complex ( IC) as a potential biomarker of chronic liver diseases. Methods Serum levels of Survivin- IgM were measured using an enzyme-linked immunoassay that had been standardized and validated in our laboratory in 262 individuals, including healthy subjects and patients with chronic viral hepatitis, cirrhosis and hepatocellular carcinoma ( HCC). Results Survivin- IgM IC was lower in healthy subjects (median, 99.39 AU/m L) than in patients with chronic viral hepatitis (median, 148.03 AU/m L; P = 0.002) or with cirrhosis (median, 371.00 AU/mL; P < 0.001). Among patients with cirrhosis, those with hepatitis C virus ( HCV) infection showed the highest level of Survivin- IgM IC (median, 633.71 AU/mL; P < 0.001). The receiver-operator curve analysis revealed that Survivin- IgM accurately distinguishes HCV correlated cirrhosis from chronic viral hepatitis (area under the curve [ AUC], 0.738; sensitivity, 74.5%; specificity, 70.7%). A multivariate logistic regression model, including Survivin- IgM IC, aspartate aminotransferase ( AST) and AST/alanine aminotransferase ( ALT) ratio increased the prediction accuracy for the identification of the cirrhotic HCV patients ( AUC, 0.818; sensitivity, 87.2%; specificity, 65.9%). Conversely, Survivin- IgM IC significantly decreased in HCC patients (median, 165.72 AU/mL; P = 0.022). Conclusion Our results suggest that Survivin- IgM immune complex may be used as a potential biomarker for liver damage, particularly for the identification of the HCV-related cirrhotic population. [ABSTRACT FROM AUTHOR]

Background and Aim The serpin squamous cell carcinoma antigen complexed with IgM ( SCCA- IgM) has been reported as a promising serological marker for hepatocellular carcinoma ( HCC). We aimed to further evaluate SCCA- IgM diagnostic accuracy and to determine its prognostic role. Methods SCCA- IgM levels were determined in 327 sera obtained from 81 HCC patients, 206 cirrhotics and 40 healthy blood donors (controls). Sensitivity, specificity, correlation with clinical and tumor parameters and with survival were evaluated. Results HCC patients had SCCA- IgM levels significantly higher than controls and cirrhotics ( P < 0.0001). Sensitivity, specificity, positive and negative predictive values for HCC were 89%, 50%, 41% and 92%, respectively. In comparison, sensitivity and specificity for alphafetoprotein were 48% and 85%. SCCA- IgM levels were not significantly correlated with clinical or biological variables. With a cut-off of 130 AU/ mL (receiver operating characteristic curves), SCCA- IgM proved efficient in the prediction of prognosis, identifying the patients with long overall survival (efficiency validated in the homogenous subgroup of patients with intermediate-stage HCC undergoing transarterial chemoembolization) and predicting progression-free survival. A Cox multivariate analysis confirmed SCCA- IgM predictive value, identifying tumor size and SCCA- IgM levels as independent predictors of survival. A reduction in SCCA- IgM levels correlated with response to treatment. Conclusions SCCA- IgM is a sensitive marker of HCC in patients with cirrhosis even though lacking in specificity. The determination of the levels of the marker in HCC patients is highly efficient in predicting the patients' prognosis, identifying those with long overall and progression-free survival and the responders and should be introduced in the clinical practice. [ABSTRACT FROM AUTHOR]

Abstract: miRNAs are small non-coding RNAs which target complementary mRNA sequences, usually resulting in gene silencing. They can exhibit oncogenic or tumor suppressor properties, modulating cell homeostasis. Several data have documented that miRNAs are typically deregulated in different types of cancers, including hepatocellular carcinoma (HCC). Some of the miRNAs such as miR-122, miR-221, miR-1 and miR-21 have been found to repress post-transcriptionally the expression of genes involved in cell cycle regulation, cell proliferation, apoptosis, cell migration and invasion. In HCC serum levels of miR-122, miR-221 and miR-16 have been described deregulated, suggesting that they may be used as molecular targets for early detection, prognosis and treatment. The ov-serpin SerpinB3 was found previously increased in liver tumor cancers and associated with apoptosis resistance, increased cell proliferation and invasiveness. Recent data indicate that this serpin may enhance its oncogenic potential through inhibition of several tumor suppressive miRNAs, typically described in HCC. [Copyright &y& Elsevier]

