Noninvasive scores are poorly predictive of histological fibrosis in paediatric fatty liver disease.

OBJECTIVES: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children. Roughly a quarter of paediatric patients with NAFLD develop nonalcoholic steatohepatitis and fibrosis. Here, we evaluated the diagnostic accuracy of previously published noninvasive fibrosis scores to predict liver fibrosis in a large European cohort of paediatric patients with NAFLD. METHODS: The 457 patients with biopsy-proven NAFLD from 10 specialized centers were included. We assessed diagnostic accuracy for the prediction of any (F ≥ 1), moderate (F ≥ 2) or advanced (F ≥ 3) fibrosis for the AST/platelet ratio (APRI), Fibrosis 4 score (FIB-4), paediatric NAFLD fibrosis score (PNFS) and paediatric NAFLD fibrosis index (PNFI). RESULTS: Patients covered the full spectrum of fibrosis (F0: n = 103; F1: n = 230; F2: n = 78; F3: n = 44; F4: n = 2). None of the scores were able to accurately distinguish the presence of any fibrosis from no fibrosis. For the detection of moderate fibrosis, area under the receiver operating characteristic curve (AUROC) were: APRI: 0.697, FIB-4: 0.663, PNFI: 0.515, PNFS: 0.665, while for detection of advanced fibrosis AUROCs were: APRI: 0.759, FIB-4: 0.611, PNFI: 0.521, PNFS: 0.712. Fibrosis scores showed no diagnostic benefit over using ALT ≤ 50/ > 50 IU/L as a cut-off. CONCLUSIONS: Established fibrosis scores lack diagnostic accuracy to replace liver biopsy for staging of fibrosis, giving similar results as compared to using ALT alone. New diagnostic tools are needed for Noninvasive risk-stratification in paediatric NAFLD.

Long-term outcomes of patients with type 1 or 2 autoimmune hepatitis presenting in childhood.

BACKGROUND & AIMS: In children with autoimmune hepatitis, uncertainties include outcomes associated with type 2 hepatitis, the possibility of and criteria for attempting withdrawal of treatment, and long-term outcomes. We report our experience on these issues. METHODS: From 1973 to 2002, 117 children with type 1 (n = 65) or type 2 (n = 52) hepatitis, excluding fulminant hepatitis, were treated, primarily with prednisone and azathioprine. Median follow-up was 20 years in survivors. RESULTS: Normalisation of aminotransferases and prothrombin ratio were observed in 93% and 84% of children, respectively; sustained remission after treatment withdrawal was recorded in 24% of the entire population, with a median follow-up of 7 years. Sustained treatment-free remission was obtained in 11 of 24 children with follow-ups of 4-22 years based on durable normalisation of aminotransferases (without histological assessment). Gastrointestinal bleeding from varices and the emergence of extrahepatic autoimmune disorders occurred in 10 and 22 patients, respectively. Liver transplantation was performed in 23 patients at a median age of 21 years. The 30-year probabilities of overall and native liver survival were 81% and 61%, respectively. No differences were observed between type 1 and 2 hepatitis for any of the component parts of outcome. In the multivariate analysis, a persistent abnormal prothrombin ratio was associated with worse probabilities of overall and native liver survival. CONCLUSIONS: In terms of liver outcome, type 2 hepatitis is not different from type 1. Withdrawal of treatment is possible without prior liver histology. A persistent abnormal prothrombin ratio identifies patients who will require liver transplantation in adolescence or early adulthood. IMPACT AND IMPLICATIONS: In children with autoimmune hepatitis, there are conflicting reports on the differences in outcome between type 1 and type 2 hepatitis, and on the possibility of treatment withdrawal, before which liver histology is required; data concerning >10-year overall and native liver survival rates are limited. In this study, we found no differences in outcomes between type 1 and 2 hepatitis; a durable treatment-free state was achieved in 19% of all patients throughout childhood and early adulthood, and in 45% of children for whom treatment withdrawal was attempted without prior liver histology; prothrombin was found to be predictive of 30-year overall and native liver survival. The results allow for a less-strict approach to treatment withdrawal in children, avoiding the risks of a liver biopsy, and they provide a tool to help anticipate the need for liver transplantation before complications occur.

