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Background: Checkpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy., Methods: CRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence., Results: We found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4 + and CD8 + T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival., Conclusions: These findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy., (© 2024. The Author(s).)

Background: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, with embryonal (ERMS) and alveolar (ARMS) representing the two most common histological subtypes. ARMS shows poor prognosis, being often metastatic at diagnosis. Thus, the discovery of novel biomarkers predictive of tumor aggressiveness represents one of the most important challenges to overcome and may help the development of tailored therapies. In the last years, miRNAs carried in extracellular vesicles (EVs), small vesicles of endocytic origin, have emerged as ideal candidate biomarkers due to their stability in plasma and their tissue specificity., Methods: EVs miRNAs were isolated from plasma of 21 patients affected by RMS and 13 healthy childrens (HC). We performed a miRNA profile using the Serum/Plasma Focus microRNA PCR panels (Qiagen), and RT-qPCR for validation analysis. Statistically significant (p < 0.05) miRNAs were obtained by ANOVA test., Results: We identified nine EVs miRNAs (miR-483-5p, miR-132-3p, miR-766-3p, miR-454-3p miR-197-3p, miR-335-3p, miR-17-5p, miR-486-5p and miR-484) highly upregulated in RMS patients compared to HCs. Interestingly, 4 miRNAs (miR-335-5p, miR-17-5p, miR-486-5p and miR-484) were significantly upregulated in ARMS samples compared to ERMS. In the validation analysis performed in a larger group of patients only three miRNAs (miR-483-5p, miR-335-5p and miR-484) were differentially significantly expressed in RMS patients compared to HC. Among these, mir-335-5p was significant also when compared ARMS to ERMS patients. MiR-335-5p was upregulated in RMS tumor tissues respect to normal tissues (p = 0.00202) and upregulated significantly between ARMS and ERMS (p = 0.04). Furthermore, the miRNA expression correlated with the Intergroup Rhabdomyosarcoma Study (IRS) grouping system, (p = 0.0234), and survival (OS, p = 0.044; PFS, p = 0.025). By performing in situ hybridization, we observed that miR-335-5p signal was exclusively in the cytoplasm of cancer cells., Conclusion: We identified miR-335-5p as significantly upregulated in plasma derived EVs and tumor tissue of patients affected by ARMS. Its expression correlates to stage and survival in patients. Future studies are needed to validate miR-335-5p as prognostic biomarker and to deeply elucidate its biological role., (© 2024. The Author(s).)

Total Quality Management (TQM) is a holistic approach widely adopted across industries to ensure quality control and management. This document examines TQM practices in the pharmaceutical, food, and nutritional supplement sectors, highlighting their vital role in public health, sustainability, and consumer acceptance. By analyzing the literature and case studies, the article demonstrates how TQM significantly ensures product safety and quality. Real-world examples and empirical evidence showcase the benefits of TQM methodologies, from rigorous quality control to efficient management processes, helping to meet and exceed regulatory standards. The article also underscores TQM's critical role in addressing sustainability challenges, integrating eco-friendly practices, reducing waste, and optimizing resources. Furthermore, TQM fosters consumer trust and loyalty through transparency, continuous improvement, and responsiveness to feedback, building lasting business-customer relationships. In conclusion, this manuscript illuminates TQM's multifaceted impact on the pharmaceutical, food, and nutritional supplement sectors, presenting it as a pivotal framework for safeguarding public health, promoting sustainability, and enhancing consumer acceptance in a dynamic global landscape.

This study aims to recover the main by-product of Citrus fruits processing, the raw pomace, known also as pastazzo , to produce plant complexes to be used in the treatment of inflammatory bowel disease (IBD). Food-grade extracts from orange (OE) and lemon (LE) pomace were obtained by ultrasound-assisted maceration. After a preliminary phytochemical and biological screening by in vitro assays, primary and secondary metabolites were characterized by proton nuclear magnetic resonance ( 1 H-NMR) and liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI-MS) analyses. The intestinal bioaccessibility and antioxidant and anti-inflammatory properties were investigated by in vitro simulated gastro-intestinal digestion followed by treatments on a lipopolysaccharide (LPS)-stimulated human colorectal adenocarcinoma cell line (Caco-2). The tight junctions-associated structural proteins (ZO-1, Claudin-1, and Occludin), transepithelial electrical resistance (TEER), reactive oxygen species (ROS)-levels, expression of some key antioxidant ( CAT , NRF2 and SOD2 ) and inflammatory ( IL-1β , IL-6 , TNF-α , IL-8 ) genes, and pNFkB p65 nuclear translocation, were evaluated. The OE and LE digesta, which did not show any significant difference in terms of phytochemical profile, showed significant effects in protecting against the LPS-induced intestinal barrier damage, oxidative stress and inflammatory response. In conclusion, both OE and LE emerged as potential candidates for further preclinical studies on in vivo IBD models.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare skin fragility disorder caused by mutations in COL7A1. RDEB is hallmarked by trauma-induced unremitting blistering, chronic wounds with inflammation, and progressive fibrosis, leading to severe disease complications. There is currently no cure for RDEB-associated fibrosis. Our previous studies and increasing evidence highlighted the profibrotic role of NOTCH pathway in different skin disorders, including RDEB. In this study, we further investigated the role of NOTCH signaling in RDEB pathogenesis and explored the effects of its inhibition by γ-secretase inhibitors DAPT and PF-03084014 (nirogacestat). Our analyses demonstrated that JAG1 and cleaved NOTCH1 are upregulated in primary RDEB fibroblasts (ie, RDEB-derived fibroblasts) compared with controls, and their protein levels are further increased by TGF-β1 stimulation. Functional assays unveiled the involvement of JAG1/NOTCH1 axis in RDEB fibrosis and demonstrated that its blockade counteracts a variety of fibrotic traits. In particular, RDEB-derived fibroblasts treated with PF-03084014 showed (i) a significant reduction of contractility, (ii) a diminished secretion of TGF-β1 and collagens, and (iii) the downregulation of several fibrotic proteins. Although less marked than PF-03084014-treated cells, RDEB-derived fibroblasts exhibited a reduction of fibrotic traits also upon DAPT treatment. This study provides potential therapeutic strategies to antagonize RDEB fibrosis onset and progression., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Hepatoblastoma (HB) is the most common pediatric hepatic malignancy. Despite the progress in HB treatment, investigating HB pathomechanisms to optimize stratification and therapies remains a focal point to improve the outcome for high-risk patients., Methods: Here, we pointed to explore the impact of these mechanisms in HB. An observational study was performed on liver samples from a cohort of 17 patients with a diagnosis of HB and two normal liver samples. The in vitro experiments were executed on the Huh6 human HB cell line treated with the FAK inhibitor TAE226., Results: Our results highlight a significant up-regulation of mRNA and protein expression of FAK in livers from HB with respect to normal livers. The increased protein expression of total and Tyr397 phosphorylated FAK (pTyr397FAK) was significantly correlated with the expression of some epigenetic regulators of histone H3 methylation and acetylation. Of note, the expression of pTyr397FAK, N-methyltransferase enzyme (EZH2) and tri-methylation of the H3K27 residue correlated with tumor size and alpha-fetoprotein (AFP) levels. Finally, TAE226 caused a significant reduction of pTyr397FAK, epigenetic regulators, AFP , EPCAM , OCT4 , and SOX2 , in association with anti-proliferative and pro-apoptotic effects on HB cells., Conclusion: Our results suggest a role of FAK in HB that requires further investigations mainly focused on the exploration of its effective diagnostic and therapeutic translatability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Braghini, De Stefanis, Tiano, Castellano, Cicolani, Pezzullo, Tocco, Spada, Alaggio, Alisi and Francalanci.)

