Objective: Liver biopsy is still needed for fibrosis staging in many patients with non-alcoholic fatty liver disease. The aims of this study were to evaluate the individual diagnostic performance of liver stiffness measurement by vibration controlled transient elastography (LSM-VCTE), Fibrosis-4 Index (FIB-4) and NAFLD (non-alcoholic fatty liver disease) Fibrosis Score (NFS) and to derive diagnostic strategies that could reduce the need for liver biopsies. Design: Individual patient data meta-analysis of studies evaluating LSM-VCTE against liver histology was conducted. FIB-4 and NFS were computed where possible. Sensitivity, specificity and area under the receiver operating curve (AUROC) were calculated. Biomarkers were assessed individually and in sequential combinations. Results: Data were included from 37 primary studies (n=5735; 45% women; median age: 54 years; median body mass index: 30 kg/m²; 33% had type 2 diabetes; 30% had advanced fibrosis). AUROCs of individual LSM-VCTE, FIB-4 and NFS for advanced fibrosis were 0.85, 0.76 and 0.73. Sequential combination of FIB-4 cut-offs (<1.3; ≥2.67) followed by LSM-VCTE cut-offs (<8.0; ≥10.0 kPa) to rule-in or rule-out advanced fibrosis had sensitivity and specificity (95% CI) of 66% (63–68) and 86% (84–87) with 33% needing a biopsy to establish a final diagnosis. FIB-4 cut-offs (<1.3; ≥3.48) followed by LSM cut-offs (<8.0; ≥20.0 kPa) to rule out advanced fibrosis or rule in cirrhosis had a sensitivity of 38% (37–39) and specificity of 90% (89–91) with 19% needing biopsy. Conclusion: Sequential combinations of markers with a lower cut-off to rule-out advanced fibrosis and a higher cut-off to rule-in cirrhosis can reduce the need for liver biopsies.

Background: Intestinal microbiota and their metabolites (e.g. short‐chain fatty acids (SCFAs)) may influence nonalcoholic fatty liver disease (NAFLD).Objective: The objective of this article is to analyze gut bacterial diversity together with fecal SCFA concentrations and immunophenotyping of peripheral blood in histology‐proven NAFLD patients.Methods: Thirty‐two NAFLD patients (14 nonalcoholic fatty liver (NAFL), 18 nonalcoholic steatohepatitis (NASH)) and 27 healthy controls (HCs)) were included in this study. Bacterial communities in feces were profiled by 16S ribosomal RNA gene sequencing of the V3–V4 region. Fecal SCFA levels were analyzed by high‐performance liquid chromatography. Fluorescence‐activated cell sorting analysis was performed of peripheral blood mononuclear cells.Results: NASH patients were characterized by higher abundance of Fusobacteria and Fusobacteriaceae compared to NAFL and HCs. Conforming to our finding that NAFLD patients had higher fecal acetate and propionate levels, taxonomical differences of fecal bacteria were dominated by SCFA‐producing bacteria. Higher fecal propionate and acetate levels were associated with lower resting regulatory T‐cells (rTregs) (CD4+CD45RA+CD25++) as well as higher Th17/rTreg ratio in peripheral blood as immunological characteristics of NASH patients.Conclusions: NASH patients are characterized by a different gut microbiome composition with higher fecal SCFA levels and higher abundance of SCFA‐producing bacteria in NAFLD. These changes are associated with immunological features of disease progression. Our data suggest an important role of the intestinal microbiome and immunomodulatory bacterial metabolites in human NAFLD.

Nonalcoholic fatty liver disease is increasing in prevalence. It can be subdivided into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). Five to twenty percent of cases progress from NAFL to NASH. Increased hepatic Th17 cells and IL-17 expression were observed in NASH mice and patients, respectively. We analyzed CD4⁺ effector T cells and regulatory T cells (Tregs) from peripheral blood and livers of NAFL and NASH patients. A total of 51 NAFL patients, 30 NASH patients, 31 nonalcoholic fatty liver disease patients (without histology), and 43 healthy controls were included. FACS analysis was performed on PBMCs and intrahepatic lymphocytes. Compared with healthy controls, a lower frequency of resting Tregs (rTregs; CD4⁺CD45RA⁺CD25⁺⁺) and higher frequencies of IFN-γ⁺ and/or IL-4⁺ cells were detected among CD4⁺ T cells of peripheral blood in NASH, and to a lesser degree in NAFL. In hepatic tissue, NAFL to NASH progression was marked by an increase in IL-17⁺ cells among intrahepatic CD4⁺ T cells. To define immunological parameters in peripheral blood to distinguish NAFL from NASH, we calculated different ratios. Th17/rTreg and Th2/rTreg ratios were significantly increased in NASH versus NAFL. The relevance of our findings for NASH pathogenesis was highlighted by the normalization of all of the changes 1 y after bariatric surgery. In conclusion, our data indicate that NAFL patients show changes in their immune cell profile compared with healthy controls. NAFL to NASH progression is marked by an increased frequency of IL-17⁺ cells among intrahepatic CD4⁺ T cells and higher Th17/rTreg and Th2/rTreg ratios in peripheral blood.

