Noreen Hossain

Independent Predictors of Fibrosis in Patients With Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. We investigated factors associated with advanced fibrosis in NAFLD. METHODS The study included 432 patients with histologically proven NAFLD (26.8% with nonalcoholic steatohepatitis [NASH] and 17.4% with moderate-to severe fibrosis). NASH was defined as steatosis, lobular inflammation, and ballooning degeneration with or without Mallory-Denk bodies and/or fibrosis. Fibrosis was classified into 2 groups: those with no or minimal fibrosis and those with moderate-to-severe fibrosis. Groups were compared using Mann-Whitney and chi-square method analyses. A model was constructed using a stepwise bidirectional method; its predictive power was measured using a 10-fold cross-validation technique. RESULTS Patients with NASH were more likely to be male (P < .0001); have lower hip-to-waist ratios (P = .03); were less likely to be African American (P = .06); have higher levels of alanine aminotransferase (ALT; P < .0001), aspartate aminotransferase (AST; P < .0001), and serum triglycerides (P = .0154), but lower levels of high-density lipoprotein cholesterol (P < .0001). Patients with moderate-to-severe fibrosis were older (P = .0245); more likely to be male (P = .0189), Caucasian (P = .0382), have diabetes mellitus (P = .0238), and hypertension (P = .0375); and have a lower hip-to-waist ratio (P = .0077) but higher serum AST (P < .0001) and ALT (P < .0001) levels. The multivariate analysis model to predict moderate-to-severe fibrosis included male sex, Caucasian ethnicity, diabetes mellitus, and increased AST and ALT levels (model P value < .0001). CONCLUSIONS In patients with NAFLD, diabetes mellitus and aminotransferase levels are independent predictors of moderate-to-severe fibrosis. They can be used to identify NAFLD patients at risk for advanced fibrosis.

A Biomarker Panel for Non-alcoholic Steatohepatitis (NASH) and NASH-Related Fibrosis

BackgroundPatients with biopsy-proven NASH and especially those with fibrosis are at risk for progressive liver disease, emphasizing the clinical importance of developing non-invasive biomarkers for NASH and NASH-related fibrosis.AimThis study examines the performance of a new biomarker panel for NASH and NASH-related fibrosis with a combination of clinical and laboratory variables.MethodsEnrolled patients had biopsy-proven NAFLD. Clinical data, laboratory data, and serum samples were collected at the time of biopsy. Fasting serum was assayed for adiponectin, resistin, glucose, M30, M65, Tissue inhibitor of metalloproteinases-1 (Timp-1), ProCollagen 3 N-terminal peptide (PIIINP), and hyaluronic acid (HA). Regression models predictive of NASH, NASH-related fibrosis, and NASH-related advanced fibrosis were designed and cross-validated.ResultsOf the 79 enrolled NAFLD patients, 40 had biopsy-proven NASH and 39 had non-NASH NAFLD. Clinical and laboratory data were from this cohort were used to develop a NAFLD Diagnostic Panel that includes three models (models for NASH, NASH-related fibrosis, and NASH-related advanced fibrosis). The model for predicting NASH includes diabetes, gender, BMI, triglycerides, M30 (apoptosis), and M65–M30 (necrosis) [AUC: 0.81, 95% CI, 0.70–0.89, 300 p value <9E 301 −06]. The NASH-related fibrosis prediction model includes the same predictors [AUC: 0.80, 95% CI 0.68–0.88, 307 p value <0.00014]. Finally, the NASH-related advanced fibrosis model includes type 2 diabetes, serum triglycerides, Timp-1, and AST [AUC: 0.81, 95% CI, 0.70–0.89; p value, 0.000062].ConclusionsThis NAFLD Diagnostic Panel based on a clinical and laboratory data has good performance characteristics and is easy to use. This biomarker panel could become useful in the management of patients with NAFLD.

Differential expression of miRNAs in the visceral adipose tissue of patients with non‐alcoholic fatty liver disease

Aliment Pharmacol Ther 2010; 32: 487–497

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

BackgroundGiven the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals.ResultsCross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes.ConclusionsWe recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

Expression of Cytokine Signaling Genes in Morbidly Obese Patients with Non-Alcoholic Steatohepatitis and Hepatic Fibrosis