Allograft Fibrosis After Pediatric Liver Transplantation: Incidence, Risk Factors, and Evolution.

Allograft fibrosis (AF) after pediatric liver transplantation (pLT) is frequent, but its dynamics are unclear. Our aim was to assess the evolution and risk factors of AF after pLT. A retrospective single-center analysis of pLT patients with a follow-up of ≥5 years who underwent protocol liver biopsies at 6 months, 1 year, 2 years, 5 years, and 10 years was performed. Fibrosis was assessed using the METAVIR and Ishak systems and the liver allograft fibrosis score (LAFs). Of 219 pLTs performed from 2008 to 2018, 80 (36.5%) pLTs were included, and 320 biopsies were reviewed. At 6 months after pLT, fibrosis was found in 54 (67.5%) patients by the METAVIR/Ishak systems and in 59 (73.8%) by the LAFs (P = 0.65). By 5 years, AF was detected in 67 (83.8%), 69 (86.3%), and 72 (90%) specimens using the METAVIR, Ishak, and LAFs systems, respectively (P = 0.54); mild (METAVIR, 51 [63.8%]; Ishak, 60 [75%]; LAFs, 65 [81.2%]) and moderate (METAVIR, 16 [20%]; Ishak, 9 [11.9%]; LAFs, 7 [8.8%]) stages were detected, but severe fibrosis was not found (P = 0.09). In the LAFs, fibrosis involved the portal (85%), sinusoidal (15%), and centrolobular (12%) areas. Of 18 patients with 10-year protocol biopsies, AF was present in 16 (90%), including 1 (5.5%) with severe fibrosis. In all systems, 36.3% of patients showed fibrosis progression from 2 years to 5 years after LT, but they remained stable at the 10-year biopsies without clinical implications. In multivariate analysis, only donor age >40 years was a risk factor for moderate AF at 5 years after LT (odds ratio, 8.3; 95% confidence interval, 1.6-42.1, P = 0.01). Cold ischemia time (CIT) >8 hours was associated with portal (P < 0.001)/sinusoidal fibrosis (P = 0.04), donor age >40 years was associated with sinusoidal (P = 0.01)/centrilobular (P = 0.04) fibrosis, and low tacrolimus trough level within 1 year after LT was associated with centrilobular fibrosis (P = 0.02). AF has a high incidence after pLT, occurring early after transplantation. In most cases, AF is mild or moderate and remains stable in the long run without clinical implications. Donor selection, short CIT, and immunosuppression adherence are crucial to reducing the risk of advanced AF.

LncOb rs10487505 variant is associated with leptin levels in pediatric non-alcoholic fatty liver disease.

BACKGROUND: Low and high leptin levels are associated with non-alcoholic fatty liver disease (NAFLD). The LncOb rs10487505 variant has been associated with body mass index (BMI), and the C allele was reported as leptin-lowering. We evaluated the association of rs10487505 with leptin levels, liver histology, and surgery-induced weight loss in youths with NAFLD. METHODS: One-hundred five obese youths with NAFLD, of whom 19 undergoing laparoscopic sleeve gastrectomy (LSG), were analyzed for rs10487505 and leptin circulating levels. RESULTS: The G allele frequency was lower in youths with NAFLD than in controls (p = 0.049). No difference was found in anthropometrics, biochemistry and histology between G allele carriers and CC homozygotes, except for leptin levels (p = 0.016). Leptin correlated with body weight, BMI, BMI-z score, waist circumference, insulin resistance/sensitivity, and triglycerides (p ≤ 0.01). A multivariable regression model including body weight and homeostasis model assessment of insulin resistance was a good predictor of plasma leptin (R2 = 0.45), and the addition of genotype to the model increased the R2 to 0.50. Following LSG, leptin levels and body weight were more reduced in G allele carriers (p < 0.05). CONCLUSIONS: LncOb rs10487505 variant was associated with pediatric NAFLD and high leptin levels, and with weight and leptin reduction after LSG in youths. IMPACT: The interplay of environment, genetics and epigenetics is crucial inflating the risk of non-alcoholic fatty liver disease (NAFLD). Several long non-coding RNA (LncRNAs) are found associated with NAFLD pathogenesis. Here, we evaluated the impact of the genetic variant rs10487505 in LncOb which is involved in the regulation of leptin gene expression. The LncOb rs10487505 is associated with increased levels of leptin, but not with liver histology, in youths with NAFLD. The LncOb rs10487505 was also associated with the significant decrease of leptin and body weight after bariatric surgery.