Marcin Krawczyk, Monika Rau, Jörn M. Schattenberg, Heike Bantel, Anita Pathil, Münevver Demir, Johannes Kluwe, Tobias Boettler, Frank Lammert, Andreas Geier, for the NAFLD Clinical Study Group

Liver biopsies are the current gold standard in non-alcoholic steatohepatitis (NASH) diagnosis. Their invasive nature, however, still carries an increased risk for patients’ health. The development of non-invasive diagnostic tools to differentiate between bland steatosis (NAFL) and NASH remains crucial. The aim of this study is the evaluation of investigated circulating microRNAs in combination with new targets in order to optimize the discrimination of NASH patients by non-invasive serum biomarkers. Serum profiles of four microRNAs were evaluated in two cohorts consisting of 137 NAFLD patients and 61 healthy controls. In a binary logistic regression model microRNAs of relevance were detected. Correlation of microRNA appearance with known biomarkers like ALT and CK18-Asp396 was evaluated. A simplified scoring model was developed, combining the levels of microRNA in circulation and CK18-Asp396 fragments. Receiver operating characteristics were used to evaluate the potential of discriminating NASH. The new finding of our study is the different profile of circulating miR-21 in NASH patients (p<0.0001). Also, it validates recently published results of miR-122 and miR-192 to be differentially regulated in NAFL and NASH. Combined microRNA expression profiles with CK18-Asp396 fragment level scoring model had a higher potential of NASH prediction compared to other risk biomarkers (AUROC = 0.83, 95% CI = 0.754–0.908; p<0.001). Evaluation of score model for NAFL (Score = 0) and NASH (Score = 4) had shown high rates of sensitivity (91%) and specificity (83%). Our study defines candidates for a combined model of miRNAs and CK18-Asp396 levels relevant as a promising expansion for diagnosis and in turn treatment of NASH.

Previously, we identified needle-like and filamentous, putatively “anhydrous” cholesterol crystallization in vitro at very low phospholipid concentrations in model and native biles. Our aim now was to address whether spontaneous gallstone formation occurs in Mdr2 (Abcb4) knockout mice that are characterized by phospholipid-deficient bile. Biliary phenotypes and cholesterol crystallization sequences in fresh gallbladder biles and non-fixed liver sections were determined by direct and polarizing light microscopy. The physical chemical nature and composition of crystals and stones were determined by sucrose density centrifugation and before mass and infrared spectroscopy. Gallbladder biles of Mdr2 ^−/− mice precipitate needle-like cholesterol crystals at 12 weeks of age on chow. After 15 weeks, more than 50% of Mdr2 ^−/− mice develop gallbladder stones, with female mice displaying a markedly higher gallstone-susceptibility. Although gallbladder biles of Mdr2 ^−/− mice contain only traces (≤ 1.1 mM) of phospholipid and cholesterol, they become supersaturated with cholesterol and plot in the left 2-phase zone of the ternary phase diagram, consistent with “anhydrous” cholesterol crystallization. Furthermore, more than 40% of adult female Mdr2 ^−/− mice show intra- and extrahepatic bile duct stones. In conclusion, spontaneous gallstone formation is a new consistent feature of the Mdr2 ^−/− phenotype. The Mdr2 ^−/− mouse is therefore a model for low phospholipid-associated cholelithiasis recently described in humans with a dysfunctional mutation in the orthologous ABCB4 gene. The mouse model supports the concept that this gene is a monogenic risk factor for cholesterol gallstones and a target for novel therapeutic strategies. (Hepatology 2004;39:117-128.)