Is There a Safe Alcohol Consumption Limit for the General Population and in Patients with Liver Disease?

Excessive alcohol consumption represents an important burden for health systems worldwide and is a major cause of liver- and cancer-related deaths. Alcohol consumption is mostly assessed by self-report that often underestimates the amount of drinking. While alcohol use disorders identification test - version C is the most widely used test for alcohol use screening, in patients with liver disease the use of alcohol biomarker could help an objective assessment. The amount of alcohol that leads to significant liver disease depends on gender, genetic background, and coexistence of comorbidities (i.e., metabolic syndrome factors). All patients with alcohol-associated liver disease are recommended to follow complete abstinence and they should be treated within multidisciplinary teams. Abstinence slows down and even reverses the progression of liver fibrosis and can help recompensate patients with complicated cirrhosis. Whether there is a safe amount of alcohol in the general population is a matter of intense debate. Large epidemiological studies showed that the safe amount of alcohol to avoid overall health-related risks is lower than expected even in the general population. Even one drink per day can increase cancer-related death. In patients with any kind of chronic liver disease, especially in those with metabolic-associated steatotic liver disease, no alcohol intake is recommended. This review article discusses the current evidence supporting the deleterious effects of small-to-moderate amounts of alcohol in the general population and in patients with underlying chronic liver disease.

Impaired anti-HBV vaccine response in non-cirrhotic chronic HCV is not overcome by double dose regimen: randomized control trial.

INTRODUCTION AND OBJECTIVES: Some studies suggest chronic HCV infection diminishes responses to the anti-HBV vaccine. We evaluated the efficacy of double versus standard dose HBV vaccination among HCV patients without cirrhosis. PATIENTS AND METHODS: 141 adults with untreated chronic HCV were randomized to HBV vaccination with double dose (40µg) or standard dose (20µg) at 0, 1 and 6 months; 70 healthy HCV-negative patients given standard dose served as controls. Vaccine response was defined by anti-HBs ≥10 mIU/mL. RESULTS: 128 patients (60 double, 68 standard doses) completed the study. Patients were of median age 52 years, 61% female, 60% fibrosis <2 of 4, and 76% genotype 1 with median 6-log 10 IU/mL HCV RNA. Overall seroprotection rate was 76.7% (95% CI: 65-87) in the 40µg versus 73.5% (95% CI: 63-84) in the 20µg dose HCV-positive groups (p =0.68) and 91.2% (95%CI:84-99) in HCV-negative controls (p =0.011 and 0.003, respectively). In multivariate logistic regression, vaccine dose (double vs. standard dose) was not associated with vaccine response (OR=0.63, p =0.33). Of 32 HCV-infected patients who were non-responders to 3- doses, 25 received the fourth dose of vaccine. The fourth dose seroconversion rate for the 40µg and 20µg groups were 45.5% and 21.4%, respectively. CONCLUSIONS: In HCV-infected patients without cirrhosis, impaired responses to HBV vaccination cannot be overcome by the use of double dose HBV vaccination, but adding a fourth dose of vaccine for non-responders may be an effective strategy. Other adjuvant measures are needed to enhance seroconversion rates in these patients. TRIAL REGISTER: U 1111-1264-2343 (www.ensaiosclinicos.gov.br).

Interaction of Microbiome, Diet, and Hospitalizations Between Brazilian and American Patients With Cirrhosis.

