Serum ferritin levels can predict long-term outcomes in patients with metabolic dysfunction-associated steatotic liver disease.

OBJECTIVE: Hyperferritinaemia is associated with liver fibrosis severity in patients with metabolic dysfunction-associated steatotic liver disease (MASLD), but the longitudinal implications have not been thoroughly investigated. We assessed the role of serum ferritin in predicting long-term outcomes or death. DESIGN: We evaluated the relationship between baseline serum ferritin and longitudinal events in a multicentre cohort of 1342 patients. Four survival models considering ferritin with confounders or non-invasive scoring systems were applied with repeated five-fold cross-validation schema. Prediction performance was evaluated in terms of Harrell's C-index and its improvement by including ferritin as a covariate. RESULTS: Median follow-up time was 96 months. Liver-related events occurred in 7.7%, hepatocellular carcinoma in 1.9%, cardiovascular events in 10.9%, extrahepatic cancers in 8.3% and all-cause mortality in 5.8%. Hyperferritinaemia was associated with a 50% increased risk of liver-related events and 27% of all-cause mortality. A stepwise increase in baseline ferritin thresholds was associated with a statistical increase in C-index, ranging between 0.02 (lasso-penalised Cox regression) and 0.03 (ridge-penalised Cox regression); the risk of developing liver-related events mainly increased from threshold 215.5 µg/L (median HR=1.71 and C-index=0.71) and the risk of overall mortality from threshold 272 µg/L (median HR=1.49 and C-index=0.70). The inclusion of serum ferritin thresholds (215.5 µg/L and 272 µg/L) in predictive models increased the performance of Fibrosis-4 and Non-Alcoholic Fatty Liver Disease Fibrosis Score in the longitudinal risk assessment of liver-related events (C-indices>0.71) and overall mortality (C-indices>0.65). CONCLUSIONS: This study supports the potential use of serum ferritin values for predicting the long-term prognosis of patients with MASLD.

Caucasian lean subjects with non-alcoholic fatty liver disease share long-term prognosis of non-lean: time for reappraisal of BMI-driven approach?

OBJECTIVE: The full phenotypic expression of non-alcoholic fatty liver disease (NAFLD) in lean subjects is incompletely characterised. We aimed to investigate prevalence, characteristics and long-term prognosis of Caucasian lean subjects with NAFLD. DESIGN: The study cohort comprises 1339 biopsy-proven NAFLD subjects from four countries (Italy, UK, Spain and Australia), stratified into lean and non-lean (body mass index (BMI) 10 483 person-years), 4.7% of lean vs 7.7% of non-lean patients reported liver-related events (p=0.37). No difference in survival was observed compared with non-lean NAFLD (p=0.069). CONCLUSIONS: Caucasian lean subjects with NAFLD may progress to advanced liver disease, develop metabolic comorbidities and experience cardiovascular disease (CVD) as well as liver-related mortality, independent of longitudinal progression to obesity and PNPLA3 genotype. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD where the disease manifests at lower overall BMI thresholds. LAY SUMMARY: NAFLD may affect and progress in both obese and lean individuals. Lean subjects are predominantly males, have a younger age at diagnosis and are more prevalent in some geographic areas. During the follow-up, lean subjects can develop hepatic and extrahepatic disease, including metabolic comorbidities, in the absence of weight gain. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD.

