The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis.

OBJECTIVE: The serine protease inhibitor SerpinB3 has been described as critical mediator of liver fibrosis and it has been recently proposed as an additional hepatokine involved in NASH development and insulin resistance. Protease Activated Receptor 2 has been identified as a novel regulator of hepatic metabolism. A targeted therapeutic strategy for NASH has been investigated, using 1-Piperidine Propionic Acid (1-PPA), since this compound has been recently proposed as both Protease Activated Receptor 2 and SerpinB3 inhibitor. METHODS: The effect of SerpinB3 on inflammation and fibrosis genes was assessed in human macrophage and stellate cell lines. Transgenic mice, either overexpressing SerpinB3 or carrying Serpinb3 deletion and their relative wild type strains, were used in experimental NASH models. Subgroups of SerpinB3 transgenic mice and their controls were also injected with 1-PPA to assess the efficacy of this compound in NASH inhibition. RESULTS: 1-PPA did not present significant cell and organ toxicity and was able to inhibit SerpinB3 and PAR2 in a dose-dependent manner. This effect was associated to a parallel reduction of the synthesis of the molecules induced by endogenous SerpinB3 or by its paracrine effects both in vitro and in vivo, leading to inhibition of lipid accumulation, inflammation and fibrosis in experimental NASH. At mechanistic level, the antiprotease activity of SerpinB3 was found essential for PAR2 activation, determining upregulation of the CCAAT Enhancer Binding Protein beta (C/EBP-ß), another pivotal regulator of metabolism, inflammation and fibrosis, which in turn determined SerpinB3 synthesis. CONCLUSIONS: 1-PPA treatment was able to inhibit the PAR2 - C/EBP-ß - SerpinB3 axis and to protect from NASH development and progression, supporting the potential use of a similar approach for a targeted therapy of NASH.

SerpinB3/4 Expression Is Associated with Poor Prognosis in Patients with Cholangiocarcinoma.

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.

The polymorphic variant of SerpinB3 (SerpinB3-PD) is associated with faster cirrhosis decompensation.

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-ß1 expression than controls. The addition of recombinant SB3-PD induced an up-regulation of TGF-ß1 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-ß1 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.

Galad Score as a Prognostic Marker for Patients with Hepatocellular Carcinoma.

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.

1-Piperidine Propionic Acid as an Allosteric Inhibitor of Protease Activated Receptor-2.

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.

SerpinB3 Upregulates Low-Density Lipoprotein Receptor-Related Protein (LRP) Family Members, Leading to Wnt Signaling Activation and Increased Cell Survival and Invasiveness.

Abnormal activation of the Wnt-ß-catenin signaling cascade is involved in tumor growth and dissemination. SerpinB3 has been shown to induce ß-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-ß-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 ß-catenin, and c-Myc, which are associated with increased cell lifespan and proliferation. In liver tumors in mice, the expression of ß-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.

Epitope-Specific Anti-SerpinB3 Antibodies for SerpinB3 Recognition and Biological Activity Inhibition.

SerpinB3 is a serine protease inhibitor that plays a relevant role in disease progression and cancer by increasing fibrosis, cell proliferation, and invasion, besides conferring resistance to apoptosis. The mechanisms underlying these biological activities are not yet fully understood. The aim of this study was to generate antibodies directed against different SerpinB3 epitopes to better investigate their biological role. Five exposed epitopes were identified using the software DNASTAR Lasergene and the corresponding synthetic peptides were used for NZW rabbit immunization. Anti-P#2 and anti-P#4 antibodies were able to recognize both SerpinB3 and SerpinB4 by ELISA. Anti-P#5 antibody, produced against the reactive site loop of SerpinB3, showed the greatest specific reactivity for human SerpinB3. This antibody was able to recognize SerpinB3 at nuclear level, while anti-P#3 antibody recognized SerpinB3 only at cytoplasmic level, both by immunofluorescence and by immunohistochemistry. The biological activity of each antibody preparation was assessed in HepG2 cells overexpressing SerpinB3 and anti-P#5 antibody reduced proliferation by 12% cell and cell invasion by 75%, while trivial results were obtained with the other antibody preparations. These findings indicate that the reactive site loop of SerpinB3 is essential for the invasiveness features induced by this serpin and it could become a novel druggable target.

SerpinB3 administration protects liver against ischemia-reperfusion injury.

We have investigated the change in SerpinB3 during hepatic ischemia and the potential role of its antiprotease activity in cell protection by the administration of wild-type SerpinB3 (SerpinB3-WT) or active loop-deleted recombinant SerpinB3 protein (SerpinB3-D) in a rat model of ischemia (60 min)/reperfusion (60 min) (I/R). A time-dependent increase of SerpinB3, both at transcription and protein level, was found in ischemic livers after 60, 120 and 180 min. SerpinB3-WT decreased polymorphonuclear cell infiltration and serum enzymes and increased ATP when compared with I/R group. These events were not obtained using SerpinB3-D. No significant changes in both liver SerpinB3 mRNA and protein were found in all I/R groups considered. The present data show that the administration of SerpinB3-WT reduced the I/R injury and this effect appears to be dependent on its anti-protease activity.