BACKGROUND & AIMS: Gut microbiota are affected by diet, country, and affect outcomes in cirrhosis. Western diets are associated with dysbiosis. Comparisons with other diets is needed. We aimed to compare cirrhosis patients from the United States with cirrhosis patients from Brazil with respect to diet, microbiota, and impact on hospitalizations. METHODS: Healthy controls and compensated/decompensated outpatients with cirrhosis from the United States and Brazil underwent dietary recall and stool for 16S ribosomal RNA sequencing. Demographics and medications/cirrhosis details were compared within and between countries. Patients with cirrhosis were followed up for 90-day hospitalizations. Regression for Shannon diversity was performed within cirrhosis. Regression for hospitalizations adjusting for clinical and microbial variables was performed. RESULTS: Model for end-stage liver disease (MELD), diabetes, ascites, and albumin were similar, but more Americans were men, had higher hepatic encephalopathy and alcohol/hepatitis C etiology, with lower nonalcoholic fatty liver disease than Brazilians. Brazilians had higher cereal, rice, and yogurt intake vs the United States. As disease progressed, cereals, rice/beans, coffee, and chocolate consumption was reduced. Microbial diversity was higher in Brazilians. Within cirrhosis, high diversity was related to Brazilian origin (P < .0001), age, and cereal intake (P = .05), while high MELD scores (P = .009) and ascites (P = .05) did the reverse. Regardless of stage, beneficial taxa and taxa associated with grant and yogurt intake were higher (Ruminococcaceae, Christensenellacae, and Prevotellaceae), while pathobionts (Porphyromonadaceae, Sutterellaceae, and Enterobacteriaceae) were lower in Brazilians. More Americans were hospitalized vs Brazilians (P = .002). On regression, MELD (P = .001) and ascites (P = .001) were associated with higher hospitalizations, while chocolate (P = .03) and Brazilian origin (P = .001) were associated with lower hospitalizations with/without microbiota inclusion. CONCLUSIONS: Brazilian cirrhotic patients follow a diet richer in cereals and yogurt, which is associated with higher microbial diversity and beneficial microbiota and could contribute toward lower hospitalizations compared with a Western-diet-consuming American cohort.

Non-Alcoholic Fatty Liver Disease-Related Hepatocellular Carcinoma: Immunohistochemical Assessment of Markers of Cancer Cell Metabolism.

INTRODUCTION: Hepatocellular carcinoma (HCC) has been associated to non-alcoholic fatty liver disease (NAFLD). We sought to investigate the immunoexpression of several glycolytic metabolism-associated markers in patients with HCC associated to NAFLD and associate these factors to their clinical-pathological characteristics. METHODS: We evaluated 35 HCC specimens from 21 patients diagnosed with non-alcoholic steatohepatitis (NASH) undergoing liver resection (12 patients), liver transplantation (8 patients), or both (1 patient). Histological features, clinical aspects, demographic and biochemical data, as well as the immunohistochemical reactivity for monocarboxylate transporters 1, 2, and 4; their chaperone CD147; carbonic anhydrase IX; and glucose transporter-1 (GLUT1) were assessed. RESULTS: Metabolic-associated cirrhosis was present in 12 of the 21 patients (8 child A and 4 child B scores). From 9 patients without cirrhosis, 3 presented NASH F3 and 6 NASH F2. Sixteen (76%) had diabetes mellitus, 17 (81%) arterial hypertension, and 19 (90%) body mass index above 25 kg/m2; 8 (38%) had dyslipidemia. From 35 nodules, steatosis was found in 26, ballooning in 31 nodules, 25 of them diagnosed as steatohepatitic subtype of HCC. MCT4 immunoexpression was associated with extensive intratumoral fibrosis, advanced clinical stages, and shorter overall survival. GLUT1 was noticeable in nodules with extensive intratumoral steatosis, higher intratumoral fibrosis, and advanced clinical stages. Immunohistochemical expression of the metabolic biomarkers MCT4 and GLUT1 was higher in patients with Barcelona-clinic liver cancer B or C. GLUT1 correlated with higher degree of steatosis, marked ballooning, intratumoral fibrosis, and higher parenchymal necroinflammatory activity. CONCLUSION: Our data indicate that the expression of the glycolytic phenotype of metabolic markers, especially GLUT1 and MCT4, correlates with a more severe course of HCC occurring in NASH patients.

African genetic ancestry is associated with lower frequency of PNPLA3 G allele in non-alcoholic fatty liver in an admixed population.