Long-term outcomes and predictive ability of non-invasive scoring systems in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-invasive scoring systems (NSS) are used to identify patients with non-alcoholic fatty liver disease (NAFLD) who are at risk of advanced fibrosis, but their reliability in predicting long-term outcomes for hepatic/extrahepatic complications or death and their concordance in cross-sectional and longitudinal risk stratification remain uncertain. METHODS: The most common NSS (NFS, FIB-4, BARD, APRI) and the Hepamet fibrosis score (HFS) were assessed in 1,173 European patients with NAFLD from tertiary centres. Performance for fibrosis risk stratification and for the prediction of long-term hepatic/extrahepatic events, hepatocarcinoma (HCC) and overall mortality were evaluated in terms of AUC and Harrell's c-index. For longitudinal data, NSS-based Cox proportional hazard models were trained on the whole cohort with repeated 5-fold cross-validation, sampling for testing from the 607 patients with all NSS available. RESULTS: Cross-sectional analysis revealed HFS as the best performer for the identification of significant (F0-1 vs. F2-4, AUC = 0.758) and advanced (F0-2 vs. F3-4, AUC = 0.805) fibrosis, while NFS and FIB-4 showed the best performance for detecting histological cirrhosis (range AUCs 0.85-0.88). Considering longitudinal data (follow-up between 62 and 110 months), NFS and FIB-4 were the best at predicting liver-related events (c-indices>0.7), NFS for HCC (c-index = 0.9 on average), and FIB-4 and HFS for overall mortality (c-indices >0.8). All NSS showed limited performance (c-indices <0.7) for extrahepatic events. CONCLUSIONS: Overall, NFS, HFS and FIB-4 outperformed APRI and BARD for both cross-sectional identification of fibrosis and prediction of long-term outcomes, confirming that they are useful tools for the clinical management of patients with NAFLD at increased risk of fibrosis and liver-related complications or death. LAY SUMMARY: Non-invasive scoring systems are increasingly being used in patients with non-alcoholic fatty liver disease to identify those at risk of advanced fibrosis and hence clinical complications. Herein, we compared various non-invasive scoring systems and identified those that were best at identifying risk, as well as those that were best for the prediction of long-term outcomes, such as liver-related events, liver cancer and death.

Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia.

Livers from donors after circulatory death (DCD) are a promising option to increase the donor pool, but their use is associated with higher complication rate and inferior graft survival. Normothermic machine perfusion (NMP) keeps the graft at 37°C, providing nutrients and oxygen supply. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) are able to reduce liver injury and promote regeneration. We investigated the efficacy of a reconditioning strategy with HLSC-EVs in an experimental model of NMP. Following total hepatectomy, rat livers were divided into 4 groups: (i) healthy livers, (ii) warm ischemic livers (60 min of warm ischemia), (iii) warm ischemic livers treated with 5 × 108 HLSC-EVs/g-liver, and (iv) warm ischemic livers treated with a 25 × 108 HLSC-EVs/g-liver. NMP lasted 6 h and HLSC-EVs (Unicyte AG, Germany) were administered within the first 15 min. Compared to controls, HLSC-EV treatment significantly reduced transaminases release. Moreover, HLSC-EVs enhanced liver metabolism by promoting phosphate utilization and pH self-regulation. As compared to controls, the higher dose of HLSC-EV was associated with significantly higher bile production and lower intrahepatic resistance. Histologically, this group showed reduced necrosis and enhanced proliferation. In conclusion, HLSC-EV treatment during NMP was feasible and effective in reducing injury in a DCD model with prolonged warm ischemia.

Cooperative Role of Thrombopoietin and Vascular Endothelial Growth Factor-A in the Progression of Liver Cirrhosis to Hepatocellular Carcinoma.

Primary thrombopoietic mediator thrombopoietin (THPO) is mainly produced by the liver; it may act as a growth factor for hepatic progenitors. Principal angiogenesis inducer vascular endothelial growth factor-A (VEGF-A) is critical for the complex vascular network within the liver architecture. As a cross-regulatory loop between THPO and VEGF-A has been demonstrated in the hematopoietic system, the two growth factors were hypothesized to cooperatively contribute to the progression from liver cirrhosis (LC) to hepatocellular carcinoma (HCC). The mRNA and protein expression levels of THPO, VEGF-A, and their receptors were examined, compared, and correlated in paired cancerous and LC tissues from 26 cirrhosis-related HCC patients, using qRT-PCR and immunohistochemistry. THPO and VEGF-A were alternatively silenced by small interfering RNA (siRNA) in human liver cancer cell lines Huh7 and HepG2. THPO and VEGF-A expressions significantly increased in tumor versus LC tissues. HCC and paired LC cells expressed similar levels of THPO receptor (R), whereas vascular endothelial growth factor receptor (VEGFR) -1 and VEGFR-2 levels were higher in HCC than in corresponding LC tissue samples. A significant linear correlation emerged between THPO and VEGF-A transcripts in HCC and, at the protein level, THPO and THPOR were significantly correlated with VEGF-A in tumor tissues. Both HCC and LC expressed similar levels of gene and protein hypoxia inducible factor (HIF)-1α. Positive cross-regulation occurred with the alternative administration of siRNAs targeting THPO and those targeting VEGF-A in hypoxic liver cancer cell lines. These results suggest THPO and VEGF-A might act as interdependently regulated autocrine and/or paracrine systems for cellular growth in HCC. This might be clinically interesting, since new classes of THPOR agonistic/antagonistic drugs may provide novel therapeutic options to correct the frequent hemostatic abnormality seen in HCC patients.

