Metabolic Associated Fatty Liver Disease in Children and Adolescents: Mechanisms of a Silent Epidemic and Therapeutic Options.

Non-alcoholic fatty liver disease (NAFLD) is now identified as a hepatic sign of metabolic syndrome and is the most frequent cause of chronic liver disease in all ages. It is assumed that a genetic predisposition associated with epigenetic factors participates in the evolution of this condition. Visceral obesity and insulin resistance (IR) have always been considered the most important causative factors of Metabolic Syndrome (MetS) and NAFLD, but currently, the interaction between genetic heritage and environmental factors is increasingly considered fundamental in the genesis of metabolic disorders associated with NAFLD. In fact, in patients with NAFLD, insulin resistance, arterial hypertension, abdominal obesity, dyslipidemia and reduced intestinal permeability have often been found, as well as a higher prevalence of coronary artery disease, obstructive sleep apnea, polycystic ovary syndrome and osteopenia, which define a MetS framework. Early diagnosis is needed to prevent disease progression through primarily lifestyle interventions. Unfortunately, at present, there are no molecules recommended for pediatric patients. However, several new drugs are in clinical trials. For this reason, targeted studies on the interaction between genetics and environmental factors involved in the development of NAFLD and MetS and on the pathogenetic mechanisms that determine the evolution in non-alcoholic steatohepatitis (NASH), should be implemented. Therefore, it is desirable that future studies may be useful in identifying patients at risk of developing NAFLD and MetS early.

Profiling of cell-free DNA methylation and histone signatures in pediatric NAFLD: A pilot study.

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in children and adolescents, increasing the risk of its progression toward nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. There is an urgent need for noninvasive early diagnostic and prognostic tools such as epigenetic marks (epimarks), which would replace liver biopsy in the future. We used plasma samples from 67 children with biopsy-proven NAFLD, and as controls we used samples from 20 children negative for steatosis by ultrasound. All patients were genotyped for patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), membrane bound O-acyltransferase domain containing 7 (MBOAT7), and klotho-ß (KLB) gene variants, and data on anthropometric and biochemical parameters were collected. Furthermore, plasma cell-free DNA (cfDNA) methylation was quantified using a commercially available kit, and ImageStream(X) was used for the detection of free circulating histone complexes and variants. We found a significant enrichment of the levels of histone macroH2A1.2 in the plasma of children with NAFLD compared to controls, and a strong correlation between cfDNA methylation levels and NASH. Receiver operating characteristic curve analysis demonstrated that combination of cfDNA methylation, PNPLA3 rs738409 variant, coupled with either high-density lipoprotein cholesterol or alanine aminotransferase levels can strongly predict the progression of pediatric NAFLD to NASH with area under the curve >0.87. Conclusion: Our pilot study combined epimarks and genetic and metabolic markers for a robust risk assessment of NAFLD development and progression in children, offering a promising noninvasive tool for the consistent diagnosis and prognosis of pediatric NAFLD. Further studies are necessary to identify their pathogenic origin and function.

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.

Antioxidant activity of Hydroxytyrosol and Vitamin E reduces systemic inflammation in children with paediatric NAFLD.

BACKGROUND: The rise in paediatric non-alcoholic fatty liver disease (NAFLD) is particularly alarming. We recently reported that Hydroxytyrosol (HXT) and Vitamin E (VitE) may improve oxidative stress, insulin resistance, and steatosis in children with biopsy-proven NAFLD. AIM: Here, we investigated if HXT+VitE may reduce systemic inflammation in the above-mentioned patients. METHODS: This study analysed the plasma levels of IL (interleukin)-6, IL-1ß, IL-10, tumour necrosis factor (TNF)-α, 4­hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2'deoxyguanosine (8-OHdG) in children enrolled in the HXT+VitE trial (ClinicalTrials.gov, NCT02842567). RESULTS: Changes in markers of systemic inflammation were found in both placebo (Pla) and HXT+VitE. In particular, after four months, the levels of IL-1ß and TNF-α were reduced in both groups, while IL-6 decreased, and IL-10 increased significantly only in the group treated with HXT+VitE. Children treated with HXT+VitE showed a significant decrease of 4-HNE and 8-OHdG that correlated with the improvement of triglyceride levels. Noticeably, only the 8-OHdG decrease correlated with steatosis amelioration and with the increase of IL-10 levels. CONCLUSION: The treatment with HXT and VitE reduced the NAFLD-related systemic inflammation in children, mainly by an increase of IL-10 circulating levels that occurred in response to DNA damage recovery, ultimately improving steatosis and hypertriglyceridemia.