Purpose: Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects., Experimental Design: GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9., Results: GD2 was expressed in 82.68% of MB tumors. The SHH and G3-G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood-brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells., Conclusions: Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients., (©2024 The Authors; Published by the American Association for Cancer Research.)

Digital pathology (DP) has begun to play a key role in the evaluation of liver specimens. Recent studies have shown that a workflow that combines DP and artificial intelligence (AI) applied to histopathology has potential value in supporting the diagnosis, treatment evaluation, and prognosis prediction of liver diseases. Here, we provide a systematic review of the use of this workflow in the field of hepatology. Based on the PRISMA 2020 criteria, a search of the PubMed, SCOPUS, and Embase electronic databases was conducted, applying inclusion/exclusion filters. The articles were evaluated by two independent reviewers, who extracted the specifications and objectives of each study, the AI tools used, and the results obtained. From the 266 initial records identified, 25 eligible studies were selected, mainly conducted on human liver tissues. Most of the studies were performed using whole-slide imaging systems for imaging acquisition and applying different machine learning and deep learning methods for image pre-processing, segmentation, feature extractions, and classification. Of note, most of the studies selected demonstrated good performance as classifiers of liver histological images compared to pathologist annotations. Promising results to date bode well for the not-too-distant inclusion of these techniques in clinical practice.

Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27 Kip1 and p57 Kip2 , respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS., (© 2023. The Author(s).)

The present research deals with sentiment analysis performed with Microsoft Azure Machine Learning Studio to classify Facebook posts on the Greek National Public Health Organization (EODY) from November 2021 to January 2022 during the pandemic. Positive, negative and neutral sentiments were included after processing 300 reviews. This approach involved analyzing the words appearing in the comments and exploring the sentiments related to daily surveillance reports of COVID-19 published on the EODY Facebook page. Moreover, machine learning algorithms were implemented to predict the classification of sentiments. This research assesses the efficiency of a few popular machine learning models, which is one of the initial efforts in Greece in this domain. People have negative sentiments toward COVID surveillance reports. Words with the highest frequency of occurrence include government, vaccinated people, unvaccinated, telephone communication, health measures, virus, COVID-19 rapid/molecular tests, and of course, COVID-19. The experimental results disclose additionally that two classifiers, namely two class Neural Network and two class Bayes Point Machine, achieved high sentiment analysis accuracy and F1 score, particularly 87% and over 35%. A significant limitation of this study may be the need for more comparison with other research attempts that identified the sentiments of the EODY surveillance reports of COVID in Greece. Machine learning models can provide critical information combating public health hazards and enrich communication strategies and proactive actions in public health issues and opinion management during the COVID-19 pandemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stefanis, Giorgi, Kalentzis, Tselemponis, Nena, Tsigalou, Kontogiorgis, Kourkoutas, Chatzak, Dokas, Constantinidis and Bezirtzoglou.)

In this study, machine learning models were implemented to predict the classification of coastal waters in the region of Eastern Macedonia and Thrace (EMT) concerning Escherichia coli ( E. coli ) concentration and weather variables in the framework of the Directive 2006/7/EC. Six sampling stations of EMT, located on beaches of the regional units of Kavala, Xanthi, Rhodopi, Evros, Thasos and Samothraki, were selected. All 1039 samples were collected from May to September within a 14-year follow-up period (2009-2021). The weather parameters were acquired from nearby meteorological stations. The samples were analysed according to the ISO 9308-1 for the detection and the enumeration of E. coli . The vast majority of the samples fall into category 1 (Excellent), which is a mark of the high quality of the coastal waters of EMT. The experimental results disclose, additionally, that two-class classifiers, namely Decision Forest, Decision Jungle and Boosted Decision Tree, achieved high Accuracy scores over 99%. In addition, comparing our performance metrics with those of other researchers, diversity is observed in using algorithms for water quality prediction, with algorithms such as Decision Tree, Artificial Neural Networks and Bayesian Belief Networks demonstrating satisfactory results. Machine learning approaches can provide critical information about the dynamic of E. coli contamination and, concurrently, consider the meteorological parameters for coastal waters classification.

Background: Lymphopenia, particularly when restricted to the T-cell compartment, has been described as one of the major clinical hallmarks in patients with coronavirus disease 2019 (COVID-19) and proposed as an indicator of disease severity. Although several mechanisms fostering COVID-19-related lymphopenia have been described, including cell apoptosis and tissue homing, the underlying causes of the decline in T-cell count and function are still not completely understood., Objective: Given that viral infections can directly target thymic microenvironment and impair the process of T-cell generation, we sought to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on thymic function., Methods: We performed molecular quantification of T-cell receptor excision circles and κ-deleting recombination excision circles to assess, respectively, T- and B-cell neogenesis in SARS-CoV-2-infected patients. We developed a system for in vitro culture of primary human thymic epithelial cells (TECs) to mechanistically investigate the impact of SARS-CoV-2 on TEC function., Results: We showed that patients with COVID-19 had reduced thymic function that was inversely associated with the severity of the disease. We found that angiotensin-converting enzyme 2, through which SARS-CoV-2 enters the host cells, was expressed by thymic epithelium, and in particular by medullary TECs. We also demonstrated that SARS-CoV-2 can target TECs and downregulate critical genes and pathways associated with epithelial cell adhesion and survival., Conclusions: Our data demonstrate that the human thymus is a target of SARS-CoV-2 and thymic function is altered following infection. These findings expand our current knowledge of the effects of SARS-CoV-2 infection on T-cell homeostasis and suggest that monitoring thymic activity may be a useful marker to predict disease severity and progression., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the gastrointestinal tract affecting millions of patients worldwide. The gut microbiome partly determines the pathogenesis of both diseases. Even though probiotics have been widely used as a potential treatment, their efficacy in inducing and maintaining remission is still controversial. Our study aims to review the present-day literature about the possible role of probiotics in treating inflammatory bowel diseases in adults. This research was performed according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. We included studies concerning adult patients who compared probiotics with placebo or non-probiotic intervention. We identified thirty-three studies, including 2713 patients from fourteen countries. The role of probiotics in Crohn's disease was examined in eleven studies. Only four studies presented statistically significant results in the remission of disease, primarily when used for three to six months. On the other hand, in twenty-one out of twenty-five studies, probiotics proved effective in achieving or maintaining remission in ulcerative colitis. Supplementation with Bifidobacterium sp . or a combination of probiotics is the most effective intervention, especially when compared with a placebo. There is strong evidence supporting the usage of probiotic supplementation in patients with ulcerative colitis, yet more research is needed to justify their efficacy in Crohn's disease.