Presence of Serum Antinuclear Antibodies Does Not Impact Long-Term Outcomes in Nonalcoholic Fatty Liver Disease.

INTRODUCTION: We investigated the longitudinal impact of antinuclear antibody (ANA) on clinical outcomes and survival in nonalcoholic fatty liver disease (NAFLD). METHODS: ANA were found in 16.9% of 923 biopsy-proven NAFLD patients, but none of them had histologic autoimmune hepatitis (AIH) or developed AIH after a mean follow-up of 106±50 months. RESULTS: Although ANA-positive cases had a higher prevalence of nonalcoholic steatohepatitis at baseline, the occurrence of liver-related events, hepatocellula carcinoma, cardiovascular events, extrahepatic malignancy, and overall survival were similar to ANA-negative. DISCUSSION: Once AIH has been ruled out, the long-term outcomes and survival are unaffected by the presence of ANA in patients with NAFLD.

Hypoxia-inducible factor 2α drives nonalcoholic fatty liver progression by triggering hepatocyte release of histidine-rich glycoprotein.

Mechanisms underlying progression of nonalcoholic fatty liver disease (NAFLD) are still incompletely characterized. Hypoxia and hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of chronic liver diseases, but the actual role of HIF-2α in the evolution of NAFLD has never been investigated in detail. In this study, we show that HIF-2α is selectively overexpressed in the cytosol and the nuclei of hepatocytes in a very high percentage (>90%) of liver biopsies from a cohort of NAFLD patients at different stages of the disease evolution. Similar features were also observed in mice with steatohepatitis induced by feeding a methionine/choline-deficient diet. Experiments performed in mice carrying hepatocyte-specific deletion of HIF-2α and related control littermates fed either a choline-deficient L-amino acid-defined or a methionine/choline-deficient diet showed that HIF-2α deletion ameliorated the evolution of NAFLD by decreasing parenchymal injury, fatty liver, lobular inflammation, and the development of liver fibrosis. The improvement in NAFLD progression in HIF-2α-deficient mice was related to a selective down-regulation in the hepatocyte production of histidine-rich glycoprotein (HRGP), recently proposed to sustain macrophage M1 polarization. In vitro experiments confirmed that the up-regulation of hepatocyte HRGP expression was hypoxia-dependent and HIF-2α-dependent. Finally, analyses performed on specimens from NAFLD patients indicated that HRGP was overexpressed in all patients showing hepatocyte nuclear staining for HIF-2α and revealed a significant positive correlation between HIF-2α and HRGP liver transcript levels in these patients. CONCLUSIONS: These results indicate that hepatocyte HIF-2α activation is a key feature in both human and experimental NAFLD and significantly contributes to the disease progression through the up-regulation of HRGP production. (Hepatology 2018;67:2196-2214).

Transient elastography for non-invasive evaluation of post-transplant liver graft fibrosis in children.