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.

Nonalcoholic Fatty Liver Disease in Italian Children with Down Syndrome: Prevalence and Correlation with Obesity-Related Features.

OBJECTIVE: To assess the prevalence of overweight/obesity in a cohort of Italian children with Down syndrome (DS) and to investigate the correlation of both obesity and DS with nonalcoholic fatty liver disease (NAFLD). STUDY DESIGN: We enrolled 280 children with DS (age range 5-18 years), who were referred to the DS outpatient clinic of the Bambino Gesù Children's Hospital in Rome. For all children, we collected the clinical history and measured anthropometric variables. Eighty-four of 280 children with DS were selected to undergo liver ultrasound scanning to evaluate the presence of NAFLD. RESULTS: Italian children with DS exhibited a prevalence of 19.64% for overweight and 12.14% for obesity. The prevalence of NAFLD in nonobese (45%) and overweight/obese (82%) children with DS is greater than in the European pediatric nonobese (5.7%) or obese population (33%). Moreover, the severity of liver brightness on ultrasound scan correlated positively with body mass index, triglycerides, low-density lipoprotein-cholesterol, and leptin levels and negatively with adiponectin. CONCLUSIONS: We demonstrated that, independently from the obese phenotype, children with DS display a greater risk to develop NAFLD than the general pediatric population.

Beverage consumption and paediatric NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and adolescents, due to the increased worldwide incidence of obesity among children. It is now clear enough that of diet high in carbohydrates and simple sugars are associated with hepatic steatosis and non-alcoholic steatohepatitis (NASH). Several studies have shown that an increased consumption of simple sugars is also positively associated with overweight and obesity, and related co-morbidities, such as type 2 diabetes, metabolic syndrome and NAFLD. It is difficult to define the role of the various components of soft drinks and energy drinks in the pathogenesis of NAFLD and its progression in NASH, but the major role is played by high calorie and high sugar consumption, mainly fructose. In addition, other components of these beverages (e.g. xanthine) seem to have an important role in the pathogenesis of metabolic disorders, crucial pathways involved in NAFLD/NASH. The drastic reduction in the consumption of energy drinks and soft drinks is an appropriate intervention for the prevention of obesity and NAFLD in young people.

Autoimmune hepatitis in children: an overview of the disease focusing on current therapies.

Autoimmune hepatitis (AIH) is an immune-mediated necroinflammatory disease of the liver characterized by elevation of IgG, presence of characteristic autoantibodies, and histological features of interface hepatitis. Two types of juvenile AIH have been recognized according to seropositivity for smooth muscle and/or antinuclear antibody (AIH type 1) or liver kidney microsomal antibody (AIH type 2). The exact pathogenesis of AIH is still unclear, but it is known that unidentified environmental factors, and occasionally drugs, might trigger disease in genetically susceptible individuals. The clinical spectrum of this disease is very wide, ranging from asymptomatic individuals with abnormal liver function to those with fulminant liver failure. The diagnosis is based on a combination of biochemical and histological parameters and on exclusion of other liver diseases. It is a relatively rare but devastating disease, which progresses rapidly unless immunosuppressive treatment is started promptly. Standard therapy consists of a combination of corticosteroids and azathioprine, which is efficacious in 80% of patients. Alternative therapies are increasingly being explored in patients who do not respond to standard treatment and/or have intolerable side-effects. The purpose of this paper is to review our current knowledge about AIH in children, evaluating mainly the therapeutic options for its treatment, considering also the newer immunosuppressant agents used in difficult-to-treat cases.

Celiac disease in pediatric patients with autoimmune hepatitis: etiology, diagnosis, and management.