Comparison of the Lipidomic Signature of Fatty Liver in Children and Adults: A Cross-Sectional Study.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition in children characterised by insulin resistance and altered lipid metabolism. Affected patients are at increased risk of cardiovascular disease (CVD) and children with NAFLD are likely to be at risk of premature cardiac events. Evaluation of the plasma lipid profile of children with NAFLD offers the opportunity to investigate these perturbations and understand how closely they mimic the changes seen in adults with cardiometabolic disease. METHODS: We performed untargeted liquid chromatography-mass spectrometry (LC-MS) plasma lipidomics on 287 children: 19 lean controls, 146 from an obese cohort, and 122 NAFLD cases who had undergone liver biopsy. Associations between lipid species and liver histology were assessed using regression adjusted for age and sex. Results were then replicated using data from 9500 adults with metabolic phenotyping. RESULTS: More severe paediatric NAFLD was associated with lower levels of long chain, polyunsaturated phosphatidylcholines (pC) and triglycerides (TG). Similar trends in pC and TG chain length and saturation were seen in adults with hepatic steatosis; however, many of the specific lipids associated with NAFLD differed between children and adults. Five lipids replicated in adults (including PC(36:4)) have been directly linked to death and cardiometabolic disease, as well as indirectly via genetic variants. CONCLUSION: These findings suggest that, whilst similar pathways of lipid metabolism are perturbed in paediatric NAFLD as in cardiometabolic disease in adults, the specific lipid signature in children is different.

Pediatric Non-Alcoholic Fatty Liver Disease Is Affected by Genetic Variants Involved in Lifespan/Healthspan.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in both adults and children. Along with obesity and metabolic syndrome, genetic predisposition influences the progression of NAFLD. Here, we investigated the effect of lifespan/healthspan-related single nucleotide polymorphisms (SNPs) on metabolically associated fatty liver disease in children. METHODS: We evaluated the impact of 10 SNPs involved in both human liver/metabolic diseases and healthspan (interleukin-6 [IL-6] rs1800795, antisense non coding RNA in the INK4 locus (ANRIL) rs1556516, SH2B3/ATXN2 rs7137828, FURIN rs17514846, TP53 rs1042522, APOC3 rs2542052, KL rs9536314, KL rs9527025, SIRT6 rs107251, FOXO3 rs2802292) on NAFLD-related metabolic and liver features in 177 pediatric patients with biopsy-proven NAFLD, by comparing them to 146 healthy controls. We then applied a multidimensional reduction (MDR) case-control analysis of SNP-SNP interactions, to identify the joint effect of analyzed SNPs in predicting NAFLD and associated features. RESULTS: Discrete SNPs were significantly associated with individual metabolic NAFLD features, but none of them significantly associated with NAFLD diagnosis. By testing potential synergies using the MDR approach, the best combination to diagnose NAFLD (P = 0.0011) resulted in the one encompassing IL-6 rs1800795 and ANRIL rs1556516. Consistently, the risk combinations suggested by SNP-SNP analysis strongly associated with a higher level of fasting plasma blood glucose level (P = 0.024). CONCLUSION: In conclusion, here we demonstrated a synergic interaction between IL-6 rs1800795 and ANRIL rs1556516 in the diagnosis of NAFLD, and NAFLD-associated hyperglycemia in children. Larger studies are required to confirm our findings and to elucidate mechanisms by which the genetic interaction between these two genes influences healthspan in pediatric NAFLD.

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.

Relationship Between PNPLA3 rs738409 Polymorphism and Decreased Kidney Function in Children With NAFLD.