INTRODUCTION AND OBJECTIVES: PNPLA3 (rs738409) and TM6SF2 (rs58542926) variants, interindividual and ethnic differences may be risk factors for non-alcoholic fatty liver disease (NAFLD). The PNPLA3 G allele is associated with worse NAFLD evolution in Hispanics and Caucasians. TM6SF2 is associated with hypertriglyceridemia, NAFLD, and cardiovascular disease. We aimed to evaluate the association between genetic ancestry by Ancestry Informative Markers (AIM), PNPLA3 and TM6SF2 polymorphisms in patients with biopsy-proven NAFLD in an admixed population. METHODS: We included adults with biopsy-proven NAFLD and excluded patients with the presence of other chronic liver disease, alcohol intake >100g/week, HIV, drug-induced fatty liver disease, or liver transplantation. We classified NAFLD using the Non-Alcoholic Steatohepatitis Clinical Research Network (NASH-CRN) histological scoring system. The PNPLA3 (rs738409 c.444C>G) and TM6SF2 (rs58542926 c.449C>T) genotyping were performed by RT-PCR. Genetic ancestry was determined using 46 insertion-deletion AIM; α<0.05 was considered significant. RESULTS: A total of 248 patients with NAFLD were enrolled [34 with simple steatosis (NAFL); 214 with NASH]. Overall, we detected a greater European ancestry contribution (0.645), followed by African (0.173), Amerindian (0.095), and East Asian (0.087) ancestry contribution, without differences between NAFL and NASH patients. However, we found a higher African genetic ancestry contribution among patients with NAFL who had the PNPLA3 C/C genotype than those with the G allele (0.216 ± 0.205 versus 0.105 ± 0.101, respectively; p=0.047). Ancestry contributions did not differ among TM6SF2 genotypes. CONCLUSION: Among NAFL patients, greater African genetic ancestry was associated to a lower frequency of the PNPLA3 G allele, demonstrating a possible NASH ancestry-related protective factor.

Synbiotic Supplementation Modulates Gut Microbiota, Regulates ß-Catenin Expression and Prevents Weight Gain in ob/ob Mice: Preliminary Findings.

BACKGROUND: Obesity is one of the main health problems in the world today, and dysbiosis seems to be one of the factors involved. The aim of this study was to examine the impact of synbiotic supplementation on obesity and the microbiota in ob/ob mice. Twenty animals were divided into four groups: obese treated (OT), obese control (OC), lean treated (LT) and lean control (LC). All animals received a standard diet for 8 weeks. The treated groups received a synbiotic (Simbioflora-Invictus Farmanutrição Ltd., Sao Paulo, Brazil) in water, while the nontreated groups received only water. After 8 weeks, all animals were sacrificed, and gut tissue and stool samples were collected for mRNA isolation and microbiota analysis, respectively. ß-Catenin, occludin, cadherin and zonulin in the gut tissue were analyzed via RT-qPCR. Microbiome DNA was extracted from stool samples and sequenced using an Ion PGM Torrent platform. RESULTS: Synbiotic supplementation reduced body weight gain in the OT group compared with the OC group (p = 0.0398) and was associated with an increase in Enterobacteriaceae (p = 0.005) and a decrease in Cyanobacteria (p = 0.047), Clostridiaceae (p = 0.026), Turicibacterales (p = 0.005) and Coprococcus (p = 0.047). On the other hand, a significant reduction in Sutterella (p = 0.009) and Turicibacter (p = 0.005) bacteria was observed in the LT group compared to the LC group. Alpha and beta diversities were different among all treated groups. ß-Catenin gene expression was significantly decreased in the gut tissue of the OT group (p ≤ 0.0001) compared to the other groups. No changes were observed in occludin, cadherin or zonulin gene expression in the gut tissue. CONCLUSIONS: Synbiotic supplementation prevents excessive weight gain, modulates the gut microbiota, and reduces ß-catenin expression in ob/ob mice.

Molecular characterisation of hepatocellular carcinoma in patients with non-alcoholic steatohepatitis.

BACKGROUND AND AIMS: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies. METHODS: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays. RESULTS: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-ß proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved. CONCLUSIONS: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature. LAY SUMMARY: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC.

The role of PNPLA3 and TM6SF2 polymorphisms on liver fibrosis and metabolic abnormalities in Brazilian patients with chronic hepatitis C.