Research attention has been drawn to honey's nutritional status and beneficial properties for human health. This study aimed to provide a bibliometric analysis of honey's antioxidant and antimicrobial properties. The research advancements within this field from 2001 to 2022 were addressed using the Scopus database, R, and VOSviewer. Of the 383 results, articles (273) and reviews (81) were the most common document types, while the annual growth rate of published manuscripts reached 17.5%. The most relevant topics about honey's antimicrobial and antioxidant properties were related to the agricultural and biological sciences, biochemistry, and pharmacology. According to a keyword analysis, the most frequent terms in titles, abstracts, and keywords were honey, antimicrobial, antioxidant, bee, propolis, phenolic compounds, wound, antibacterial, anti-inflammatory, and polyphenols. A trend topic analysis showed that the research agenda mainly encompassed antioxidants, pathogens, and anti-infection and chemical agents. In a co-occurrence analysis, antioxidants, anti-infection agents, and chemistry were connected to honey research. The initial research focus of this domain was primarily on honey's anti-inflammatory and antineoplastic activity, wound healing, and antibacterial agents. The research agenda was enriched in the subsequent years by pathogens, propolis, oxidative stress, and flavonoids. It was possible to pinpoint past trends and ongoing developments and provide a valuable insight into the field of honey research.

Non-alcoholic fatty liver disease (NAFLD)-related liver fibrosis results in the encapsulation of injured liver parenchyma by a collagenous scar mainly imputable to hepatic stellate cells' activation. Approved pharmacological treatments against NAFLD-related fibrosis are still lacking, but natural compounds such as hydroxytyrosol (HXT) and vitamin E (VitE), are emerging as promising therapeutic opportunities. In this study, the potential anti-fibrotic effect of HXT + VitE combination therapy was investigated in vitro and in vivo. In particular, tumor growth factor (TGF)-β-activated LX-2 cells as an in vitro model, and carbon tetrachloride plus a Western diet as a mice model were employed. The effect of HXT + VitE on fibrosis was also investigated in children with biopsy-proven NAFLD. Our results demonstrated that HXT + VitE caused a reduction of proliferation, migration, contractility, and expression of pro-fibrogenic genes in TGF-β-activated LX-2 cells. HXT + VitE treatment also antagonized TGF-β-dependent upregulation of pro-oxidant NOX2 by interfering with nuclear translocation/activation of SMAD2/3 transcription factors. The mouse model of NAFLD-related fibrosis treated with HXT + VitE showed a marked reduction of fibrosis pattern by histology and gene expression. Accordingly, in children with NAFLD, HXT + VitE treatment caused a decrease of circulating levels of PIIINP and NOX2 that was supported over time. Our study suggests that HXT + VitE supplementation may improve NAFLD-related fibrosis.

Background: Diffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy., Methods: Immunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function., Results: GD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo., Conclusion: Our study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Purpose: Paediatric obesity is a well-known risk factor for metabolic-associated fatty liver disease (MAFLD). The aim of this study was to evaluate the effects of laparoscopic sleeve gastrectomy (LSG) on the levels of total homocysteine (tHcy) and total glutathione (tGSH) plasma levels in children with MAFLD., Material and Methods: Twenty-four children with severe obesity who underwent LSG were included in the study. The metabolic parameters, systemic inflammatory markers, one-carbon metabolism products, ultrasound and histological improvement were evaluated at baseline (T0M) and after 12 months from LSG (T12M)., Results: The patients exhibited a significant amelioration of several metabolic parameters at T12M. A significant reduction of steatosis was observed at ultrasound (from 72.7% of moderate-severe grade to 0% severe steatosis), accompanied by a statistically significant improvement of ballooning, portal and lobular inflammation and fibrosis. A statistically significant decrease of tumour necrosis factor circulating levels was also observed (T0M median = 290.3, IQR = 281.0-317.0 pg/mL; T12M median = 260.4, IQR = 240.0-279.0 pg/mL; p < 0.0001). After 12 months from LSG, a significant increase of mean plasma levels of tHcy(T0M mean = 15.7 ± 4.1 μmol/L; T12M mean = 21.1 ± 9.3 μmol/L; p = 0.0146) was also observed. The increase of tHcy showed no causal link with the improvement of MAFLD-related inflammatory, metabolic and histological pattern., Conclusion: LSG in children with obesity induces an improvement of MAFLD-related metabolic derangement and liver damage, but also a mild hyperhomocysteinemia that should be avoided to prevent cardiovascular risk., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nephropathic cystinosis is a rare disease caused by mutations of the CTNS gene that encodes for cystinosin, a lysosomal cystine/H+ symporter. The disease is characterized by early-onset chronic kidney failure and progressive development of extra-renal complications related to cystine accumulation in all tissues. At the cellular level, several alterations have been demonstrated, including enhanced apoptosis, altered autophagy, defective intracellular trafficking, and cell oxidation, among others. Current therapy with cysteamine only partially reverts some of these changes, highlighting the need to develop additional treatments. Among compounds that were identified in a previous drug-repositioning study, disulfiram (DSF) was selected for in vivo studies. The cystine depleting and anti-apoptotic properties of DSF were confirmed by secondary in vitro assays and after treating Ctns -/- mice with 200 mg/kg/day of DSF for 3 months. However, at this dosage, growth impairment was observed. Long-term treatment with a lower dose (100 mg/kg/day) did not inhibit growth, but failed to reduce cystine accumulation, caused premature death, and did not prevent the development of renal lesions. In addition, DSF also caused adverse effects in cystinotic zebrafish larvae. DSF toxicity was significantly more pronounced in Ctns -/- mice and zebrafish compared to wild-type animals, suggesting higher cell toxicity of DSF in cystinotic cells.

Background: Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors (FAKi), alone or in combination with SOR, using in vitro and in vivo models of HCC., Methods: The effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods., Results: TAE226 was the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of FAK nuclear interactome. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation caused a decrease in the nuclear amount of HDAC1/2 and a consequent increase of the histone H3 lysine 27 acetylation, thus counteracting histone H3 lysine 27 trimethylation., Conclusions: Altogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduces HCC growth in vitro and in vivo. Also, our data highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising targets for HCC therapy., (© 2021. The Author(s).)

Cleft lip and palate (CL/P) is the most prevalent craniofacial birth defect in humans. None of the surgical procedures currently used for CL/P repair lead to definitive correction of hard palate bone interruption. Advances in tissue engineering and regenerative medicine aim to develop new strategies to restore palatal bone interruption by using tissue or organ-decellularized bioscaffolds seeded with host cells. Aim of this study was to set up a new natural scaffold deriving from a decellularized porcine mucoperiosteum, engineered by an innovative micro-perforation procedure based on Quantum Molecular Resonance (QMR) and then subjected to in vitro recellularization with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Our results demonstrated the efficiency of decellularization treatment gaining a natural, non-immunogenic scaffold with preserved collagen microenvironment that displays a favorable support to hMSC engraftment, spreading and differentiation. Ultrastructural analysis showed that the micro-perforation procedure preserved the collagen mesh, increasing the osteoinductive potential for mesenchymal precursor cells. In conclusion, we developed a novel tissue engineering protocol to obtain a non-immunogenic mucoperiosteal scaffold suitable for allogenic transplantation and CL/P repair. The innovative micro-perforation procedure improving hMSC osteogenic differentiation potentially impacts for enhanced palatal bone regeneration leading to future clinical applications in humans., (© 2021. The Author(s).)