As graft survival in pediatric LT is often affected by progressive fibrosis, numerous centers carry out protocol liver biopsies. Follow-up biopsy protocols differ from center to center, but all biopsies are progressively spaced out, as time from transplant increases. Therefore, there is a need for non-invasive techniques to evaluate graft fibrosis progression in those children who have no clinical or serological signs of liver damage. Indirect markers, such as the APRI, should be relied on with caution because their sensitivity in predicting fibrosis can be strongly influenced by the etiology of liver disease, severity of fibrosis, and patient age. A valid alternative could be TE, a non-invasive technique already validated in adults, which estimates the stiffness of the cylindrical volume of liver tissue, 100-fold the size of a standard needle biopsy sample. The aims of this study were to evaluate the reliability of TE in children after LT and to compare both the TE and the APRI index results with the histological scores of fibrosis on liver biopsies. A total of 36 pediatric LT recipients were studied. All patients underwent both TE and biopsy within a year (median interval -0.012 months) at an interval from LT of 0.36 to 19.47 years (median 3.02 years). Fibrosis was assessed on the biopsy specimens at histology and staged according to METAVIR. There was a statistically significant correlation between TE stiffness values and METAVIR scores (P = .005). The diagnostic accuracy of TE for the diagnosis of significant fibrosis (F ≥ 2) was measured as the area under the curve (AUROC = 0.865), and it demonstrated that the method had a good diagnostic performance. APRI was not so accurate in assessing graft fibrosis when compared to METAVIR (AUROC = 0.592). A liver stiffness cutoff value of 5.6 kPa at TE was identified as the best predictor for a significant graft fibrosis (METAVIR F ≥ 2) on liver biopsy, with a 75% sensitivity, a 95.8% specificity, a 90% positive predictive value, and an 88.5% negative predictive value. These data suggest that TE may represent a non-invasive, reliable tool for the assessment of graft fibrosis in the follow-up of LT children, alerting the clinicians to the indication for a liver biopsy, with the aim of reducing the number of protocol liver biopsies.

The macrophage activation marker sCD163 is associated with morphological disease stages in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Macrophage activation plays a key pathogenic role in experimental non-alcoholic fatty liver disease (NAFLD) and contributes to the progression of steatohepatitis (NASH) and fibrosis. We studied macrophage activation in human NAFLD by measuring soluble (s)CD163, a specific macrophage activation marker, hypothesizing that sCD163 would be associated with the patients' morphological disease grade. Furthermore, we investigated an association between sCD163 and the apoptosis marker cytokeratin-18 (CK-18) to explore a link between macrophage activation and apoptosis. METHODS: sCD163 associations with biochemical and histological measures of NAFLD were investigated in two independent cohorts of 157 Australian and 174 Italian NAFLD patients, with liver biopsies graded for NAFLD severity, steatosis and fibrosis. sCD163 and CK-18 were measured by enzyme-linked immunosorbent assay. RESULTS: In both cohorts sCD163 increased in parallel with the patients' morphological disease grading, being independently associated with the Kleiner fibrosis score (P < 0.001). A high sCD163 predicted advanced fibrosis {F ≥ 3; Australian cohort: area under receiver-operating characteristics curve 0.77 [95% confidence interval (CI): 0.76-0.87], Italian cohort: 0.80 (95% CI: 0.72-0.88)}. In both groups, sCD163 was independently associated with CK-18 (P < 0.001). CONCLUSION: Soluble CD163 reflecting macrophage activation is associated with morphological features of NAFLD suggesting their involvement in the pathogenesis of NAFLD, NASH and particularly fibrosis. An independent association between sCD163 and cytokeratin-18 suggests that apoptosis may contribute to macrophage activation in NAFLD/NASH.

The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular carcinoma.