BACKGROUND: Despite the growing body of knowledge about TM6SF2 and PNPLA3 polymorphisms in non-alcoholic fatty liver disease, their influence in the spectrum of HCV liver disease is not yet fully defined. Besides that, admixed populations, such as Brazilians, were not included in most of the studies. METHODS: This cross-sectional study enrolled 365 treatment-naïve patients with HCV and 134 healthy individuals. TM6SF2 (rs58542926 c.499C > T) and PNPLA3 (rs738409 c.444C > G) polymorphisms were evaluated regarding their association with clinical and laboratory data, histological liver steatosis and fibrosis, and with components of the metabolic syndrome. RESULTS: In HCV subjects, the frequencies of TM6SF2 CC and CT + TT were 89% and 11%, while PNPLA3 frequencies of CC and CG + GG were 51.4% and 48.6%. In the univariate logistic regression analysis, the TM6SF2 CT + TT genotype in HCV was associated with significant liver fibrosis (p = 0.047; OR 1.953; 95% CI 1.009-3.788). In comparison to the CT + TT genotype, the TM6SF2 CC genotype in HCV was associated with older age (p = 0.002), higher frequency of arterial hypertension (p = 0.032), obesity (p = 0.030), metabolic syndrome (p = 0.014) and lower total cholesterol levels (p = 0.036). The PNPLA3 GG subjects had lower body mass index than CG/ CC individuals (p = 0.047). None of the polymorphisms, or their combinations, was independently associated with hepatic steatosis or fibrosis. On the other hand, older age, lower serum levels of total cholesterol, and higher serum levels of alanine aminotransferase and alkaline phosphatase were associated with liver fibrosis in the multivariate logistic regression analysis. CONCLUSION: In this evaluation of an admixed HCV population, neither TM6SF2 nor PNPLA3 polymorphisms were independently associated with hepatic steatosis or fibrosis. Other factors seem more influential than these specific polymorphisms in isolation. More studies are warranted to clarify the role of the TM6SF2 and PNPLA3 polymorphisms in Brazilians with HCV.

Usefulness of collagen type IV in the detection of significant liver fibrosis in nonalcoholic fatty liver disease.

INTRODUCTION/AIMS: Liver fibrosis assessment is a key issue in the evaluation of nonalcoholic fatty liver disease (NAFLD) patients. In the present study, we aimed to validate a noninvasive marker panel to assess significant and advanced fibrosis in these patients. METHOD: 126 biopsy-proven NAFLD patients were included. NAFLD diagnosis was based on histological criteria. Fibrosis stages were determined according to NASH-Clinical Research Network criteria. Clinical and laboratorial data were collected during the interval of three months before or after liver biopsy. Histological fibrosis stages were classified as significant fibrosis (F2-F4) and advanced fibrosis (F3-F4). Five serum biomarkers [hyaluronic acid (HA), collagen type IV (cIV), procollagen type III (PC III), laminin (LN) and cholylglycine (CG)] were assessed by chemiluminescence immunoassays. RESULTS: Most patients were female (61.61%), mean age: 55.7 ±â€¯9.13 years old and mean BMI was 32.1 ±â€¯5.9 kg/m2. Prevalence of diabetes, dyslipidemia, arterial hypertension, and metabolic syndrome was 68.75%, 82.29%, 63.54% and 81.05%, respectively. Patients with cIV above 30 ng/mL had a 5.57-times (IC: 1.86-16.69) the chance of having significant fibrosis and 7.61-times (IC: 2.27-25.54) the chance of having advanced fibrosis versus patients with values below 30 ng/mL. HA, PC III, LN and CG did not detect the presence of significant and advanced fibrosis. The AUROC of clV for detection of significant (0.718) and advanced fibrosis (0.791) was better than that of other serum biomarkers. CONCLUSION: Type 4 collagen could predict the presence of significant and advanced fibrosis in NAFLD patients and it would be a useful tool in routine clinical practice.

NAFLD: Challenges and opportunities to address the public health problem in Latin America.