Emerging evidence suggests that patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 genotype (the major genetic variant associated with susceptibility to nonalcoholic fatty liver disease [NAFLD]) is associated with decreased kidney function in adults. Currently, it is uncertain whether this association also occurs in children/adolescents and whether any association is independent of liver disease severity. We enrolled a sample of 142 Caucasian children and adolescents with biopsy-proven NAFLD, presenting to the Liver Unit of the "Bambino Gesù" Children's Hospital. The glomerular filtration rate (e-GFR) was estimated using the Bedside Schwartz equation, whereas 24-hour proteinuria was measured using a radioimmunoassay method. Genotyping for the PNPLA3 rs738409 genotype was undertaken using the single-nucleotide polymorphism genotyping allelic discrimination method. Overall, 45 children had G/G, 56 had G/C, and 41 had C/C PNPLA3 rs738409 genotype, respectively. Children with G/G genotype had significantly lower e-GFR (107.5 ± 20 versus 112.8 ± 18 versus 125.3 ± 23 mL/min/1.73 m2 , P = 0.002) and higher 24-hour proteinuria (58.5 ± 21 versus 53.9 ± 22 versus 42.9 ± 20 mg/day, P = 0.012) compared with those with either G/C or C/C genotypes. After adjustment for age, sex, systolic blood pressure, measures of adiposity, homeostasis model assessment-estimated insulin resistance and biopsy-confirmed nonalcoholic steatohepatitis and stage of liver fibrosis, the presence of rs738409 G/G genotype was independently associated with both lower e-GFR (ß coefficient: -23.6; 95% confidence interval [CI]: -36.3 to -10.8; P < 0.001) and higher 24-hour proteinuria (ß coefficient: 15.3; 95% CI: 1.12 to 30.5; P = 0.046). Conclusion: Regardless of established renal risk factors and the histological severity of NAFLD, the PNPLA3 G/G genotype was strongly associated with decreasing kidney function and increasing 24-hour proteinuria in children/adolescents with histologically confirmed NAFLD.

Plasma methylcitric acid and its correlations with other disease biomarkers: The impact in the follow up of patients with propionic and methylmalonic acidemia.

Methylcitric acid (MCA) analysis has been mainly utilized for the diagnosis of propionate disorders or as a second-tier test in newborn screening, but its utility for patients monitoring still needs to be established. We explored the potential contribution of MCA in the long-term management of organic acidurias. We prospectively evaluated plasma MCA and its relationship with disease biomarkers, clinical status, and disease burden in 22 patients, 13 with propionic acidemia (PA) and nine with methylmalonic acidemia (MMA) on standard treatment and/or after transplantation. Samples were collected at scheduled routine controls or during episodes of metabolic decompensation (MD), 10 patients were evaluated after transplantation (six liver, two combined liver and kidney, 2 kidney). MCA levels were higher in PA compared to MMA and its levels were not influenced by the clinical status (MD vs well state). In MMA, MCA was higher in elder patients and, along with fibroblast growth factor 21 (FGF21) and plasma methylmalonic acid, negatively correlated with GFR. In both diseases, MCA correlated with ammonia, glycine, lysine, C3, and the C3/C2, C3/C16 ratios. The disease burden showed a direct correlation with MCA and FGF21, for both diseases. All transplanted patients showed a significant reduction of MCA in comparison to baseline values, with some differences dependent on the type of transplantation. Our study provided new insights in understanding the disease pathophysiology, showing similarities between MCA and FGF21 in predicting disease burden, long-term complications and in evaluating the impact of organ transplantation.

Does the Treatment After Kasai Procedure Influence Biliary Atresia Outcome and Native Liver Survival?

OBJECTIVES: Biliary atresia (BA) is a rare and progressive idiopathic disease affecting the biliary tract that can lead to end-stage liver disease. The main treatment is Kasai portoenterostomy (KP). The use of adjuvant therapy (AT; prophylactic antibiotics and steroids) after KP aims to prevent cholangitis and reduce the need for liver transplantation (LT), but there is a lack of evidence on their effectiveness. We investigated the impact of significant changes in the post-KP protocol on the overall outcomes of BA. METHODS: We enrolled 43 consecutive infants undergoing KP at Bambino Gesù Children's Hospital between July 2012 and October 2018. We compared AT (AT group; n=25) against no treatment (AT-free group; n = 18). RESULTS: No significant differences in anthropometric and laboratory parameters were shown between the 2 groups at baseline and every study evaluation (1, 3, and 6 months). The incidences of clinical complications of liver disease were similar. Six months post-KP, the achievement of serum total bilirubin ≤1.5 mg/dL and satisfactory Pediatric End-Stage Liver Disease scores were not significantly different between the 2 groups. Cholangitis was observed in 30% of patients in the first 6 months postoperatively: 33% and 28% in the AT-free and AT groups, respectively (P = 0.18). Survival to LT listing at 12 months and without LT at 24 months were not significantly different between the 2 groups (P > 0.05). CONCLUSIONS: AT after KP confirmed conflicting results; therefore, multicentered, prospective, randomized control studies are needed to better understand its utility after KP, especially in the multidrug resistance spread era.