Celiac disease (CD) is defined as a permanent intolerance to ingested wheat gliadins and other cereal prolamins, occurring in genetically susceptible people. Persistent elevation of serum aminotransferase activity is expression of liver damage related to CD, which occurs in two distinctive forms. The most frequent is a mild asymptomatic liver injury, with a moderate increase of serum aminotransferase activities and a mild inflammatory portal and lobular infiltrate on liver biopsy (celiac hepatitis), reversible on a gluten-free diet (GFD). More rarely, severe and progressive inflammatory liver damage, induced by an autoimmune process and identified as autoimmune hepatitis (AIH), can develop and it is generally unaffected by gluten withdrawal. Surveys that included only pediatric patients report a wide range of prevalence of CD in AIH of 11.5-46% (mean 21.5%). CD and AIH share selected combinations of genes coding for class II human leukocyte antigens, which could explain their coexistence. Increased intestinal permeability and circulation of anti-tissue transglutaminase (tTG) have also been considered as further potential causes of liver damage in CD patients. tTG in the liver and in other extraintestinal tissues could modify other external- or self-antigens and generate different neo-antigens, which are responsible for liver injury in patients with CD. Patients with AIH represent a population at high risk for developing CD; screening for CD should be integrated into the diagnostic routine of all patients with AIH, with or without gastrointestinal manifestations, before starting immunosuppressive treatments. The only currently available treatment for CD is the GFD and the supportive nutritional care for iron, calcium, and vitamin deficiencies. Due to the difficulties of a GFD, in the past decade researchers have become increasingly interested in therapeutic alternatives to continuous or intermittent use of a GFD in patients with CD. Interventions addressed to correct the defect in the intestinal barrier are currently at the most advanced stage of clinical trials. The impact of a GFD on the outcome of AIH is not clear but it seems to be ineffective in the treatment of AIH. The early detection and treatment of CD, however, may prevent progression to end-stage liver failure.

Successful tenofovir treatment for fulminant hepatitis B infection in an infant.

Fulminant hepatic failure is defined by the presence of severe impairment of liver function, with or without encephalopathy, in patients with no underlying chronic liver disease. We report the case of a 4-month-old infant who developed fulminant hepatitis B infection and recovered concomitant with tenofovir therapy without liver transplantation.

Lifestyle intervention and antioxidant therapy in children with nonalcoholic fatty liver disease: a randomized, controlled trial.

UNLABELLED: No proven treatment exists for nonalcoholic fatty liver disease (NAFLD) in children and adolescents. We sought to determine the efficacy of lifestyle intervention with or without antioxidant therapy in pediatric NAFLD. A total of 53 patients (age 5.7-18.8 years, 37 boys) were included. Lifestyle intervention consisting of a diet tailored to the patient's calorie needs, and increased physical activity was prescribed in all. Patients were concomitantly randomized to alpha-tocopherol 600 IU/day plus ascorbic acid 500 mg/day (n = 25) or placebo (n = 28), and treated for 24 months. The study was an extension of a previous study aimed at evaluating the effect of 12-month lifestyle intervention and antioxidant therapy on serum levels of aminotransferases. The primary end point of the present study was change in liver histology on repeated biopsy at 24 months. Secondary end points were changes in body weight, liver enzymes, and insulin sensitivity indices on 2-hour oral glucose tolerance test. The amount of weight lost at 24 months was similar in the placebo and antioxidant groups (-4.75 [range, -16-4.0] versus -5.5 [range, -12.2-0.4] kg, respectively, P = 0.9). A significant improvement occurred in the grade of steatosis, lobular inflammation, and hepatocyte ballooning, and in the NAFLD activity score in both groups. Levels of aminotransferases, triglycerides, cholesterol, fasting glucose, and insulin, and insulin sensitivity indices improved significantly as well. The improvement in all these parameters was not significantly different between the two groups. CONCLUSION: Lifestyle intervention with diet and increased physical activity induces weight loss and is associated with a significant improvement in liver histology and laboratory abnormalities in pediatric NAFLD. Alpha-tocopherol plus ascorbic acid does not seem to increase the efficacy of lifestyle intervention alone.

NAFLD in children: a prospective clinical-pathological study and effect of lifestyle advice.

Nonalcoholic fatty liver disease (NAFLD), a common cause of chronic liver disease in adults, is incompletely characterized in children. We conducted a prospective study to better characterize the clinical presentation of NAFLD in children and to determine the effect of lifestyle advice in the management of pediatric NAFLD. From June 2001 to April 2003, 84 children (age 3-18.8 yr) who had elevated aminotransferases and the diagnosis of NAFLD confirmed via liver biopsy underwent a 2-hour oral glucose tolerance test and a 12-month program of lifestyle advice consisting of diet and physical exercise. Thirty-four (40.5%) patients were obese (body mass index [BMI] >97th percentile), and 43 (51.2%) were overweight (BMI 85th-97th percentile). Ten (12%) had abnormal glucose tolerance; 10 (12%) had elevated triglycerides, cholesterol, or both; and all had normal blood pressure. Most children (67/84, 80%) were insulin-resistant, including the 7 children with normal BMI (<85th percentile). Increased liver fibrosis was present in 49 (58.1%) patients and was independently associated with obesity (OR 2.7, 95% CI 1.2-6.2) and age (1-year increase; OR 1.2, 95% CI 1.04-1.5). A 12-month program with diet and physical exercise resulted in a significant decrease in BMI, and levels of fasting glucose, insulin, lipids, and liver enzymes, as well as liver echogenicity on ultrasonography. In conclusion, children with NAFLD are almost always insulin-resistant regardless of BMI. Obesity and older age are independently associated with increased liver fibrosis. A simple lifestyle advice program significantly improves insulin resistance, and the liver disease in pediatric NAFLD.