Background In the setting of liver injury hepatic progenitor cells are activated, counterbalancing the inhibited regenerative capacity of mature hepatocytes. Chronic activation of this compartment may give rise to a subset of liver tumours with poor prognosis. SerpinB3, a serpin over-expressed in injured liver and in primary liver cancer, has been shown to induce apoptosis resistance, epithelial to mesenchymal transition and to increase TGF-beta and Myc expression. Aim of the present study was to explore the presence of SerpinB3 in hepatic progenitor cells in human livers and in a mouse model of liver stem/progenitor cell activation. Hepatic progenitor cells were analysed in foetal and adult livers at protein and transcriptional levels. To induce experimental activation of the liver stem/progenitor compartment, C57BL/6J mice were injected with lipopolysaccharide plus D-galactosamine and were sacrificed at different time points. Liver cDNA was amplified using specific primers for mouse-homologous SerpinB3 isoforms and automatically sequenced. Results The presence of SerpinB3 in the progenitor cell compartment was detected in sorted human foetal and adult epithelial cell adhesion molecule (EpCAM) positive liver cells. By immunohistochemistry SerpinB3 was found in human cirrhotic livers in portal areas with progenitor cell activation showing ductular proliferation. CK-7, CK-19, EpCAM and CD-90 positive cell were also positive for SerpinB3. In the animal model, time course analysis in liver specimens revealed a progressive increase of SerpinB3 and a parallel decrease of activated caspase 3, which was barely detectable at 20 hours. Transcription analysis confirmed the presence of SerpinB3-homologous only in the liver of injured mice and sequence analysis proved its belonging to mouse Serpinb3b. Conclusion SerpinB3 is highly expressed in hepatic stem/progenitor cell compartment of both foetal and adult livers. [ABSTRACT FROM AUTHOR]

The physiological roles of the protease inhibitor SERPINB3 (SB3) are still largely unknown. The study was addressed to assess the biological effects of this serpin in vivo using a SB3 transgenic mouse model. Two colonies of mice (123 transgenic for SB3 and 148 C57BL/6J controls) have been studied. Transgenic (TG) mice showed longer survival than controls and the difference was more remarkable in males than in females (18.5% vs 12.7% life span increase). In TG mice decreased IL-6 in serum and lower p66shc in the liver were observed. In addition, TG males showed higher expression of mTOR in the liver. Liver histology showed age-dependent increase of steatosis and decrease of glycogen storage in both groups and none of the animals developed neoplastic lesions. In conclusion, the gain in life span observed in SB3-transgenic mice could be determined by multiple mechanisms, including the decrease of circulating IL-6 and the modulation of ageing genes in the liver. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and is characterized by poor clinical outcomes, with the majority of patients not being eligible for curative therapy and treatments only being applicable for early-stage tumors. CD44 is a receptor for hyaluronic acid (HA) and is involved in HCC progression. The aim of this work is to propose HA- and PEGylated-liposomes as promising approaches for the treatment of HCC. It has been found, in this work, that CD44 transcripts are up-regulated in HCC patients, as well as in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Cell culture experiments indicate that HA-liposomes are more rapidly and significantly internalized by Huh7 cells that over-express CD44, compared with HepG2 cells that express low levels of the receptor, in which the uptake seems due to endocytic events. By contrast, human and murine macrophage cell lines (THP-1, RAW264.7) show improved and rapid uptake of PEG-modified liposomes without the involvement of the CD44. Moreover, the internalization of PEG-modified liposomes seems to induce polarization of THP1 towards the M1 phenotype. In conclusion, data reported in this study indicate that this strategy can be proposed as an alternative for drug delivery and one that dually and specifically targets liver cancer cells and infiltrating tumor macrophages in order to counteract two crucial aspect of HCC progression. [ABSTRACT FROM AUTHOR]