PNPLA3 gene polymorphism is associated with liver steatosis in children with Down syndrome.

BACKGROUND AND AIMS: We previously demonstrated that children with Down syndrome (DS) exhibited a greater risk of steatosis than the general pediatric population. This trend was independent of obese phenotype, thus suggesting a role of genetic predisposition. Therefore, we investigated the prevalence of non-alcoholic fatty liver disease (NAFLD) and metabolic syndrome (MetS) in function of genetic susceptibility and adipocytokine levels in children with DS. METHODS AND RESULTS: A total of 84 Caucasian children with DS (age range 5-17 years), were included in this study. For all children, we collected data on anthropometric and biochemical parameters, and liver ultrasound (US). We also measured adipocytokines circulating levels and specific polymorphisms closed to NAFLD. We found a prevalence of 64.3% of liver steatosis at US, with a severe steatosis of about 4% in children with DS. The presence of steatosis in children with DS was associated with the presence of patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 variant, which also correlated with interleukin (IL)-6 levels. Moreover, we found that the 52.4% had a waist circumference > 90th percentile, 21.4% were hypertensive, 7.14% had hyperglycemia, 9.5% had hypertriglyceridemia, and 17.9% showed high-density lipoprotein cholesterol ≤ 40 mg/dl. Finally, the IL-6 and adiponectin levels correlated with steatosis, and several adipocytokines correlated with single MetS traits in children with DS. CONCLUSION: The present study explores for the first time potential pathomechanisms connecting pediatric NAFLD and MetS in DS. We found that the PNPLA3 variant is associated with steatosis, but not with MetS, in children with DS.

Prevalence of prediabetes and diabetes in children and adolescents with biopsy-proven non-alcoholic fatty liver disease.

BACKGROUND & AIMS: We undertook a cross-sectional study of children/adolescents with and without non-alcoholic fatty liver disease (NAFLD) to compare the prevalence of prediabetes and diabetes, and to examine the role of abnormal glucose tolerance as a predictor of liver disease severity. METHODS: We recruited a cohort of 599 Caucasian children/adolescents with biopsy-proven NAFLD, and 118 children/adolescents without NAFLD, who were selected to be similar for age, sex, body mass index and waist circumference to those with NAFLD. The diagnosis of prediabetes and diabetes was based on either hemoglobin A1c, fasting plasma glucose or 2 h post-load glucose concentrations. RESULTS: Children/adolescents with NAFLD had a significantly higher prevalence of abnormal glucose tolerance (prediabetes or diabetes) than those without NAFLD (20.6% vs. 11%, p = 0.02). In particular, 124 (20.6%) children/adolescents with NAFLD had abnormal glucose tolerance, with 19.8% (n = 119) satisfying the diagnostic criteria for prediabetes and 0.8% (n = 5) satisfying the criteria for diabetes. The combined presence of prediabetes and diabetes was associated with a nearly 2.2-fold increased risk of non-alcoholic steatohepatitis (NASH; unadjustedodds ratio 2.19; 95% CI 1.47-3.29; p <0.001). However, this association was attenuated (but remained significant) after adjustment for age, sex, waist circumference (adjustedodds ratio 1.69, 95% CI 1.06-2.69, p = 0.032), and the PNPLA3 rs738409 polymorphism. Both this PNPLA3 polymorphism and waist circumference were strongly associated with NASH. CONCLUSIONS: Abnormal glucose tolerance (especially prediabetes) is highly prevalent among children/adolescents with biopsy-proven NAFLD. These children also have a higher risk of NASH, though central adiposity is the factor that is most strongly associated with NASH. LAY SUMMARY: Children with biopsy-proven non-alcoholic fatty liver disease (NAFLD) have a higher prevalence of abnormal glucose tolerance (prediabetes or type 2 diabetes) than children without NAFLD. Children with biopsy-proven NAFLD and abnormal glucose tolerance also have a higher prevalence of the progressive form of disease, non-alcoholic steatohepatitis, than those with normal glucose tolerance, though central adiposity is the factor that is most strongly associated with non-alcoholic steatohepatitis.