OBJECTIVE: The extracellular signal-regulated kinase 5 (ERK5 or BMK1) is involved in tumour development. The ERK5 gene may be amplified in hepatocellular carcinoma (HCC), but its biological role has not been clarified. In this study, we explored the role of ERK5 expression and activity in HCC in vitro and in vivo. DESIGN: ERK5 expression was evaluated in human liver tissue. Cultured HepG2 and Huh-7 were studied after ERK5 knockdown by siRNA or in the presence of the specific pharmacological inhibitor, XMD8-92. The role of ERK5 in vivo was assessed using mouse Huh-7 xenografts. RESULTS: In tissue specimens from patients with HCC, a higher percentage of cells with nuclear ERK5 expression was found both in HCC and in the surrounding cirrhotic tissue compared with normal liver tissue. Inhibition of ERK5 decreased HCC cell proliferation and increased the proportion of cells in G0/G1 phase. These effects were associated with increased expression of p27 and p15 and decreased CCND1. Treatment with XMD8-92 or ERK5 silencing prevented cell migration induced by epidermal growth factor or hypoxia and caused cytoskeletal remodelling. In mouse xenografts, the rate of tumour appearance and the size of tumours were significantly lower when Huh-7 was silenced for ERK5. Moreover, systemic treatment with XMD8-92 of mice with established HCC xenografts markedly reduced tumour growth and decreased the expression of the proto-oncogene c-Rel. CONCLUSIONS: ERK5 regulates the biology of HCC cells and modulates tumour development and growth in vivo. This pathway should be investigated as a possible therapeutic target in HCC.

Liver resection for hepatocellular carcinoma in patients with metabolic syndrome: A multicenter matched analysis with HCV-related HCC.

BACKGROUND & AIMS: The incidence of metabolic syndrome-related hepatocellular carcinoma (MS-HCC) is increasing worldwide. High resection risks are anticipated because of underlying steatohepatitis, but long-term results are unknown. To clarify the outcomes following liver resection in patients with MS-HCC and to compare the outcomes of MS-HCC to HCV-related HCC (HCV-HCC). METHODS: All the consecutive patients undergoing liver resection for HCC in six high-volume HPB units between 2000 and 2012 were retrospectively considered. The patients with MS-HCC were identified and matched one-to-one with HCV-HCC patients without metabolic syndrome. Matching was based on age, cirrhosis, Child-Pugh class, portal hypertension, HCC number and diameter and liver resection extension. RESULTS: Among 1563 patients undergoing liver resection for HCC in the study period, 96 (6.1%) had MS-HCC. They were matched with 96 HCV-HCC patients. All patients were Child-Pugh class A, 22.9% had cirrhosis. Forty-one patients per group (42.7%) required major hepatectomy. The MS-HCC group had a higher prevalence of steatohepatitis (25.0% vs. 9.4%, p=0.004). Operative mortality was 2.1% (1 MS-HCC, 3 HCV-HCC, p=0.621). Morbidity and liver failure rates were similar between the two groups. In the multivariate analysis, cirrhosis, major hepatectomy, and MELD >8, but not steatohepatitis, impacted severe morbidity and liver failure rates. The MS-HCC group had better 5-year overall survival (65.6% vs. 61.4%, p=0.031) and recurrence-free survival (37.0% vs. 27.5%, p=0.077). Independent negative prognostic factors were HCV-HCC, multiple HCC, microvascular invasion, and satellite nodules. CONCLUSIONS: Liver resection is safe for MS-HCC, as for HCV-HCC. Cirrhosis, but not steatohepatitis, affects short-term outcomes. MS-HCC is associated with excellent long-term outcomes, better than HCV-HCC.

Prevalence and prognostic value of hepatic histological alterations in patients with Crohn's disease.

UNLABELLED: Variable degrees of liver histological changes in patients with Crohn's disease (CD) have been reported. OBJECTIVE: To evaluate the liver histological alterations and their prognostic significance in patients affected by CD without abnormalities of liver biochemical parameters and ultrasound features. MATERIAL AND METHODS: A prospective, single-blind study, including 35 consecutive patients with CD that underwent bowel resection with a contemporary performance of liver biopsy from 1992 to 2003. EXCLUSION CRITERIA: the presence of standard causes of liver disease, such as alcohol consumption exceeding 20 g/day, primary sclerosing cholangitis, viral infections, celiac disease, metabolic syndrome and alterations of the metabolism. Patients were followed up with regular evaluation of hepatic cytolysis, cholestasis, synthesis and ultrasound performance. After a mean interval of 14 years (from May to December 2013), liver fibrosis was assessed by Fibroscan®. RESULTS: Histological alterations were shown in 60% of patients, without serious liver injuries (no case of inflammation or significant fibrosis). Fibroscan® was performed in 33 subjects and no significant changes were observed (mean value of liver stiffness: 5.2 ± 1.2 kPa). The minimal microscopic damage did not evolve either in patients with a normal histology or in those with an altered histology at baseline (p = 0.9). Only patients who took azathioprine had a statistically significant increase of liver stiffness values (5.7 ± 1.5 kPa vs 4.7 ± 1.3 kPa, p = 0.017). CONCLUSIONS: Patients with CD do not need additional examinations compared to the general population, unless clinical or biochemical abnormalities are found.