Background: Epidemiological studies indicate that a growing number of cirrhotic patients will develop hepatocellular carcinoma (HCC) in the next decade. Recent findings have demonstrated that Squamous cell carcinoma antigen 1 (SCCA1) and 2 (SCCA2) isoforms, now classified as serpinB3 and serpinB4, are over-expressed in HCC, but not in normal liver. As reported, high levels of circulating SCCA-IgM immunocomplexes in patients with cirrhosis are significantly associated with HCC development. Aim: To ascertain whether IgM-linked SCCA isoforms circulate in patients with chronic liver disease, compared to total SCCA-IgM levels. Methodology and Findings: 79 patients with chronic liver disease were studied, including 17 patients with chronic hepatitis, 36 patients with cirrhosis and 26 with HCC. 28 blood donors were used as control. Monoclonal antibodies against serpinB3 and serpinB4 were used as catcher antibodies to set up specific ELISA assays, while total SCCA-IgM immunocomplexes were detected by commercially available ELISA assay. Overall, the results revealed a better diagnostic sensitivity of total SCCA-IgM assay, compared to both serpinB3 and serpinB4 IgM-linked assays. SerpinB4-IgM median values obtained with SCC103 antibody were moderately higher in patients with cirrhosis than in those with HCC, median values: 0.168 (IQR 0.140-0.427) vs. 0.140 (IQR 0.140-0.278), (p = 0.177). A trend toward decreasing serpinB4-IgM/serpinB3-IgM median ratio was observed in patients with advanced liver disease, being 1.08 in patients with HCC, 1.10 in patients with cirrhosis and 1.40 in patients with chronic hepatitis (p = 0.079). Conclusions: IgM-linked SCCA isoforms in serum of patients with chronic liver diseases were quantified for the first time. Although the number of patients was limited, this preliminary study reveals that the relative balance of the two serpin isoforms is altered in HCC and it is characterized by a lower serpinB4-IgM/serpinB3-IgM ratio, determined by lower serpinB4 levels. [ABSTRACT FROM AUTHOR]

Abstract: Background: The serpin SERPINB3 (SB3) found over-expressed in human hepatocellular carcinoma and in regenerating liver in mice has been shown to induce apoptosis resistance, epithelial-to-mesenchymal transition and increasing cellular invasion. It has also been hypothesised that SB3 may provide a pro-proliferative stimulus for liver cells in vivo. No information is available on SB3 in hepatoblastoma (HB). Aims of the study were to analyse SB3 expression in HB specimens and to investigate its possible correlation with Myc expression and tumour extension at diagnosis as evaluated by the pre-treatment extent of disease evaluation system (PRETEXT). Methods: Frozen tumour specimens from 42 children with HB were analysed for SB3 and Myc expression by real-time PCR. SB3 localisation in tumour specimens was assessed by immunohistochemistry. Results: At transcription level SB3 was positive in 79% of the cases. By immunohistochemistry, SB3 expression was found mainly in the embryonic, blastemal, small cell undifferentiated (SCUD) components of HB, while it was not detectable in normal hepatocytes. High SB3 reactivity was also detected in neoplastic cell clusters of portal vein tumour thrombosis. A direct correlation was observed between SB3 gene expression, the up-regulation of Myc (r =0.598, p <0.0001) and tumour extension (PRETEXT III/IV versus I/II, p =0.013). Conclusions: SB3 is over-expressed in HB and its expression is positively correlated with Myc expression and high tumour stage. The role of SB3 in the genesis of HB and in defining the risk profile of children affected by this tumour is hypothesised. [Copyright &y& Elsevier]

Cirrhotic portal hypertension is characterized by mesenteric arterial vasodilation and hyporeactivity to vasoconstrictors. We evaluated the role of epoxyeicosatrienoic acid (EET) and of myoendothelial gap junctions (GJ) in the haemodynamic alterations of experimental cirrhosis. Thirty-five control rats and 35 rats with carbon tetrachloride (CCl)-induced cirrhosis were studied. Small resistance mesenteric arteries (diameter <350 μm) were connected to a pressure servo controller in a video-monitored perfusion system. Concentration-response curves to acetylcholine (ACh) were evaluated in mesenteric arteries pre-incubated with indomethacin, N-nitro--arginine-methyl-ester and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one before and after the epoxygenase inhibitor miconazole or 18α-glycyrrhetinic acid (18α-GA) (GJ inhibitor). EC was calculated. Concentration-response curves to 11,12-EET were also evaluated. mRNA and protein expression of connexins (Cxs) in the mesenteric arteries was evaluated by real-time PCR and immunohistochemistry. The ACh response was increased in cirrhotic rats (EC: −6.55±0.10 vs. −6.01±0.10 log[M]; P<0.01) and was blunted by miconazole only in cirrhotic animals. 18α-GA blunted the response to ACh more in cirrhotic than that in control rats ( P<0.05). Concentration-response curves to 11,12-EET showed an increased endothelium-dependent vasodilating response in cirrhotic rats ( P<0.05); the BK inhibitor Iberiotoxin (25 nM) blocked the response in normal rats but not in cirrhotic rats, while 18α-GA blunted the response in cirrhotic rats but not in control rats. An increased mRNA and protein expression of Cx40 and Cx43 in cirrhotic arteries was detected ( P<0.05). The increased nitric oxide/PGI-independent vasodilation of mesenteric arterial circulation in cirrhosis is because of, at least in part, hyperreactivity to 11,12-EET through an increased expression of myoendothelial GJs. [ABSTRACT FROM AUTHOR]