The Role of Genetic Predisposition, Programing During Fetal Life, Family Conditions, and Post-natal Diet in the Development of Pediatric Fatty Liver Disease.

OBJECTIVE: To evaluate, in patients with nonalcoholic fatty liver disease (NAFLD), the role of lifetime exposures associated with genetic predisposition, family history (parental obesity, economic income), programming during fetal life (gestational age, birthweight), being breastfed or not, and later biomarkers of dietary habits and lifestyle in the development of fibrosis. STUDY DESIGN: In total, 182 children with overweight/obesity diagnosed with NAFLD proven by biopsy results were enrolled in our study and evaluated for liver fibrosis. We estimated prevalence ORs of fibrosis according to genetics, parental obesity, occupational socioeconomic status (SES), birth weight, breastfeeding, fructose intake (indicator of junk food consumption), and vitamin D status (inflammatory indicator) using logistic regression models, adjusted for age and children's body mass. RESULTS: One hundred thirty-seven patients (75.3%) had liver fibrosis, and 45 patients (24.7%) did not have liver fibrosis. The ORs of fibrosis were significant (P < .05) for patatin like phospholipase domain-containing 3-GG genotype (OR 2.1), parental obesity (OR 2.9), not being breastfed (OR 3.1), vitamin D status (<20 mg/dL) (OR 1.24), and fructose consumption (OR 1.6 per 1 g/day increase), whereas a high SES maternal occupation was inversely associated with fibrosis (OR 0.30). CONCLUSIONS: Our results show independent roles of the patatin like phospholipase domain-containing 3 gene, parental obesity, maternal SES, and postnatal diet and lifestyle in the development of progressive liver disease secondary to NAFLD.

Plasma N-terminal propeptide of type III procollagen accurately predicts liver fibrosis severity in children with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: We examined the diagnostic performance of plasma N-terminal propeptide of type III procollagen (PIIINP) levels, aspartate aminotransferase to platelet ratio index (APRI) and Fibrosis-4 (FIB-4) score for predicting non-alcoholic steatohepatitis (NASH) and liver fibrosis stage in children/adolescents with non-alcoholic fatty liver disease (NAFLD). METHODS: We enrolled 204 children/adolescents with biopsy-proven NAFLD at the "Bambino Gesù" Children's Hospital. We measured plasma PIIINP levels using a commercially available enzyme-linked immunosorbent assay kit and calculated APRI and FIB-4 scores using standard methods. RESULTS: Children with NASH had higher plasma PIIINP levels, APRI and FIB-4 scores compared with those without NASH (all P < .001). However, PIIINP levels had much better diagnostic performance and accuracy than APRI and FIB-4 scores for predicting liver fibrosis stage. PIIINP levels correlated with the total NAFLD activity score (NAS) and its constituent components (P < .0001). The risk of either NASH or F ≥ 2 fibrosis progressively increased with increasing PIIINP levels (P < .0001), independent of age, gender, adiposity measures, insulin resistance, NAS score and the patatin-like phospholipase domain-containing protein-3 rs738409 polymorphism. For every 3.6 ng/mL increase in PIIINP levels, the likelihood of having F ≥ 2 fibrosis increased by ~14-fold (adjusted-odds ratio 14.1, 95% CI 5.50-35.8, P < .0001) after adjustment for the aforementioned risk factors. The area under the receiver operating characteristics curve was 0.921 (95% CI 0.87-0.97) for F ≥ 2 fibrosis, and 0.993 (95% CI 0.98-1.0) for F3 fibrosis respectively. CONCLUSIONS: Unlike APRI and FIB-4 scores, plasma PIIINP levels are a promising, non-invasive biomarker for diagnosing liver fibrosis stage in children/adolescents with biopsy-proven NAFLD.

Eosinophilic esophagitis in children: current knowledge to open new horizons.

