GD2-Targeting CAR T-cell Therapy for Patients with GD2+ Medulloblastoma.

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.

The Use of Artificial Intelligence in the Liver Histopathology Field: A Systematic Review.

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.

MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2 targeting.

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 p27Kip1 and p57Kip2, 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.

Combination Treatment with Hydroxytyrosol and Vitamin E Improves NAFLD-Related Fibrosis.

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)-ß-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-ß-activated LX-2 cells. HXT + VitE treatment also antagonized TGF-ß-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.

Changes in Total Homocysteine and Glutathione Levels After Laparoscopic Sleeve Gastrectomy in Children with Metabolic-Associated Fatty Liver Disease.

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.

Dual IGF1R/IR inhibitors in combination with GD2-CAR T-cells display a potent anti-tumor activity in diffuse midline glioma H3K27M-mutant.

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.

Benefits and Toxicity of Disulfiram in Preclinical Models of Nephropathic Cystinosis.

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.

Focal adhesion kinase inhibitor TAE226 combined with Sorafenib slows down hepatocellular carcinoma by multiple epigenetic effects.

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.

Establishment and Characterization of a Cell Line (S-RMS1) Derived from an Infantile Spindle Cell Rhabdomyosarcoma with SRF-NCOA2 Fusion Transcript.

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.

Oncolytic adenovirus and gene therapy with EphA2-BiTE for the treatment of pediatric high-grade gliomas.

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.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

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) pathway1,2. 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.

Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma.

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.

CD28.OX40 co-stimulatory combination is associated with long in vivo persistence and high activity of CAR.CD30 T-cells.

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.

Neuroblastoma-secreted exosomes carrying miR-375 promote osteogenic differentiation of bone-marrow mesenchymal stromal cells.

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.

Focal Adhesion Kinase (FAK) Over-Expression and Prognostic Implication in Pediatric Hepatocellular Carcinoma.

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 ß-Catenin gene. Conclusion: FAK gene/protein is over-expressed in pediatric HCCs concomitantly to EZH2 protein and ß-Catenin gene, with a more significant up-regulation in a cirrhotic background. This triad of interactors deserves further investigations for translational application.

The Contribution of the Adipose Tissue-Liver Axis in Pediatric Patients with Nonalcoholic Fatty Liver Disease after Laparoscopic Sleeve Gastrectomy.

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-1ß 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.

ß-Klotho gene variation is associated with liver damage in children with NAFLD.

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 ß-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-1ß 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.

The Number of Liver Galectin-3 Positive Cells Is Dually Correlated with NAFLD Severity in Children.

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 ß-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.

Expression of insulin-like growth factor I and its receptor in the liver of children with biopsy-proven NAFLD.

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.