Multiparametric Gd-EOB-DTPA magnetic resonance in diagnosis of HCC: dynamic study, hepatobiliary phase, and diffusion-weighted imaging compared to histology after orthotopic liver transplantation.

PURPOSE: To evaluate Gd-EOB-DTPA-enhanced magnetic resonance (MR) performance during dynamic (DYN) phases, hepatobiliary (HB) phase and diffusion-weighted imaging (DWI) compared with pathological findings in patients undergoing orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC) associated with different conditions, such as stage of chronic liver disease, histological grading, nodule size, and occurrence of previous treatments. METHODS: Retrospective analysis of 64 nodules reported as HCC at pathological analysis on 28 explanted livers, examined about 3 months before OLT using a 1.5 T device and 16 channels array after intravenous GD-EOB-DTPA injection. Lesions features and hepatic functional values were recorded for each patient. Two radiologists performed in consensus the analysis of nodules on DYN, HB, and DWI. MR findings were compared with those of pathological anatomy. Diagnostic indicators were calculated for each technique. RESULTS: DYN and HB showed no statistically significant difference in sensitivity (88% and 98%, respectively), diagnostic accuracy (90.6% and 99.9%), and specificity (both 100%), for all Child-Pugh scores, gradings, sizes, and presence or absence of previous treatments. DWI had a statistically significant lower sensitivity compared to DYN (p = 0.001) and HB (p < 0.0001); its sensitivity was significantly inferior for Child-Pugh Class B nodules than for Child-Pugh Class A ones (p = 0.00005). DWI sensitivity presented a significant increase (p = 0.03) with grading and presence of previous treatments (p = 0.0006). ADC values showed no statistically significant changes with Child-Pugh score, grading and nodules size; statistically significant increase was instead found for treated vs. untreated nodules (p = 0.016). CONCLUSIONS: In a multiparametric HCC MRI assessment, DYN and HB play the leading role, with DWI faring acceptably well for Child-Pugh Class A nodules and treated ones.

The biphasic nature of hypoxia-induced directional migration of activated human hepatic stellate cells.

Liver fibrogenesis is sustained by pro-fibrogenic myofibroblast-like cells (MFs), mainly originating from activated hepatic stellate cells (HSC/MFs) or portal (myo)fibroblasts, and is favoured by hypoxia-dependent angiogenesis. Human HSC/MFs were reported to express vascular-endothelial growth factor (VEGF) and VEGF-receptor type 2 and to migrate under hypoxic conditions. This study was designed to investigate early and delayed signalling mechanisms involved in hypoxia-induced migration of human HSC/MFs. Signal transduction pathways and intracellular generation of reactive oxygen species (ROS) were evaluated by integrating morphological, cell, and molecular biology techniques. Non-oriented and oriented migration were evaluated by using wound healing assay and the modified Boyden's chamber assay, respectively. The data indicate that hypoxia-induced migration of HSC/MFs is a biphasic process characterized by the following sequence of events: (a) an early (15 min) and mitochondria-related increased generation of intracellular ROS which (b) was sufficient to switch on activation of ERK1/2 and JNK1/2 that were responsible for the early phase of oriented migration; (c) a delayed and HIF-1α-dependent increase in VEGF expression (facilitated by ROS) and its progressive, time-dependent release in the extracellular medium that (d) was mainly responsible for sustained migration of HSC/MFs. Finally, immunohistochemistry performed on HCV-related fibrotic/cirrhotic livers revealed HIF-2α and haem-oxygenase-1 positivity in hepatocytes and α-SMA-positive MFs, indicating that MFs were likely to be exposed in vivo to both hypoxia and oxidative stress. In conclusion, hypoxia-induced migration of HSC/MFs involves an early, mitochondrial-dependent ROS-mediated activation of ERK and JNK, followed by a delayed- and HIF-1α-dependent up-regulation and release of VEGF.