Background : Spindle cell rhabdomyosarcoma (S-RMS) is a rare tumor that was previously considered as an uncommon variant of embryonal RMS (ERMS) and recently reclassified as a distinct RMS subtype with NCOA2, NCOA1, and VGLL2 fusion genes. In this study, we established a cell line (S-RMS1) derived from a four-month-old boy with infantile spindle cell RMS harboring SRF-NCOA2 gene fusion. Methods : Morphological and molecular characteristics of S-RMS1 were analyzed and compared with two RMS cell lines, RH30 and RD18. Whole genome sequencing of S-RMS1 and clinical exome sequencing of genomic DNA were performed. Results : S-RMS1 showed cells small in size, with a fibroblast-like morphology and positivity for MyoD-1, myogenin, desmin, and smooth muscle actin. The population doubling time was 3.7 days. Whole genome sequencing demonstrated that S-RMS1 retained the same genetic profile of the tumor at diagnosis. A Western blot analysis showed downregulation of AKT-p and YAP-p while RT-qPCR showed upregulation of endoglin and GATA6 as well as downregulation of TGFßR1 and Mef2C transcripts. Conclusion : This is the first report of the establishment of a cell line from an infantile spindle cell RMS with SRF-NCOA2 gene fusion. S-RMS1 should represent a useful tool for the molecular characterization of this rare and almost unknown tumor.

Background: Pediatric high-grade gliomas (pHGGs) are among the most common and incurable malignant neoplasms of childhood. Despite aggressive, multimodal treatment, the outcome of children with high-grade gliomas has not significantly improved over the past decades, prompting the development of innovative approaches., Methods: To develop an effective treatment, we aimed at improving the suboptimal antitumor efficacy of oncolytic adenoviruses (OAs) by testing the combination with a gene-therapy approach using a bispecific T-cell engager (BiTE) directed towards the erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2), conveyed by a replication-incompetent adenoviral vector (EphA2 adenovirus (EAd)). The combinatorial approach was tested in vitro, in vivo and thoroughly characterized at a molecular level., Results: After confirming the relevance of EphA2 as target in pHGGs, documenting a significant correlation with worse clinical outcome of the patients, we showed that the proposed strategy provides significant EphA2-BiTE amplification and enhanced tumor cell apoptosis, on coculture with T cells. Moreover, T-cell activation through an agonistic anti-CD28 antibody further increased the activation/proliferation profiles and functional response against infected tumor cells, inducing eradication of highly resistant, primary pHGG cells. The gene-expression analysis of tumor cells and T cells, after coculture, revealed the importance of both EphA2-BiTE and costimulation in the proposed system. These in vitro observations translated into significant tumor control in vivo, in both subcutaneous and a more challenging orthotopic model., Conclusions: The combination of OA and EphA2-BiTE gene therapy strongly enhances the antitumor activity of OA, inducing the eradication of highly resistant tumor cells, thus supporting the clinical translation of the approach., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway 1,2 . Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

The prognosis of many patients with chemotherapy-refractory or multiply relapsed CD30+ non-Hodgkin Lymphoma (NHL) or Hodgkin lymphoma (HL) still remains poor, and novel therapeutic approaches are warranted to address this unmet clinical need. In light of this consideration, we designed and pre-clinically validated a Chimeric Antigen Receptor (CAR) construct characterized by a novel anti-CD30 single-chain variable-fragment cassette, linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. We found that CAR.CD30 T-cells exhibit remarkable cytolytic activity in vitro against HL and NHL cell lines, with sustained proliferation and pro-inflammatory cytokine production, even after multiple and sequential lymphoma cell challenges. CAR.CD30 T-cells also demonstrated anti-lymphoma activity in two in vivo xenograft immune-deficient mouse models of metastatic HL and NHL. We observed that administration of CAR.CD30 T-cells, incorporating the CD28.OX40 costimulatory domains and manufactured in the presence of IL7 and IL15, were associated with the best overall survival in the treated mice, along with the establishment of a long-term immunological memory, able to protect mice from further tumor re-challenge. Our data indicate that, in the context of in vivo systemic metastatic xenograft mouse models, the costimulatory machinery of CD28.OX40 is crucial for improving persistence, in vivo expansion and proliferation of CAR.CD30 T-cells upon tumor encounter. CD28.OX40 costimulatory combination is ultimately responsible for the antitumor efficacy of the approach, paving the way to translate this therapeutic strategy in patients with CD30+ HL and NHL.

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.

Background and Aims: Liver X receptors (LXRs) exert anti-inflammatory effects even though their hepatic activation is associated with hypertriglyceridemia and hepatic steatosis. Selective induction of LXRs in the gut might provide protective signal(s) in the aberrant wound healing response that induces fibrosis during chronic liver injury, without hypertriglyceridemic and steatogenic effects., Methods: Mice with intestinal constitutive LXRα activation (iVP16-LXRα) were exposed to intraperitoneal injection of carbon tetrachloride (CCl 4 ) for 8 weeks, and in vitro cell models were used to evaluate the beneficial effect of high-density lipoproteins (HDL)., Results: After CCl 4 treatment, the iVP16-LXRα phenotype showed reduced M1 macrophage infiltration, increased expression M2 macrophage markers, and lower expression of hepatic pro-inflammatory genes. This anti-inflammatory effect in the liver was also associated with decreased expression of hepatic oxidative stress genes and reduced expression of fibrosis markers. iVP16-LXRα exhibited increased reverse cholesterol transport in the gut by ABCA1 expression and consequent enhancement of the levels of circulating HDL and their receptor SRB1 in the liver. No hepatic steatosis development was observed in iVP16-LXRα. In vitro, HDL induced a shift from M1 to M2 phenotype of LPS-stimulated Kupffer cells, decreased TNFα-induced oxidative stress in hepatocytes and reduced NF-kB activity in both cells. SRB1 silencing reduced TNFα gene expression in LPS-stimulated KCs, and NOX-1 and IL-6 in HepG2., Conclusions: Intestinal activation of LXRα modulates hepatic response to injury by increasing circulating HDL levels and SRB1 expression in the liver, thus suggesting this circuit as potential actionable pathway for therapy., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Focal adhesion kinase (FAK) is over-expressed and is correlated with aggressiveness in adult hepatocellular carcinoma (HCC). Inhibition of FAK decreases HCC invasiveness by down-regulating Enhancer of Zeste homolog 2 (EZH2), an epigenetic controller, that acts in transcriptional repression of a large number of genes via histone 3 methylation of lysine 27 (H3K27me3). Here, we investigated the hepatic expression of total FAK, EZH2, H3K27me3, and proliferating cell nuclear antigen (PCNA) in 17 pediatric HCCs and 8 healthy livers (CTRL). Quantitative imaging analysis showed that FAK gene/protein expression is up-regulated in HCCs compared to CTRL and, among tumor samples the levels of this gene/protein are significantly higher in cirrhotic HCCs than in a healthy milieu. Accordingly, the protein levels of EZH2 were also significantly increased in HCCs from a cirrhotic background. Intriguingly, the protein expression of FAK, EZH2, and PCNA significantly inversely correlated with tumor size. Finally, in HCC samples, mainly in cirrhotic background, the up-regulation of FAK gene positively correlated with that observed in β-Catenin gene. Conclusion: FAK gene/protein is over-expressed in pediatric HCCs concomitantly to EZH2 protein and β-Catenin gene, with a more significant up-regulation in a cirrhotic background. This triad of interactors deserves further investigations for translational application.

Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement., Competing Interests: The authors declare no potential conflicts of interest., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.)

Background & Aim: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in adults and children. Along with obesity, diabetes and insulin resistance, genetic factors strongly impact on NAFLD development and progression. Dysregulated bile acid metabolism and the fibroblast growth factor 19 (FGF19) pathway play a pivotal role in NAFLD pathogenesis. However, the mechanism through which the FGF19 receptor system is associated with liver damage in NAFLD remains to be defined., Methods: We evaluated the impact of the rs17618244 G>A β-Klotho (KLB) variant on liver damage in 249 pediatric patients with biopsy-proven NAFLD and the association of this variant with the expression of hepatic and soluble KLB. In vitro models were established to investigate the role of the KLB mutant., Results: The KLB rs17618244 variant was associated with an increased risk of ballooning and lobular inflammation. KLB plasma levels were lower in carriers of the rs17618244 minor A allele and were associated with lobular inflammation, ballooning and fibrosis. In HepG2 and Huh7 hepatoma cell lines, exposure to free fatty acids caused a severe reduction of intracellular and secreted KLB. Finally, KLB downregulation obtained by the expression of a KLB mutant in HepG2 and Huh7 cells induced intracellular lipid accumulation and upregulation of p62, ACOX1, ACSL1, IL-1β and TNF-α gene expression., Conclusion: In conclusion, we showed an association between the rs17618244 KLB variant, which leads to reduced KLB expression, and the severity of NAFLD in pediatric patients. We can speculate that the KLB protein may exert a protective role against lipotoxicity and inflammation in hepatocytes., Lay Summary: Genetic and environmental factors strongly impact on the pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD). The FGF19/FGFR4/KLB pathway plays a pivotal role in the pathogenesis of NAFLD. The aim of the study was to investigate the impact of a genetic variant in the KLB gene on the severity of liver disease. Our data suggest that the KLB protein plays a protective role against lipotoxicity and inflammation in hepatocytes., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Objective: To evaluate the histopathologic modifications in liver and visceral adipose tissue (VAT), and to correlate these changes with clinical measures, adipokine production, and proinflammatory cytokines in a population of adolescents with obesity with nonalcoholic fatty liver disease (NAFLD) who underwent laparoscopic sleeve gastrectomy (LSG)., Study Design: Twenty adolescents with obesity who underwent LSG and with biopsy-proven NAFLD were included. Patients underwent clinical evaluation and blood tests at baseline and 1 year after the surgical procedure. Liver and VAT specimens were processed for routine histology, immunohistochemistry, and immunofluorescence., Results: In adolescents with obesity and NAFLD, hepatic histologic alterations were uncorrelated with VAT inflammation. LSG induced in both liver and VAT tissue histopathology amelioration and macrophage profile modification that were correlated with body mass index and improvement in insulin resistance. The adipokine profile in liver and VAT was associated with weight loss and histologic improvement after LSG. Serum proinflammatory cytokines were correlated with liver and VAT histopathology and IL-1β and IL-6 levels were independently predicted by liver necroinflammatory grade., Conclusions: This study suggests a unique adipose tissue/fatty liver crosstalk in pediatric patients. LSG induces a similar pattern of histologic improvement in the liver and in VAT. Besides VAT, our results strengthen the role of the liver in adipocytokine production and its contribution to systemic inflammation in pediatric patients with NAFLD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: Clinical recognition of vascular acrosyndromes is often challenging. The term Raynaud's phenomenon (RP) is commonly overused to describe any form of cold-related disorder. This study aims to formally evaluate peripheral vascular symptoms affecting the population, aged ≤ 40 years, and identify any correlations to joint hypermobility (JH)., Patients and Methods: Fifty patients (31 males, 19 females) with vasomotor symptoms enrolled in this five-year prospective observational study. Clinical examination by a rheumatologist and a vascular surgeon was performed along with cardiology, echocardiographic and Doppler evaluation. Patients underwent blood cell count, biochemistry, thyroid and selectively immunologic testing. Twenty-four (48%) of them performed nailfold capillaroscopy. The SPSS for Windows, v.17.0, Chicago, USA, was used for the statistical analyses., Results: Twenty-eight patients (56%) presented with erythromelalgia (EM), 6 (12%) with acrocyanosis (AC) and 9 (18%) as a combination of the above disorder. RP diagnosed in five (10%) while two patients (4%) presented as a mix of EM-RP. There was no correlation with abnormal laboratory tests. Increased incidence of JH was found in EM and AC patients. Among those who were tested with nailfold capillaroscopy, 75% had abnormalities ranged from mild to autoimmune-like diseases., Conclusions: Erythromelalgia is the commonest functional vasculopathy in young population followed by acrocyanosis and a combination of these conditions. Joint hypermobility is markedly increased, indicating that dysautonomy may be considered the causative factor following a trigger event. Overall, RP was observed in 14% of patients. Clinical recognition of these disorders avoids unnecessary investigation. Key Points • Vascular acrosyndromes in young adults are commonly functional disorders resembling vascular algodystrophy induced by thermic stress. • Dysautonomy of joint hypermobility is the co-factor influencing the appearance of the vascular disorders. • Raynaud's phenomenon accounts to approximately 14% of vascular acrosyndromes presented in the young adult population.

Non-alcoholic fatty liver disease (NAFLD) is a complex disease ranging from steatosis to non-alcoholic steatohepatitis (NASH). Galectin-3 (Gal-3), which is a β-galactoside binding protein, has been associated with liver fibrosis, but its role in NAFLD remains elusive. We investigated the expression of Gal-3 in liver resident cells and its potential association with liver damage in 40 children with biopsy-proven NAFLD. We found that several liver cells expressed Gal-3. The number of total Gal-3 positive cells decreased with the severity of disease and the cells were correlated with the presence of steatosis and the diagnosis of NASH. CD68 macrophages expressed Gal-3 but the number CD68/Gal-3 positive cells was significantly reduced in patients diagnosed with steatosis and NASH. Triple CD68/CD206/Gal-3, which represented the subpopulation of M2 macrophages, were mainly present in patients without NASH, and clearly reduced in patients with steatosis and NASH. On the contrary, the number of α-smooth muscle actin (SMA)/Gal-3 positive cells increased with the severity of fibrosis in children with NAFLD. Our data demonstrated that the number of Gal-3 positive cells was associated with tissue damage in different ways, which suggests a dual role of this protein in the pathogenesis of pediatric NAFLD, even if the role of Gal-3 deserves further studies.