Non-alcoholic fatty liver disease (NAFLD) is reaching epidemic proportions worldwide. Collectively, Latin American countries have some of the highest obesity rates in the world and the fastest-growing prevalence of type 2 diabetes mellitus (T2DM). Since obesity and T2DM are intrinsically linked with NAFLD, epidemiological projections are worrisome. In addition to this adverse epidemiological setting, the region of Latin America faces unique challenges and obstacles to addressing the growing burden of NAFLD. In this article, on the occasion of the International NASH Day on June 10, 2021, we describe the main challenges and opportunities to improve care of people living with NAFLD in Latin America. Among the major challenges to be tackled are: lack of disease awareness, limited educational opportunities for healthcare personnel and general public, health system fragmentation, and lack of effective strategies for the prevention and effective treatment of NAFLD and common comorbidities, namely obesity and T2DM. Wide dissemination of current concepts on NAFLD, and extensive collaboration between scientific societies, governments, non-governmental organizations, pharmaceutical industry, and other stakeholders is urgently needed to advance the NAFLD public health policies agenda that allows us to address this disease with a whole of society approach.

A new definition for metabolic dysfunction-associated fatty liver disease: An international expert consensus statement.

The exclusion of other chronic liver diseases including "excess" alcohol intake has until now been necessary to establish a diagnosis of metabolic dysfunction-associated fatty liver disease (MAFLD). However, given our current understanding of the pathogenesis of MAFLD and its rising prevalence, "positive criteria" to diagnose the disease are required. In this work, a panel of international experts from 22 countries propose a new definition for the diagnosis of MAFLD that is both comprehensive and simple, and is independent of other liver diseases. The criteria are based on evidence of hepatic steatosis, in addition to one of the following three criteria, namely overweight/obesity, presence of type 2 diabetes mellitus, or evidence of metabolic dysregulation. We propose that disease assessment and stratification of severity should extend beyond a simple dichotomous classification to steatohepatitis vs. non-steatohepatitis. The group also suggests a set of criteria to define MAFLD-associated cirrhosis and proposes a conceptual framework to consider other causes of fatty liver disease. Finally, we bring clarity to the distinction between diagnostic criteria and inclusion criteria for research studies and clinical trials. Reaching consensus on the criteria for MAFLD will help unify the terminology (e.g. for ICD-coding), enhance the legitimacy of clinical practice and clinical trials, improve clinical care and move the clinical and scientific field of liver research forward.

Diagnostic performance of three non-invasive fibrosis scores (Hepamet, FIB-4, NAFLD fibrosis score) in NAFLD patients from a mixed Latin American population.

INTRODUCTION AND AIMS: Several non-invasive scoring systems have been developed and validated worldwide to predict the risk of liver fibrosis in nonalcoholic fatty liver disease (NAFLD). However, information about the performance of these systems in Latin American populations is scarce. Our aim was to evaluate the performance of the Hepamet Fibrosis Score, Fibrosis-4 (FIB-4) and the NAFLD Fibrosis Score (NFS) in a mixed Latin American group of NAFLD patients. METHODS: Clinical, laboratory and liver biopsy data collected from 379 biopsy-proven NAFLD patients from Latin American tertiary health centers were reviewed. Histological fibrosis stages were classified using the Kleiner score. Accuracy was determined, and new fibrosis score thresholds were calculated to better compare the performances of non-invasive tests and to explore their usefulness in excluding fibrosis. RESULTS: The distribution of fibrosis stages among the sample population was as follows: F0 (45%), F1 (27%), F2 (8%), F3 (16%) and F4 (4%). Using modified thresholds, the areas under the ROC curves (AUROC) for Hepamet and FIB-4 (0.73 and 0.74, respectively) to detect significant fibrosis were higher than that of NFS (0.58). However, the AUROCs of the three scores were not significantly different in advanced fibrosis and cirrhosis. To exclude fibrosis, we calculated lower cutoffs than standard thresholds for Hepamet, FIB-4 and NFS with similar performances. CONCLUSION: Thresholds of non-invasive fibrosis scores (Hepamet, FIB-4 and NFS) can be modified to maximize diagnostic accuracy in Latin American patients with NAFLD.

Microbiota and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH).