Evaluation of Graft Fibrosis, Inflammation, and Donor-specific Antibodies at Protocol Liver Biopsies in Pediatric Liver Transplant Patients: A Single-center Experience.

BACKGROUND: The impact of graft fibrosis and inflammation on the natural history of pediatric liver transplants is still debated. Our objectives were to evaluate the evolution of posttransplant fibrosis and inflammation over time at protocol liver biopsies (PLBs), risk factors for fibrosis, presence of donor-specific antibodies (DSAs), and/or their correlation with graft and recipient factors. METHODS: A single-center, retrospective (2000-2019) cross-sectional study on pediatric liver transplant recipients who had at least 1 PLB, followed by a longitudinal evaluation in those who had at least 2 PLBs, was conducted. Fibrosis was assessed by the Liver Allograft Fibrosis Semiquantitative score, inflammation by the rejection activity index, DSAs by Luminex. RESULTS: A total of 134 PLBs from 94 patients were included. Fibrosis was detected in 87% (30% mild, 45% moderate, and 12% severe), 80% in the portal tracts. There was an increase in fibrosis between the 1-3 and the 4-6 y group (P = 0.01), then it was stable. Inflammation was observed in 44% (30% mild, 13% moderate, and 1% severe), 90% in the portal tracts. Anti-HLA II (IgG) DSAs were detected in 14 of 40 (35%). Portal fibrosis was associated with portal inflammation in the 1-3 y group (P = 0.04). Low immunosuppression levels were correlated with sinusoidal fibrosis (P = 0.04) and DSA positivity (P = 0.006). There was no statistically significant correlation between DSA positivity and the presence of graft fibrosis or inflammation. CONCLUSIONS: This study corroborates the concept of an early evolution of silent graft fibrosis. Suboptimal immunosuppression may play a role in the development of fibrosis and DSAs.

Intracellular reactive oxygen species are required for directional migration of resident and bone marrow-derived hepatic pro-fibrogenic cells.

BACKGROUND & AIMS: Liver fibrogenesis is sustained by myofibroblast-like cells originating from hepatic stellate cells (HSC/MFs), portal fibroblasts or bone marrow-derived cells, including mesenchymal stem cells (MSCs). Herein, we investigated the mechanistic role of intracellular generation of reactive oxygen species (ROS) and redox-sensitive signal transduction pathways in mediating chemotaxis, a critical profibrogenic response for human HSC/MFs and for MSC potentially engrafting chronically injured liver. METHODS: Intracellular generation of ROS and signal transduction pathways were evaluated by integrating morphological and molecular biology techniques. Chemokinesis and chemotaxis were evaluated by wound healing assay and modified Boyden's chamber assay, respectively. Additional in vivo evidence was obtained in human specimens from HCV-related cirrhosis. RESULTS: Human MSCs and HSC/MFs migrate in response to a panel of polypeptide chemoattractants and extracellularly generated superoxide anion. All polypeptides induced a NADPH-oxidase-dependent intracellular rise in ROS, resulting in activation of ERK1/2 and JNK1/2. Moreover, menadione or 2,3-dimethoxy-1,4-naphthoquinone, which generate intracellular superoxide anion or hydrogen peroxide, respectively, induced ERK1/2 and JNK1/2 activation and migration. JNK1 activation was predominant for migration as shown by specific silencing. Finally, activation of ERK1/2 and JNK1/2 was found in extracts obtained from HSC/MFs during the course of an oxidative stress-mediated model of liver injury and phosphorylated JNK1/2 isoforms were detected in α-smooth muscle actin-positive myofibroblasts lining fibrotic septa in human cirrhotic livers. CONCLUSIONS: Intracellular generation of ROS, through activation of specific signaling pathways, is a critical event for directional migration of HSC/MFs and MSCs.