Background and Aims: Nonalcoholic fatty liver disease is one of the major complications of obesity, occurring already in pediatric age. Insulin like growth factor-I has been proposed as a potential therapeutic agent for its beneficial effect in experimental liver fibrosis. The aim of this work was to investigate the expression of insulin-like growth factor-I and its receptor in the liver of children with biopsy-proven nonalcoholic fatty liver disease and relate it to liver histological features., Methods: 45 obese children and adolescents (14 females and 31 males) with nonalcoholic fatty liver disease were included. Insulin like growth factor-I and its receptor expression was evaluated in liver tissue by immunofluorescence and qPCR., Results: The expression of insulin like growth factor-I and its receptor were significantly related to fibrosis and were higher in children with stage 3 fibrosis compared to stage 1 and 2 (p<0.001 and p = 0.007 respectively). mRNA of insulin like growth factor-I receptor was higher in more advanced stages of fibrosis (p<0.001). Furthermore, the expression of insulin like growth factor-I and its receptor in hepatic stellate cells, the cell type mostly involved in fibrosis progression, was significantly increased in stage 3 fibrosis compared to stage 1 (p = 0.01 and p = 0.008 respectively)., Conclusions: We demonstrated for the first time that insulin like growth factor-I and its receptor are upregulated in children with nonalcoholic fatty liver disease. These findings give a new hint for the potential therapeutic use of insulin like growth factor-I in pediatric nonalcoholic fatty liver disease complicated by liver fibrosis., Competing Interests: The authors have declared that no competing interests exist.

Early life events are associated with the susceptibility to chronic diseases in adult life. Perturbations of endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which contributes to the development of metabolic alterations. Our aim was to evaluate liver UPR in an animal model of intrauterine growth restriction (IUGR). A significantly increased expression of X-box binding protein-1 spliced (XBP1s) mRNA (p<0.01), Endoplasmic Reticulum-localized DnaJ homologue (Erdj4) mRNA (p<0.05) and Bip/GRP78-glucose-regulated protein 78 (Bip) mRNA (p<0.05) was observed in the liver of IUGR rats at birth. Furthermore, the expression of gluconeogenesis genes and lipogenesis genes were significantly upregulated (p<0.05) in IUGR pups. At 105 d, IUGR male rats showed significantly reduced glucose tolerance (p<0.01). A significant decreased expression of XBP1s mRNA (p<0.01) and increased expression of double-stranded RNA-dependent protein kinase-like ER kinase (PERK) and Asparagine synthetase (ASNS) (p<0.05) was observed in the liver of IUGR male adult rats. Liver focal steatosis and periportal fibrosis were observed in IUGR rats. These findings show for the first time that fetal exposure to uteroplacental insufficiency is associated with the activation of hepatic UPR and suggest that UPR signaling may play a role in the metabolic risk., Competing Interests: The authors have declared that no competing interests exist.

Background & Aims: Treatment with the farnesoid X receptor (FXR) agonist obeticholic acid is ineffective in some patients with non-alcoholic steatohepatitis (NASH) but the explanation is uncertain. We investigated hepatic FXR expression, and measurements of fibroblast growth factor 19 (FGF19) and bile acids (BAs) in children with NAFLD to investigate relationships with NASH., Methods: 33 children with NAFLD who underwent diagnostic liver biopsy were studied. Hepatic FXR protein levels and circulating FGF19 concentrations were compared with those analysed in five control subjects with proven normal liver histology. NASH was defined by the Paediatric NAFLD Histological Score (PNHS). Binary logistic regression with adjustment for covariates and potential confounders was undertaken to test factors independently associated with: a) NASH and b) hepatic FXR protein levels., Results: Mean ± SD age was 13.7 ± 1.9 years. Nineteen patients had NASH (PNHS ≥ 85) and 14 did not have NASH (PNHS < 85). Hepatic FXR level and plasma FGF19 concentration varied ~10-fold and 5-fold, respectively, between groups, and was highest in control subjects, intermediate in NAFLD without NASH, and lowest in NASH (between group differences P < .001 and P < .01 respectively). NASH was independently associated with both FXR protein levels (OR = 0.18, 95% CI 0.09, 0.38) and FGF19 concentration (OR = 0.55, 95% CI 0.20, 0.89)., Conclusions: FXR protein levels vary markedly between normal liver, NAFLD without NASH, and NASH. Low levels of FXR are independently associated with NASH., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hepatocellular carcinoma (HCC) is the most common type of liver cancer in humans. The focal adhesion tyrosine kinase (FAK) is often over-expressed in human HCC and FAK inhibition may reduce HCC cell invasiveness. However, the anti-oncogenic effect of FAK knockdown in HCC cells remains to be clarified. We found that FAK depletion in HCC cells reduced in vitro and in vivo tumorigenicity, by inducing G2/M arrest and apoptosis, decreasing anchorage-independent growth, and modulating the expression of several cancer-related genes. Among these genes, we showed that FAK silencing decreased transcription and nuclear localization of enhancer of zeste homolog 2 (EZH2) and its tri-methylation activity on lysine 27 of histone H3 (H3K27me3). Accordingly, FAK, EZH2 and H3K27me3 were concomitantly upregulated in human HCCs compared to non-tumor livers. In vitro experiments demonstrated that FAK affected EZH2 expression and function by modulating, at least in part, p53 and E2F2/3 transcriptional activity. Moreover, FAK silencing downregulated both EZH2 binding and histone H3K27me3 levels at the promoter of its target gene NOTCH2. Finally, we found that pharmacological inhibition of FAK activity resembled these effects although milder. In summary, we demonstrate that FAK depletion reduces HCC cell growth by affecting cancer-promoting genes including the pro-oncogene EZH2. Furthermore, we unveil a novel unprecedented FAK/EZH2 crosstalk in HCC cells, thus identifying a targetable network paving the way for new anticancer therapies.

Ozone was used to control spoilage microorganisms during the manufacturing of dairy products. Ozone stream was applied onto the surface of freshly filled yoghurt cups just before storage for curd development in order to prevent cross contamination from spoilage airborne microorganisms. Accordingly, brine solution was bubbled with ozone for various periods of time and used for ripening of white (feta type) cheese. Both products were subjected to a continuous monitoring of microbial load and also tested for their sensorial properties. In ozonated yoghurt samples there was a reduction in mould counts of approximately 0.6Logcfu/g (25.1%) by the end of the monitoring period in relation to the control samples. In white cheese ripened with ozonated brine (1.3mg/L O 3 , NaCl 5%) it seems that ozone treatment during the two months of observation reduced some of the mould load but without offering any advantages over the use of traditional brine (NaCl 7%). However, some sensorial alterations were observed, probably due to the organic load in the brine which deactivates ozone in early stages of application. It is concluded that, if the factors of time and concentration of ozone are configured properly, ozonation could be a promising approach safeguarding the production of some dairy products., (Copyright © 2017 Elsevier B.V. All rights reserved.)