Background: Squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) is a useful biomarker for the risk of development of hepatocellular carcinoma (HCC) in patients with cirrhosis due to its progressive increase associated to HCC evolution. In patients with cirrhosis, other assays have been affected by interfering reactivities of IgM. In this study, the analytical specificity of the SCCA-IgM assay was assessed by evaluating SCCA-IgM measurement dependence on different capture phases, and by measuring the recovery of SCCA-IgM reactivity following serum fractionation. Methods: Serum samples from 82 patients with cirrhosis were analyzed. SCCA-IgM was measured using the reference test (Hepa-IC, Xeptagen, Italy) that is based on rabbit oligoclonal anti-squamous cell carcinoma antigen (SCCA) and a dedicated ELISA with a mouse monoclonal anti-SCCA as the capture antibody. Results: SCCA-IgM concentrations measured with the reference assay (median value=87 AU/mL) were higher than those measured with the mouse monoclonal test (median value=78 AU/mL). However, the differences in the SCCA-IgM distribution were not statistically significant (p>0.05). When SCCA-IgM concentrations measured with both tests were compared, a linear correlation was found (r=0.77, p<0.05). Fractionation of the most reactive sera by gel-filtration chromatography showed that total recovery of SCCA-IgM reactivity was seen only in the fractions corresponding to components with a molecular weight higher than IgM and SCCA (>2000 kDa) with both tests. Conclusions: The equivalence of both SCCA-IgM assays and the absence of reactivity not related to immune complexes support the analytical specificity of SCCA-IgM measurements. The results validate the assessment of SCCA-IgM for prognostic purposes in patients with cirrhosis. Clin Chem Lab Med 2010;48:217–23. [ABSTRACT FROM AUTHOR]

Abstract: SERPINB3 (Squamous Cell Carcinoma Antigen, SCCA1) is a member of the ov-serpins, a serine protease inhibitors family expressed in many cell types including normal epithelium, leukocytes, tumors of epithelial origin and primary liver cancer. Several studies, carried out in vitro and in vivo, have documented an important role of SERPINB3 in the modulation of programmed cell death by different mechanisms, both in inflammatory processes and in cancer. SERPINB3 significantly attenuates apoptosis by contrasting cytochrome c release from the mitochondria and by antichemotactic effect for NK cells. Mechanisms involved in apoptosis induction and regulation play a key role in the balance between cell proliferation and death. Imbalance of this equilibrium may contribute to the development of autoimmune diseases, as defective apoptosis of immune cells leads to deregulated autoreactive cell proliferation. Since defective programmed cell death represents a critical feature of autoimmunity, the involvement of SERPINB3 in this pathological field deserves further studies. [Copyright &y& Elsevier]