Genetic predisposition, the intestinal microbiota (IM) and environmental factors, such as sedentary lifestyle and inadequate diet, should be considered as critical factors for the development of nonalcoholic fatty liver disease (NAFLD). Recently, some studies have demonstrated an association between dysbiosis and NAFLD; however, the exact mechanisms that lead to intestinal membrane damage, bacterial translocation and inflammation are not well elucidated. Due to the relevance of this theme, the IM and its metabolites have received special attention in recent years in an attempt to better understand the mechanisms related to the prevention, physiopathology, and treatment of NAFLD. In this paper, we provide a review of the human IM and its role in diet, obesity, and the development/progression of NAFLD/NASH, as well as the use of prebiotics and probiotics in the modulation of IM.

Validation of PNPLA3 polymorphisms as risk factor for NAFLD and liver fibrosis in an admixed population.

INTRODUCTION AND AIM: Studies have shown that two polymorphisms were associated with steatosis and progression of non-alcoholic fatty liver disease (NAFLD) in different populations: the Patatin-like Phospholipase Domain Containing 3 (PNPLA3) and Transmembrane 6 Superfamily Member 2 (TM6SF2). However, the frequency and significance of these polymorphisms in an admixed population, i.e., Brazilian, is unknown. Therefore, we aimed to evaluate them in healthy subjects in comparison to patients with NAFLD. MATERIAL AND METHODS: This was a multicenter cross-sectional study in 248 patients with biopsy-proven NAFLD and in 134 healthy controls from two tertiary centers in Brazil. PNPLA3 (rs738409 c.444C>G) and TM6SF2 (rs58542926 c.449C>T) polymorphisms were evaluated. RESULTS: In controls, the frequencies of PNPLA3 CC and CG+GG were 49.25% and 50.74%, respectively; in NAFLD patients, this was 31.05% and 68.88% (p=0.0044, 95% CI 1.037-2.977). PNPLA3 GG subjects had an increased risk (3.29-fold) of having NAFLD when compared to CC subjects (p=0.0044, 95% CI 1.504-7.225). In patients with nonalcoholic steatohepatitis (NASH), PNPLA3 GG compared to CC was associated with higher AST levels [38.4±25.3 versus 36.7±40.1IU/L, p=0.0395)] and with the presence of liver fibrosis (≥F2 fibrosis, p=0.0272). TM6SF2 polymorphisms were not in Hardy-Weinberg equilibrium in our NAFLD group precluding further analysis. CONCLUSION: We demonstrated for the first time that PNPLA3 CG+GG increase the risk of NAFLD among Brazilian subjects. Moreover, PNPLA3 GG was associated with liver enzyme elevation and fibrosis in NASH patients.

Association between the CYBA and NOX4 genes of NADPH oxidase and its relationship with metabolic syndrome in non-alcoholic fatty liver disease in Brazilian population.

BACKGROUND: Oxidative stress has been implicated in the progression of severe forms of non-alcoholic fatty liver disease (NAFLD). NADPH oxidase produces reactive oxygen species. In the present study, we investigated for the first time two single nucleotide polymorphisms (SNPs) in the regulatory region of genes encoding NADPH oxidase 4 (NOX4) and p22phox (CYBA) in NAFLD. METHODS: A total of 207 biopsy-proven NAFLD patients [simple steatosis (n = 27); nonalcoholic steatohepatitis (NASH) (n = 180)] were evaluated. Genomic DNA was extracted from peripheral blood cells, and polymorphisms in CYBA (unregistered) and NOX4 (rs3017887) were determined by direct sequencing of PCR. RESULTS: Associations of CYBA-675 T/A with high-density lipoprotein (HDL) (TT vs TA vs AA; P < 0.01) and triglycerides (TGL) (TT vs XA; P < 0.01) were observed only in NASH patients. For polymorphisms in the NOX4 gene, NOX4 (rs3017887) CA + AA genotypes was significant associated with alanine aminotransferase (ALT) (CA + AA vs CC; P = 0.02). However, there was no association of SNPs in the CYBA and NOX4 genes encoding the NADPH oxidase system proteins and the presence of NASH. Regarding the clinical results, it was observed that the most advanced degrees of fibrosis occurred in patients diagnosed with type 2 diabetes mellitus (66.9% vs 37.5%, P < 0.01) and those who were more obese (32.2 vs 29.0 kg/m2, P < 0.01). In addition, serum glucose and insulin levels increased significantly in the presence of NASH. CONCLUSIONS: There were associations between the presence of the allele A in the NOX4 SNP and a higher concentration of ALT in the NAFLD population; between the presence of the AA genotype in the polymorphism of the CYBA-675 T/A CYBA gene and a higher level of TGL and lower HDL in NASH patients. The presence of metabolic syndrome was associated with advanced degrees of fibrosis in NAFLD patients.