Treatment of Wilson's disease with zinc from the time of diagnosis in pediatric patients: a single-hospital, 10-year follow-up study.

Wilson's disease (WD) is an inherited disorder of copper metabolism characterized by a failure of the liver to excrete copper, leading to its accumulation in the liver, brain, cornea, and kidney, with resulting chronic degenerative changes. It is generally accepted that "presymptomatic" patients--in whom WD is diagnosed in childhood and who are defined as those who, although still asymptomatic, do have liver disease, as indicated by increased serum concentrations of transaminases--should be treated prophylactically. Here we report our results in 22 children treated with continuous oral zinc therapy for 10 years. Zinc sulfate was administered at a dosage of 25 mg elemental zinc twice a day until the age of 6 years, 25 mg three times a day between the ages of 7 and 16 years or until the child attained a body weight of 125 lb, and 50 mg three times a day thereafter. Five years after the start of zinc treatment, we noted highly significant decreases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and urinary copper excretion, but white blood cell counts did not vary significantly. Six of 22 patients continued to demonstrate greater-than-normal ALT concentrations and only 1 patient demonstrated an ALT concentration more than 1.5 times the upper normal limit. Further decreases in ALT, AST, and urinary copper excretion were observed at the end of the 10-year follow-up, but these decreases were not statistically significant. Only 1 patient continued to demonstrate abnormal ALT levels. Again, white blood cells showed no significant variations. All histologic scores (steatosis, inflammation, and fibrosis) were significantly decreased after treatment. Hepatic copper content was also significantly decreased, although it remained higher than normal in all patients. The removal of toxic copper was confirmed by disappearance of Kayser-Fleischer rings in 3 patients. Zinc did not have adverse effects on growth. The efficacy of zinc in WD in presymptomatic pediatric patients has been established in previous studies, and our study adds considerably to the earlier findings because it includes a large number of very young children, as many as 11 younger than 6 years and 20 younger than 10. The excellent clinical results in all patients, coupled with the improvement in hepatic histologic findings in the vast majority, indicate convincingly that zinc treatment can control the disease effectively and safely, preventing its progression over the course of 10 years. Histologic findings reportedly improved in 3 patients treated in an earlier study, but our data are numerically much more relevant. Notably, histologic study of the liver revealed that copper concentration was reduced by treatment, suggesting that oral zinc was able not only to prevent further accumulation of copper but also to promote, at least in part, the depletion of its stores. The lack of adverse effects of zinc on growth suggests that our patients received enough anticopper therapy to prevent damage resulting from copper toxicity but an adequate amount of copper for proper growth and development. In conclusion, our findings indicate that zinc is the treatment of choice in presymptomatic pediatric patients with WD.

Glutathione metabolism and antioxidant enzymes in patients affected by nonalcoholic steatohepatitis.

BACKGROUND: Oxidative stress and accumulation of excessive fat in the liver may underlie the pathophysiology of nonalcoholic steatohepatitis (NASH). Given that glutathione blood metabolism may represent an indicator of tissue oxidative status, we analysed the blood profile of various forms of glutathione in children with NASH, and we evaluated the presence of systemic oxidative stress by calculating the oxidised/reduced glutathione ratio (GSSG/GSH). Furthermore, we analysed the catalytic activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione transferase (GST), and glutathione reductase (GR) in blood of patients. METHODS: Blood samples were obtained from 21 children with NASH and 28 controls. Total, reduced, oxidised, and protein-bound glutathione concentrations were determined by reversed-phase liquid chromatography with fluorescence detection. Antioxidant enzymes were spectrophotometrically assayed by using specific substrates. RESULTS: Our findings showed a 1.5-fold increase of GSSG in patients, resulting in a significant rise of the GSSG/GSH ratio. SOD, GPx, and GR activities were not significantly different in NASH respect to controls, whereas GST, which provides the second defence line against oxidative stress, was 17.8% increased. CONCLUSIONS: Our data demonstrate an impairment of glutathione metabolism and antioxidant enzyme activities in blood of patients with NASH, supporting a consistent role of free radical cytotoxicity in the pathophysiology of the disease.