There is evidence that nonalcoholic fatty liver disease (NAFLD) is affected by gut microbiota. Therefore, we investigated its modifications in pediatric NAFLD patients using targeted metagenomics and metabolomics. Stools were collected from 61 consecutive patients diagnosed with nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), or obesity and 54 healthy controls (CTRLs), matched in a case-control fashion. Operational taxonomic units were pyrosequenced targeting 16S ribosomal RNA and volatile organic compounds determined by solid-phase microextraction gas chromatography-mass spectrometry. The α-diversity was highest in CTRLs, followed by obese, NASH, and NAFL patients; and β-diversity distinguished between patients and CTRLs but not NAFL and NASH. Compared to CTRLs, in NAFLD patients Actinobacteria were significantly increased and Bacteroidetes reduced. There were no significant differences among the NAFL, NASH, and obese groups. Overall NAFLD patients had increased levels of Bradyrhizobium, Anaerococcus, Peptoniphilus, Propionibacterium acnes, Dorea, and Ruminococcus and reduced proportions of Oscillospira and Rikenellaceae compared to CTRLs. After reducing metagenomics and metabolomics data dimensionality, multivariate analyses indicated a decrease of Oscillospira in NAFL and NASH groups and increases of Ruminococcus, Blautia, and Dorea in NASH patients compared to CTRLs. Of the 292 volatile organic compounds, 26 were up-regulated and 2 down-regulated in NAFLD patients. Multivariate analyses found that combination of Oscillospira, Rickenellaceae, Parabacteroides, Bacteroides fragilis, Sutterella, Lachnospiraceae, 4-methyl-2-pentanone, 1-butanol, and 2-butanone could discriminate NAFLD patients from CTRLs. Univariate analyses found significantly lower levels of Oscillospira and higher levels of 1-pentanol and 2-butanone in NAFL patients compared to CTRLs. In NASH, lower levels of Oscillospira were associated with higher abundance of Dorea and Ruminococcus and higher levels of 2-butanone and 4-methyl-2-pentanone compared to CTRLs., Conclusion: An Oscillospira decrease coupled to a 2-butanone up-regulation and increases in Ruminococcus and Dorea were identified as gut microbiota signatures of NAFL onset and NAFL-NASH progression, respectively. (Hepatology 2017;65:451-464)., (© 2016 by the American Association for the Study of Liver Diseases.)

Stigma and mental health literacy affect access to and quality of treatment of major depression. Though mental health professionals seem better able to recognize major depression than the general public, they often hold similarly stigmatizing attitudes towards people suffering from the disorder. These attitudes are shaped jointly by the public stigma attached to mental illnesses as well as by the content and delivery of mental health professionals' undergraduate training. In line with this, the present study aimed to explore psychology students' ability to recognize major depression, their attitudes towards the disorder, and their views surrounding helpfulness of various interventions. A random sample of 167 undergraduate students was recruited from the psychology department of one public university in Athens. During one university hour, students were administered a vignette describing a woman fulfilling the DSM-IV criteria for major depression. A self-report questionnaire exploring students' recognition abilities, attitudes to depression and views on the helpfulness of various treatment modes was also administered. In total, 80.2% of students correctly recognized major depression from the vignette. Concerning their attitudes, students were unsure about the illness and ambivalent towards the person who suffers from it. With regard to available treatments for depression, students considered discussion with a friend to be the most helpful intervention. Counseling, cognitive behavioural therapy and psychoanalysis were also viewed in a positive light. On the contrary, antidepressants were not deemed helpful by most students. Finally, recognition of as well as attitudes towards depression and its treatments seemed to improve during the second year of undergraduate study; however they remained unchanged thereafter. Consistent with these, psychology students seem to have only a rudimentary knowledge on depression, that cannot not be qualified as mental health literacy. The core misconception espoused pertains to the view that major depression is not a medical illness; a finding which can also be interpreted in light of the lingering controversy on the medicalization of normal sadness and human predicament. The clinical implications of these findings are substantial. Mental health professionals-educators should reflect on their own beliefs and attitudes towards depression, as they may convey stigmatizing messages to their students and thus perpetuate the stigmatization of the illness. Concomitantly, psychology students' attitudes to depression and its treatment might render them incapable of understanding their patients, responding to their needs and providing them with appropriate help, while they may hinder their effective collaboration with psychiatrists.

Objective: To determine whether bariatric surgery is effective for the treatment of nonalcoholic steatohepatitis (NASH) in adolescence, we compared the efficacy of laparoscopic sleeve gastrectomy (LSG) with that of lifestyle intervention (nonsurgical weight loss [NSWL]) for NASH reversal in obese adolescents., Study Design: Obese (body mass index ≥ 35 kg/m 2 ) adolescents (13-17 years of age) with biopsy-proven NAFLD underwent LSG, lifestyle intervention plus intragastric weight loss devices (IGWLD), or only NSWL. At baseline and 1 year after treatment, patients underwent clinical and psychosocial evaluation, blood tests, liver biopsy, polysomnography, and 24-hour ambulatory blood pressure estimation., Results: Twenty patients (21%) underwent LSG, 20 (21%) underwent IGWLD, and 53 (58%) received lifestyle intervention alone (NSWL). One year after treatment, patients who underwent LSG lost 21.5% of their baseline body weight, whereas patients who underwent IGWLD lost 3.4%, and patients who underwent NSWL increase 1.7%. In patients who underwent LSG, NASH reverted completely in all patients and hepatic fibrosis stage 2 disappeared in 18 patients (90%). After IGWLD, NASH reverted in 6 patients (24%) and fibrosis in 7 (37%). Patients who received the NSWL intervention did not improve significantly. Hypertension resolved in all patients who underwent LSG with preoperative hypertension (12/12) versus 50% (4/8) of the patients who underwent IGWLD (P = .02). The cohort-specific changes in impaired glucose metabolism were similar: 100% (9/9) of affected patients who underwent LSG versus 50% (1/2) of patients who underwent IGWLD (P = .02). LSG was also more affective in resolving dyslipidemia (55% [7/12] vs 26% [10/19]; P = .05) and sleep apnea (78% [2/9] vs 30% [11/20]; P = .001)., Conclusion: LSG was more effective than lifestyle intervention, even when combined with intragastric devices, for reducing NASH and liver fibrosis in obese adolescents after 1 year of treatment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: There are no licensed treatments for non alcoholic fatty liver disease (NAFLD) in adults or children. In NAFLD, several studies have shown a benefit of omega-3 fatty acid treatment on lipid profile, insulin-sensitivity and hepatic steatosis and it has also been suggested that Vitamin D treatment has potential antifibrotic properties in liver disease., Trial Design: To date, however, there are no studies that have tested the combination of Docosahexanoic acid (DHA) and vitamin D treatment which may benefit the whole spectrum of disease in NAFLD. Our aim therefore, was to test the effect of daily DHA (500 mg) plus vitamin D (800 IU) treatment, in obese children with biopsy-proven NAFLD and vitamin D deficiency, in a randomized, double-blind placebo-controlled trial., Methods: The 41/43 patients completed the study (18-treatment, 23-placebo). At 12 months: i) the main outcome was liver histology improvement, defined by NAS; ii) the secondary outcome was amelioration of metabolic parameters., Results: DHA plus vitamin D treatment reduced the NAFLD Activity Score (NAS), in the treatment group (5.4 v1.92; p<0.001 for baseline versus end of study). There was no change in fibrosis score, but a reduction of the activation of hepatic stellate cells (HSC) and fibrillar collagen content was noted (3.51±1.66 v. 1.59±1.37; p = 0.003) in treatment group. Moreover, the triglycerides (174.5 vs. 102.15 mg/dl), ALT (40.25 vs. 24.5 UI/l) and HOMA-IR (4.59 vs. 3.42) were all decreased with treatment., Conclusion: DHA plus vitamin D treatment improved insulin-resistance, lipid profile, ALT and NAS. There was also decreased HSC activation and collagen content with treatment., Competing Interests: The authors have declared that no competing interests exist.