Age as a Confounding Factor for the Accurate Non-Invasive Diagnosis of Advanced NAFLD Fibrosis.

OBJECTIVES: Non-invasive fibrosis scores are widely used to identify/exclude advanced fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). However, these scores were principally developed and validated in patients aged between 35 and 65 years of age. The objective of this study was to assess the effect of age on the performance of non-invasive fibrosis tests in NAFLD. METHODS: Patients were recruited from European specialist hepatology clinics. The cohort was divided into five age-based groups: ≤35 (n=74), 36-45 (n=96), 46-55 (n=197), 56-64 (n=191), and ≥65 years (n=76), and the performance of the aspartate aminotransferase (AST)/alanine transaminase (ALT) ratio, fibrosis 4 (FIB-4), and NAFLD fibrosis score (NFS) for advanced fibrosis (stage F3-F4) for each group was assessed using liver biopsy as the standard. RESULTS: Six hundred and thirty-four patients were included. The diagnostic accuracy of the AST/ALT ratio was lower than NFS and FIB-4 in all the age groups. The AST/ALT ratio, NFS, and FIB-4 score performed poorly for a diagnosis of advanced fibrosis in those aged ≤35 years (area under the receiver operating characteristic curves (AUROCs 0.52, 0.52, and 0.60, respectively). For all groups >35 years, AUROCs for advanced fibrosis were similar for the NFS and FIB-4 score (range 0.77-0.84). However, the specificity for advanced fibrosis using the FIB-4 and NFS declined with age, becoming unacceptably low in those aged ≥65 years (35% for FIB-4 and 20% for NFS). New cutoffs were derived (and validated) for those aged ≥65 years, which improved specificity to 70% without adversely affecting sensitivity (FIB-4 2.0, sensitivity 77%; NFS 0.12, sensitivity 80%). CONCLUSIONS: The NFS and FIB-4 scores have similar accuracy for advanced fibrosis in patients aged >35 years. However, the specificity for advanced fibrosis is unacceptably low in patients aged ≥65 years, resulting in a high false positive rate. New thresholds for use in patients aged ≥65 years are proposed to address this issue.

NLRP3 inflammasome as a target of berberine in experimental murine liver injury: interference with P2X7 signalling.

Berberine (BRB) is commonly used in herbal medicine, but its mechanisms of action are poorly understood. In the present study, we tested BRB in steatohepatitis induced by a methionine- and choline-deficient (MCD) diet, in acute acetaminophen intoxication and in cultured murine macrophages. BRB markedly improved parameters of liver injury and necroinflammation induced by the MCD diet, although increased mortality was observed by mechanisms independent of bacterial infections or plasma levels of BRB. The MCD diet induced up-regulation of all components of the NLRP3 (NACHT, LRR and PYD domain-containing protein 3) inflammasome, and increased hepatic levels of mature IL-1ß (interleukin 1ß). All of these parameters were significantly reduced in mice treated with BRB. In mice administered an acetaminophen overdose, a model dependent on inflammasome activation, BRB reduced mortality and ALT (alanine aminotransferase) elevation, and limited the expression of inflammasome components. In vitro, LPS (lipopolysaccharide)-induced activation of NLRP3 inflammasome in RAW264.7 murine macrophages was markedly decreased by pre-incubation with BRB. BRB significantly limited the activation of the purinergic receptor P2X7, involved in the late phases of inflammasome activation. Upon P2X7 knockdown, the ability of BRB to block LPS-induced secretion of IL-1ß was lost. These data indicate that administration of BRB ameliorates inflammation and injury in two unrelated murine models of liver damage. We demonstrate for the first time that BRB interferes with activation of the NLRP3 inflammasome pathway in vivo and in vitro, through a mechanism based on interference with activation of P2X7, a purinergic receptor involved in inflammasome activation.