Semisolid Wet Sol-Gel Silica/Hydroxypropyl Methyl Cellulose Formulation for Slow Release of Serpin B3 Promotes Wound Healing In Vivo.

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.

SerpinB3 as a Pro-Inflammatory Mediator in the Progression of Experimental Non-Alcoholic Fatty Liver Disease.

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-1ß and reactive oxygen species along with that of TGFß 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.

Oncostatin M is overexpressed in NASH-related hepatocellular carcinoma and promotes cancer cell invasiveness and angiogenesis.

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 Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The protease-inhibitor SerpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma.

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 (ß-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 ß-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.

Engineered EVs for Oxidative Stress Protection.

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.

Modeling the time-related fluctuations of AFP and PIVKA-II serum levels in patients with cirrhosis undergoing surveillance for hepatocellular carcinoma.

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.

Squamous cell carcinoma antigen-IgM (SCCA-IgM) is associated with interstitial lung disease in systemic sclerosis.

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.

Serum Squamous Cell Carcinoma Antigen-Immunoglobulin M complex levels predict survival in patients with cirrhosis.

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.

PreS1 peptide-functionalized gold nanostructures with SERRS tags for efficient liver cancer cell targeting.

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.

Squamous cell carcinoma antigen 1 is associated to poor prognosis in esophageal cancer through immune surveillance impairment and reduced chemosensitivity.

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.

SERPINB3 Delays Glomerulonephritis and Attenuates the Lupus-Like Disease in Lupus Murine Models by Inducing a More Tolerogenic Immune Phenotype.

Objective: To explore the effects of SERPINB3 administration in murine lupus models with a focus on lupus-like nephritis. Methods: 40 NZB/W F1 mice were subdivided into 4 groups and intraperitoneally injected with recombinant SERPINB3 (7.5 µg/0.1 mL or 15 µg/0.1 mL) or PBS (0.1 mL) before (group 1 and 2) or after (group 3 and 4) the development of proteinuria (≥100 mg/dl). Two additional mice groups were provided by including 20 MRL/lpr mice which were prophylactically injected with SERPINB3 (10 mice, group 5) or PBS (10 mice, group 6). Time of occurrence and levels of anti-dsDNA and anti-C1q antibodies, proteinuria and serum creatinine, overall- and proteinuria-free survival were assessed in mice followed up to natural death. Histological analysis was performed in kidneys of both lupus models. The Th17:Treg cell ratio was assessed by flow-cytometry in splenocytes of treated and untreated MRL/lpr mice. Statistical analysis was performed using non parametric tests and Kaplan-Meier curves, when indicated. Results: Autoantibody levels and proteinuria were significantly decreased and time of occurrence significantly delayed in SERPINB3-treated mice vs. controls. In agreement with these findings, proteinuria-free and overall survival were significantly improved in SERPINB3-treated groups vs. controls. Histological analysis demonstrated a lower prevalence of severe tubular lesions in kidneys of group 5 vs. group 6. SERPINB3-treated mice showed an overall trend toward a reduced prevalence of severe lesions in both strains. Th17:Treg ratio was significantly decreased in splenocytes of MRL/lpr mice treated with SERPINB3, compared to untreated control mice. Conclusions: SERPINB3 significantly improves disease course and delays the onset of severe glomerulonephritis in lupus-prone mice, possibly inducing a more tolerogenic immune phenotype.

Development of a novel diagnostic algorithm to predict NASH in HCV-positive patients.

Non-alcoholic steato-hepatitis (NASH) is a severe disease characterised by liver inflammation and progressive hepatic fibrosis, which may progress to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests that in hepatitis C virus patients steatosis and NASH are associated with faster fibrosis progression and hepatocellular carcinoma. A safe and reliable non-invasive diagnostic method to detect NASH at its early stages is still needed to prevent progression of the disease. We prospectively enrolled 91 hepatitis C virus-positive patients with histologically proven chronic liver disease: 77 patients were included in our study; of these, 10 had NASH. For each patient, various clinical and serological variables were collected. Different algorithms combining squamous cell carcinoma antigen-immunoglobulin-M (SCCA-IgM) levels with other common clinical data were created to provide the probability of having NASH. Our analysis revealed a statistically significant correlation between the histological presence of NASH and SCCA-IgM, insulin, homeostasis model assessment, haemoglobin, high-density lipoprotein and ferritin levels, and smoke. Compared to the use of a single marker, algorithms that combined four, six or seven variables identified NASH with higher accuracy. The best diagnostic performance was obtained with the logistic regression combination, which included all seven variables correlated with NASH. The combination of SCCA-IgM with common clinical data shows promising diagnostic performance for the detection of NASH in hepatitis C virus patients.