Transforming growth factor-beta (TGF-beta) system is involved in the control of cell growth and extracellular matrix formation. Previous studies in patients with chronic liver disease have shown that increased TGF-beta expression significantly correlates with the degree of hepatic fibrosis. The aim of our study was to define TGF-beta system expression in hepatic parenchymal cells and its significance in patients with differing extents of chronic liver disease of viral etiology. Expression of TGF-beta 1, TGF-beta 1 type II receptor (TGF-beta RII) and the Smad signaling pathway was evaluated in consecutive liver sections of 77 patients with chronic liver disease (65 HCV positive and 12 HBV positive). Results were correlated with histological scores and apoptotic activity. TGF-beta 1 was demonstrated in the liver of 30/56 (53.6%) patients with chronic hepatitis and 20/21 (95%) patients with cirrhosis, but in none of the 20 normal livers. Positive cytokine reaction was seen both in stromal cells and hepatocytes. Expression of TGF-beta RII and Smad proteins showed a distribution pattern similar to that of TGF-beta, with a direct correlation in terms of immunoreactivity extent. A significant correlation was found between parenchymal expression of TGF-beta system and inflammatory and fibrosis scores. No correlation was found with apoptotic index and other morphological, clinical or virological parameters. The TGF-beta system is up-regulated at the ligand, receptor and signaling level in the liver of patients with more active disease. The strong expression of TGF-beta and the Smad pathway in parenchymal cells suggests that hepatocytes, in addition to mesenchymal cells, may play an important role in the progression of liver disease. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are increasingly studied as vectors for drug delivery because they can transfer a variety of molecules across biological barriers. SerpinB3 is a serine protease inhibitor that has shown a protective anti-apoptotic function in a variety of stressful conditions. The aim of this study was to evaluate protection from oxidative stress-induced damage, using extracellular vesicles that overexpress SerpinB3 (EVs-SB3) in order to enhance the effect of extracellular vesicles on cellular homeostasis. EVs-SB3s were obtained from HepG2 cells engineered to overexpress SerpinB3 and they revealed significant proteomic changes, mostly characterized by a reduced expression of other proteins compared with EVs from non-engineered cells. These EV preparations showed a significantly higher protection from H2O2 induced oxidative stress in both the hepatoma cell line and in primary cardiomyocytes, compared to cells treated with naïve EVs or SerpinB3 alone, used at the same concentration. In conclusion, the induction of SerpinB3 transgene expression results in the secretion of EVs enriched with the protein product that exhibits enhanced cytoprotective activity, compared with naïve EVs or the nude SerpinB3 protein. [ABSTRACT FROM AUTHOR]

Simple Summary: Proteasome inhibitors are currently used in the treatment of certain blood cancers, and clinical trials to treat solid tumors, including liver cancer, have also been conducted. However, different malignancies are not equally susceptible to proteasome inhibitors, and resistance to the drug may develop during the therapy. Here, we characterize the molecular mechanisms underlying the resilience of liver cancer cells to the proteasome inhibitor bortezomib. The results demonstrate that the activity of the eIF2α–ATF4 stress response pathway affects the viability of cells treated with bortezomib. We found that the pseudokinase TRIB3, an endogenous regulator of ATF4 and a gene highly expressed in liver cancer, resides predominantly at the same chromatin sites as ATF4 and constrains ATF4 activity. The survival of bortezomib-exposed hepatoma cells proved sensitive to TRIB3 overexpression and inactivation. Thus, TRIB3 is a novel factor contributing to bortezomib resistance of liver cancer cells. The proteasome is an appealing target for anticancer therapy and the proteasome inhibitor bortezomib has been approved for the treatment of several types of malignancies. However, the molecular mechanisms underlying cancer cell resistance to bortezomib remain poorly understood. In the current article, we investigate how modulation of the eIF2α–ATF4 stress pathway affects hepatoma cell response to bortezomib. Transcriptome profiling revealed that many ATF4 transcriptional target genes are among the most upregulated genes in bortezomib-treated HepG2 human hepatoma cells. While pharmacological enhancement of the eIF2α–ATF4 pathway activity results in the elevation of the activities of all branches of the unfolded protein response (UPR) and sensitizes cells to bortezomib toxicity, the suppression of ATF4 induction delays bortezomib-induced cell death. The pseudokinase TRIB3, an inhibitor of ATF4, is expressed at a high basal level in hepatoma cells and is strongly upregulated in response to bortezomib. To map genome-wide chromatin binding loci of TRIB3 protein, we fused a Flag tag to endogenous TRIB3 in HepG2 cells and performed ChIP-Seq. The results demonstrate that TRIB3 predominantly colocalizes with ATF4 on chromatin and binds to genomic regions containing the C/EBP–ATF motif. Bortezomib treatment leads to a robust enrichment of TRIB3 binding near genes induced by bortezomib and involved in the ER stress response and cell death. Disruption of TRIB3 increases C/EBP–ATF-driven transcription, augments ER stress and cell death upon exposure to bortezomib, while TRIB3 overexpression enhances cell survival. Thus, TRIB3, colocalizing with ATF4 and limiting its transcriptional activity, functions as a factor increasing resistance to bortezomib, while pharmacological over-activation of eIF2α–ATF4 can overcome the endogenous restraint mechanisms and sensitize cells to bortezomib. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]