Eosinophilic Esophagitis (EoE) is a chronic immune/antigen-mediated condition which is also driven by genetic and environmental factors. It has been deeply investigated over the last years and its incidence is widely increasing in childhood. Although atopic diseases are closely linked with EoE, it does not recognize a classical IgE-mediate immune pathogenesis but it is rather a T helper type 2 inflammatory process. Familial clustering supports genetic predisposition in EoE and recent advances in understanding the genetic basis for EoE may eventually translate into targeted management of the disease. EoE diagnosis is based on clinical symptoms, micro, and macroscopic findings along with exclusion of gastroesophageal reflux disease (GERD) evidence. Management of the disease encompasses both dietary and pharmacological solutions that need to be specifically targeted on patients' history, clinical symptoms, and diagnostic evaluations. New therapies, currently not available in children, may represent the basis for future therapeutic options in the next years.

Fighting Fatty Liver Diseases with Nutritional Interventions, Probiotics, Symbiotics, and Fecal Microbiota Transplantation (FMT).

Pediatric obesity is rising worldwide leading the worrying phenomenon of nonalcoholic fatty liver disease (NAFLD) to shift into one of the most frequent causes of chronic liver illness in childhood. Occurrence of NAFLD depends on several factors such as the geographical area and the diagnostic modalities used; overall it ranges between 3% and 10% of pediatric population, increasing up to 70% in patients with metabolic comorbidities (Manco M, Bottazzo G, DeVito R et al, J Am Coll Nutr 27:667-676, 2008).Recent findings have related the intestinal microbiota to a plethora of pathological conditions, including type 2 diabetes (T2D), obesity, and nonalcoholic steatohepatitis (NASH). One of the emerging areas of the study is the link between liver diseases and gut microbiome, which has added new information to the understanding of the so-called gut-liver axis.In order to address the role of gut microbiome in NAFLD onset and progression, it is necessary to "decipher" operational codes for microbiome investigation within the context of advanced laboratory medicine to capture microbiome features and, hence, to address the function of the intestinal microbiome within the gut microbiota-liver axis.Results of these investigations have allowed the beginning of implementing the usage of probiotics and symbiotics in the medical approach of obesity and NAFLD in adults and children. Several randomized clinical trials (RCTs) have been already published on fecal microbiota transplantation (FMT), T2D, NASH, and inflammatory bowel disease (IBD).This review proposes to describe the current state of knowledge on the ways fatty liver diseases can be addressed with nutritional interventions, probiotics, symbiotics, and FMT.

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.

Laparoscopic Sleeve Gastrectomy Improves Nonalcoholic Fatty Liver Disease-Related Liver Damage in Adolescents by Reshaping Cellular Interactions and Hepatic Adipocytokine Production.

OBJECTIVES: To investigate whether the modulation of local cellular cross-talks and the modification of hepatic adipocytokine expression could mechanistically explain the improvement of liver histopathology after laparoscopic sleeve gastrectomy (LSG) in adolescents with nonalcoholic fatty liver disease (NAFLD). STUDY DESIGN: Twenty obese (body mass index of ≥35 kg/m2) adolescents who underwent LSG and with biopsy-proven NAFLD were included. At baseline (T0) and 1 year after treatment, patients underwent clinical evaluation, blood tests, and liver biopsy. Hepatic progenitor cells, hepatic stellate cells (HSCs), macrophages, and adipocytokines were evaluated by immunohistochemistry and immunofluorescence. RESULTS: Liver biopsy samples after LSG demonstrated a significant improvement of NAFLD Activity Score and fibrosis. Immunohistochemistry indicated a significant reduction of hepatocyte cell cycle arrest, ductular reaction, activated HSC, and macrophage number after LSG compared with T0. The activation state of HSC was accompanied by modification in the expression of the autophagy marker LC3. Hepatocyte expression of adiponectin was significant higher after LSG than into T0. Moreover, LSG caused decreased resistin expression in Sox9+ hepatic progenitor cells compared with T0. The number of S100A9+ macrophages was also reduced by LSG correlating with resistin expression. Finally, serum levels of proinflammatory cytokines significantly correlated with macrophages and activated HSC numbers. CONCLUSIONS: The histologic improvement induced by LSG is associated with the reduced activation of local cellular compartments (hepatic progenitor cells, HSCs, and macrophages), thus, strengthening the role of cellular interactions and hepatic adipocytokine production in the pathogenesis of NAFLD.