Rises in unemployment as a corollary to the global economic crisis may impinge on the prevalence of depression disproportionally for the two genders. Therefore, differences in the prevalence of the disorder as a function of gender and age were explored in four nationwide surveys in Greece in 2008, 2009, 2011 and 2013. Results indicate that in 2013, men of productive age had a higher prevalence of major depression than women, in contrast to the epidemiology of the disorder. The psychiatry community should be in tune to future changes in the mental health landscape elicited by the social fermentation processes of the global recession., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Purpose: A series of repeated cross-sectional surveys conducted in 2008, 2009, 2011 and 2013 were conducted with the aim of estimating the prevalence of major depression and suicidality as well as of investigating its risk factors. The present report concentrates on the 2013 survey., Methods: A random and representative sample of 2.188 people was telephone interviewed with regard to various socio-economic indicators and the presence of major depression and suicidality, which were assessed with the germane module of the Structured Clinical Interview., Results: Findings suggest a rise in 1-month prevalence of major depression (12.3 %) and a decline in prevalence of suicidality (2.8 %). Female gender, residence in rural area, low educational attainment, unemployment and economic hardship were found to increase the odds of suffering from major depression. The influence of economic hardship and unemployment on suicidality was also substantial and independent of major depression., Conclusions: Results stress the imperative need for the design and implementation of social policies and interventions that would offset the dire impact of the sustained recession in Greece.

Non-alcoholic fatty liver disease is one of the most important causes of liver-related morbidity in children. In non-alcoholic fatty liver disease, the activation of liver resident macrophage pool is a central event in the progression of liver injury. The aims of the present study were to evaluate the polarization of liver macrophages and the possible role of Wnt3a production by macrophages in hepatic progenitor cell response in the progression of pediatric non-alcoholic fatty liver disease. 32 children with biopsy-proven non-alcoholic fatty liver disease were included. 20 out of 32 patients were treated with docosahexaenoic acid for 18 months and biopsies at the baseline and after 18 months were included. Hepatic progenitor cell activation, macrophage subsets and Wnt/β-catenin pathway were evaluated by immunohistochemistry and immunofluorescence. Our results indicated that in pediatric non-alcoholic fatty liver disease, pro-inflammatory macrophages were the predominant subset. Macrophage polarization was correlated with Non-alcoholic fatty liver disease Activity Score, ductular reaction, and portal fibrosis; docosahexaenoic acid treatment determined a macrophage polarization towards an anti-inflammatory phenotype in correlation with the reduction of serum inflammatory cytokines, with increased macrophage apoptosis, and with the up-regulation of macrophage Wnt3a expression; macrophage Wnt3a expression was correlated with β-catenin phosphorylation in hepatic progenitor cells and signs of commitment towards hepatocyte fate. In conclusion, macrophage polarization seems to have a key role in the progression of pediatric non-alcoholic fatty liver disease; the modulation of macrophage polarization could drive hepatic progenitor cell response by Wnt3a production.

Background: The financial crisis has yielded adverse effects on the population worldwide, as evidenced by elevated rates of major depression. International recommendations for offsetting the mental health impact of the recession highlight the need for effective treatment, including reduction in the stigma attached to the disorder., Aims: This study endeavoured to explore lay attitudes to depression and psychiatric medication during a period of financial crisis and to identify their correlates. Furthermore, it investigated their link to help-seeking intentions., Method: A random and representative sample of 621 respondents from Athens area participated in the study (Response Rate = 81.7%). The telephone interview schedule consisted of the Personal Stigma Scale, a self-constructed scale tapping attitudes to psychiatric medication and one question addressing help-seeking intentions., Results: The preponderant stigmatising belief about depression pertains to perceiving the disorder as a sign of personal weakness. In addition, stereotypes of unpredictability and dangerousness were popular among the sample. Nonetheless, stigmatising beliefs are much stronger with regard to psychiatric medication; perceived as addictive, capable of altering one's personality, less effective than homeopathic remedies and doing more harm than good. Help-seeking intentions were predicted by education, unemployment and attitudes to psychiatric medication solely., Conclusion: Research on the mental health effects of the global recession should encompass studies investigating the stigma attached to mental disorders and its implications., (© The Author(s) 2016.)

Lipopolysaccharide (LPS) is currently considered one of the major players in non-alcoholic fatty liver disease (NAFLD) pathogenesis and progression. Here, we aim to investigate the possible role of LPS-induced TNF-α factor (LITAF) in inducing a pro-inflammatory and pro-fibrogenic phenotype of non-alcoholic steatohepatitis (NASH).We found that children with NAFLD displayed, in different liver-resident cells, an increased expression of LITAF which correlated with histological traits of hepatic inflammation and fibrosis. Total and nuclear LITAF expression increased in mouse and human hepatic stellate cells (HSCs). Moreover, LPS induced LITAF-dependent transcription of IL-1β, IL-6 and TNF-α in the clonal myofibroblastic HSC LX-2 cell line, and this effect was hampered by LITAF silencing. We showed, for the first time in HSCs, that LITAF recruitment to these cytokine promoters is LPS dependent. However, preventing LITAF nuclear translocation by p38MAPK inhibitor, the expression of IL-6 and TNF-α was significantly reduced with the aid of p65NF-ĸB, while IL-1β transcription exclusively required LITAF expression/activity. Finally, IL-1β levels in plasma mirrored those in the liver and correlated with LPS levels and LITAF-positive HSCs in children with NASH.In conclusion, a more severe histological profile in paediatric NAFLD is associated with LITAF over-expression in HSCs, which in turn correlates with hepatic and circulating IL-1β levels outlining a panel of potential biomarkers of NASH-related liver damage. The in vitro study highlights the role of LITAF as a key regulator of the LPS-induced pro-inflammatory pattern in HSCs and suggests p38MAPK inhibitors as a possible therapeutic approach against hepatic inflammation in NASH.