Ten patients were studied serially during acute hepatitis type B for lymphocyte sensitization to the hepatitis B surface antigen (HBsAg) and to the complete hepatitis B virion (Dane particle). Using the lymphocyte transformation test, sensitization to purified HBsAg was not observed during the first 10 days of illness but became detectable later, being particularly evident during convalescence, while sensitization to antigens of the complete virion, other than HBsAg, was demonstrable as soon as at the onset of symptoms, often at the time of maximum liver cell damage. These results indicate that in the course of acute hepatitis type B. lymphocyte sensitization to other antigens of the complete virion precedes that to HBsAg and may be of greater pathogenetic importance. [ABSTRACT FROM AUTHOR]

Using the natural killer (NK) sensitive K562 cell line, enhanced NK cell cytotoxicity was demonstrable early in the course of acute hepatitis B while normal values were obtained in patients studied during convalescence. No evidence of enhanced NK activity was instead obtained in the course of acute non-A, non-B hepatitis. Serum levels of α-interferon, as determined by radioimmunoassay (RIA), were significantly increased in patients with acute hepatitis B showing enhanced NK cell activity but not in those with acute non-A, non-B hepatitis and normal NK cell activity. These results suggest that natural cytotoxicity may play a role early in the course of acute hepatitis type B, before antigen-specific T lymphocytes become fully operative. [ABSTRACT FROM AUTHOR]

Simple Summary: The cytotoxic T cell response against hepatocellular carcinoma antigens is exhausted and fails in its task of deleting tumoral cells. These cells are featured by the expression of negative immune checkpoints that can be modulated to restore T cell function. The blockade of the PD-1/PD-L1 pathway has shown promising results in rescuing hepatocellular carcinoma-specific CD8 T cells but only a reduced group of cases is sensitive to this treatment and the effect is usually temporary. Therefore, new anti-PD-1 based combinatory strategies are underway to increase the response by adding the effect of blocking neo-angiogenesis and other negative immune checkpoints, boosting positive immune checkpoints, blocking suppressive cytokines, or inducing the expression of tumoral neoantigens. The restoration of T cell responses with these anti-PD-1 based combinatory therapies will change the outcome of advanced hepatocellular carcinoma. Thirty to fifty percent of hepatocellular carcinomas (HCC) display an immune class genetic signature. In this type of tumor, HCC-specific CD8 T cells carry out a key role in HCC control. Those potential reactive HCC-specific CD8 T cells recognize either HCC immunogenic neoantigens or aberrantly expressed host's antigens, but they become progressively exhausted or deleted. These cells express the negative immunoregulatory checkpoint programmed cell death protein 1 (PD-1) which impairs T cell receptor signaling by blocking the CD28 positive co-stimulatory signal. The pool of CD8 cells sensitive to anti-PD-1/PD-L1 treatment is the PD-1dim memory-like precursor pool that gives rise to the effector subset involved in HCC control. Due to the epigenetic imprints that are transmitted to the next generation, the effect of PD-1 blockade is transient, and repeated treatments lead to tumor resistance. During long-lasting disease, besides the TCR signaling impairment, T cells develop other failures that should be also set-up to increase T cell reactivity. Therefore, several PD-1 blockade-based combinatory therapies are currently under investigation such as adding antiangiogenics, anti-TGFβ1, blockade of other negative immune checkpoints, or increasing HCC antigen presentation. The effect of these combinations on CD8+ T cells is discussed in this review. [ABSTRACT FROM AUTHOR]

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-[Ru 2 (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. Cancer-targeting strategy exploiting the "Trojan Horse" concept: nanocarriers functionalized with a cancer-targeting biomolecule and encapsulating our metal-based agents with proven anticancer activity. Unlabelled Image • PF127-based micellar carrier: valuable strategy for delivering metal-based therapeutics • Pluronic micelles increase solubility and keep activity of dithiocarbamato compounds. • Nanocarriers penetrate cancer cells membrane releasing the cytotoxic cargo inside cell. • Fluorescein-labelled micelles highlight internalization in cytoplasm of formulations in 2 h. [ABSTRACT FROM AUTHOR]