Liver zonation in children with non-alcoholic fatty liver disease: Associations with dietary fructose and uric acid concentrations.

BACKGROUND & AIMS: As dietary components are delivered directly to the periportal zone of the liver lobule, there is the potential for greater injury in this zone (zone 1) compared to the perivenous zone (zone 3). We investigated the associations between dietary fructose consumption and uric acid concentrations and differential zonal injury in periportal and perivenous zones. METHODS: A total of 271 children's histological images were scored in 5 periportal and 5 perivenous zones for steatosis, ballooning, inflammation and fibrosis severity. Dietary fructose consumption (g/d) was assessed and uric acid measured in serum. Logistic regression was undertaken to test associations between both high fructose consumption and hyperuricaemia, and histological disease in periportal and perivenous zones. RESULTS: Children with a mean age of 12.5 years were included in the study. Inflammation (mean ± SD) was increased in the periportal vs perivenous zones (0.78 ± 0.43 vs 0.41 ± 0.48, P = .041). There were non-significant trends towards greater steatosis, ballooning and fibrosis in the periportal zone. In the fully adjusted models, high fructose intake was associated with disease in both zones. Example for periportal and perivenous zones, respectively, steatosis 1.56 (1.12, 2.49) and 1.21 (1.09, 2.73); inflammation 4.29 (2.31, 5.88) and 3.69 (2.14, 4.56); and fibrosis 2.72 (1.43, 3.76) and 1.96 (1.24, 2.37). Hyperuricaemia (uric acid ≥5.9 mg/dL) was associated with inflammation in the periportal zone 1.71 (1.17, 2.35); and was associated with steatosis and fibrosis in both zones; for example, for periportal and perivenous zones, respectively, steatosis 2.98 (1.65, 3.23) and 1.14 (1.05, 1.99); and fibrosis, 2.65 (1.35, 2.99) and 1.31 (1.13, 2.17). CONCLUSIONS: High fructose consumption is associated with disease severity in both lobular zones and hyperuricaemia may be associated with more severe disease in the periportal zone.

Relationship between non-alcoholic steatohepatitis, PNPLA3 I148M genotype and bone mineral density in adolescents.

BACKGROUND & AIMS: It is uncertain whether non-alcoholic steatohepatitis (NASH) is a risk factor for low bone mineral density (BMD). Our aim was to investigate: (a) associations between NASH and BMD values and (b) associations between PNPLA3 I148M genotypes and BMD, in children with histologically proven non-alcoholic fatty liver disease (NAFLD). METHODS: BMD area (g/cm2 ) was measured using dual-energy X-ray absorptiometry (DEXA). NASH was diagnosed by a Steatosis, Activity and Fibrosis (SAF) score and FLIP algorithm. Genotyping for patatin-like phospholipase domain containing-3 (PNPLA3) I148M genotype (rs738409) (CC, CG and GG) was undertaken using the TaqMan SNP genotyping allelic discrimination method. Logistic regression was used to test associations [OR (95% CIs)] between low BMD, and both NASH and PNPLA3 I148M genotypes. RESULTS: Thirty-four adolescents (mean age 13.8 ± 1.1 years) with histologically confirmed NAFLD were studied. Subjects with NASH (n = 25) had a lower BMD (means (SDs) 0.87 ± 0.06 vs 0.97 ± 0.12, P = 0.005), compared to subjects without NASH. Subjects with PNPLA3 CG+GG genotypes had a lower BMD compared with subjects with PNPLA3-CC genotype (means (SDs) 0.79 ± 0.20 vs 0.92 ± 0.10, P = 0.009). PNPLA3 CG+GG genotypes were independently associated with NASH [OR (95% CIs 1.78, 1.24, 2.99)], and low BMD was associated with both PNPLA3 CG+GG (OR 3.62 (95% CIs 1.21, 5.53), P = 0.028) and with SAF score (OR 2.76 (95% CIs 1.12, 5.41), P = 0.045). CONCLUSIONS: Taken together the independent associations between: (a) low BMD and PNPLA3 CG+GG genotype; (b) low BMD and NASH; and (c) PNPLA3 CG+GG genotype and NASH, provide support for a causal relationship between NASH and low BMD.