Protective Effects of Human Liver Stem Cell-Derived Extracellular Vesicles in a Mouse Model of Hepatic Ischemia-Reperfusion Injury.

Hepatic ischemia-reperfusion injury (IRI) is observed in liver transplantation and hepato-biliary surgery and is associated with an inflammatory response. Human liver stem cell-derived extracellular vesicles (HLSC-EV) have been demonstrated to reduce liver damage in different experimental settings by accelerating regeneration and by modulating inflammation. The aim of the present study was to investigate whether HLSC-EV may protect liver from IRI in a mouse experimental model. Segmental IRI was obtained by selective clamping of intrahepatic pedicles for 90 min followed by 6 h of reperfusion. HLSC-EV were administered intravenously at the end of the ischemic period and histopathological and biochemical alterations were evaluated in comparison with controls injected with vehicle alone. Intra liver localization of labeled HLSC-EV was assessed by in in vivo Imaging System (IVIS) and the internalization into hepatocytes was confirmed by fluorescence analyses. As compared to the control group, administration of 3 × 109 particles (EV1 group) significantly reduced alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release, necrosis extension and cytokines expression (TNF-α, CCL-2 and CXCL-10). However, the administration of an increased dose of HLSC-EV (7.5 × 109 particles, EV2 group) showed no significant improvement in respect to controls at enzyme and histology levels, despite a significantly lower cytokine expression. In conclusion, this study demonstrated that 3 × 109 HLSC-EV were able to modulate hepatic IRI by preserving tissue integrity and by reducing transaminases release and inflammatory cytokines expression. By contrast, a higher dose was ineffective suggesting a restricted window of biological activity. Graphical abstract.

Association of cytomegalovirus infections with recurrence of humoral and cellular autoimmunity to islet autoantigens and of type 1 diabetes in a pancreas transplanted patient.

Association of type 1 diabetes and cytomegalovirus (CMV) is suspected and CMV infections have also been linked to increased risk of new onset post-transplantation diabetes. We monitored response to islet autoantigens, pancreatic endocrine function, and CMV infections in one type 1 diabetic patient receiving pancreas allograft. Time course analyses of levels of islet autoantibodies (Abs), IFN-gamma ELISPOT response, analysis of T cell function, levels of C peptide together with CMV pp65 antigenaemia and viraemia and graft biopsy histopathology were performed in comparison with a cohort of diabetic recipients. Evidence of autoimmune activation to GAD and IA2, modification of CD4(+) CD25hi T cells, loss of pancreatic function, concomitantly with multiple CMV infections and allograft rejection with peri-insulitis is provided. The parallel between metabolic outcome, initiation and progression of autoreactivity to islet autoantigens and early CMV infections after transplantation, suggests that persistent CMV infections may be relevant to the pathogenesis of type 1 diabetes.

Combined antifungal therapy, iron chelation and surgical resection as treatment of hepatic zygomycosis in a patient with haematological malignancy.

We describe the case of a 19-year-old boy with acute leukaemia who developed primary hepatic zygomycosis. The patient presented with febrile neutropenia and severe abdominal tenderness. Despite the administration of antibiotics and liposomal Amphotericin-B (L-AmB), the CT scan demonstrated an increase in the size of liver lesions. A wide surgical resection was carried out and liver specimens demonstrated a branching, filamentous fungus that was identified as Rhizomucor pusillus by both phenotypic and molecular methods. The patient was treated with L-AmB combined with posaconazole, and deferasirox was subsequently added given the potential synergistic effect of this iron chelator in combination with L-AmB. Three months after surgical intervention, an allogeneic stem-cell transplantation was successfully carried out. The present case confirms that an early surgical management combined with antifungal agents is crucial to optimise the outcome of patients with zygomycosis and the use of deferasirox is a promising alternative.