BackgroundWhite adipose tissue (WAT) from visceral adiposity plays an important role in the pathogenesis of non-alcoholic steatohepatitis (NASH). Development of NASH and its progression to fibrosis is partially due to cytokines and adipokines produced by WAT. The aim of this study was to assess the association of hepatic fibrosis and NASH by evaluating the intrinsic differences in the inflammatory cytokine signaling in the visceral adipose tissue obtained from morbidly obese patients.MethodsWe used targeted microarrays representing human genes involved in the inflammatory and fibrogenic reactions to profile visceral adipose samples of 15 well-matched NASH patients with and without fibrosis. Additionally, visceral adipose samples were subjected to real-time polymerase chain reaction profiling of 84 inflammations related genes.ResultsEight genes (CCL2, CCL4, CCL18, CCR1, IL10RB, IL15RA, and LTB) were differentially expressed in NASH with fibrosis. Additionally, an overlapping but distinct list of the differentially expressed genes were found in NASH with type II diabetes (DM; IL8, BLR1, IL2RA, CD40LG, IL1RN, IL15RA, and CCL4) as compared to NASH without DM.ConclusionsInflammatory cytokines are differentially expressed in the adipose tissue of NASH with fibrosis, as well in NASH with DM. These findings point at the interaction of adipose inflammatory cytokines, DM, hepatic fibrosis in NASH, and its progression to cirrhosis and end-stage liver disease.

Non-alcoholic steatohepatitis (NASH) in patients with polycystic ovarian syndrome (PCOS)

Abstract Background. Both non-alcoholic steatohepatitis (NASH) and polycystic ovary syndrome (PCOS) are associated with metabolic syndrome (MS) and insulin resistance (IR). Except for a few case reports, there are no systematic assessments of NASH in PCOS patients. Aim. To determine the prevalence of NASH and independent factors associated with NASH in a cohort of patients with documented PCOS. Methods. Patients with established diagnosis of PCOS and matched controls (matched for gender, age, and body mass index (BMI)) were included in the study. Causes of other liver diseases were systematically excluded by clinical and laboratory tests. Excessive alcohol use was defined as alcohol consumption of greater than 10 g/day. All liver biopsies were read by a single pathologist blinded to the clinical data. Histologic NASH was defined as steatosis with lobular inflammation and ballooning degeneration of hepatocytes with or without Mallory–Denk bodies or pericellular fibrosis. Univariate and multivariate analyses with logistic regression were performed to compare PCOS to matched controls. Results. Sixty-six patients were included in the study (34 PCOS and 32 matched controls). Of PCOS patients, 73% had a liver biopsy while 78% of the matched controls had a liver biopsy. In comparing PCOS patients to the matched controls, clinical (BMI, waist circumference, type 2 diabetes, MS, or its components, any alcohol consumption in the prior year, ethnic background, age, gender, etc.) and laboratory data (aminotransferases, ferritin, glucose, etc.) were not significantly different (p > 0.05). However, PCOS patients tended to have more histologic NASH on their liver biopsies (44.0% vs. 20.8%, p = 0.08). Independent predictors of histologic NASH in PCOS patients were elevated aspartate aminotransferase (AST), high triglycerides and small amounts of alcohol consumption (p = 0.019, 10-fold cross-validated AUC = 0.80, 95% CI = 0.56–0.94). Although about half of PCOS patients did not report any alcohol consumption, 50% did report rare alcohol use. In fact, PCOS patients with histologic NASH tended to report higher alcohol consumption per week than PCOS without NASH (3.80 ± 6.16 vs. 1.11 ± 1.87 g/week, p = 0.1). Nevertheless, these amounts of alcohol consumption were quite minimal. Conclusions. Despite similar clinical and laboratory profiles to the matched controls, PCOS patients seem to have more histologic NASH. Although alcohol consumption was rare for both PCOS and controls, even rare alcohol consumption in PCOS patients was independently associated with histologic NASH.

Hepatic Gene Expression of Caucasian and African-American Patients with Obesity-Related Non-Alcoholic Fatty Liver Disease

Background/aimThere is increasing data suggesting that African Americans with NAFLD tend to have less progressive liver disease. The aim of this study is to assess differences in the hepatic gene expression of African-American and Caucasian patients with NAFLD who had undergone bariatric surgery.MethodsA total of 94 patients (81 NAFLD and 13 weight-matched controls with normal liver biopsy) were included. Of the entire cohort, 73 were Caucasians and 21 were African Americans. All patients were undergoing bariatric surgery. Two liver biopsies were obtained at the time of surgery. One biopsy was snap-frozen for gene expression and the other biopsy was stained for pathologic assessment. Liver biopsy confirmed that 24 patients from our cohort had NASH while 57 had only simple steatosis. Snap-frozen liver biopsy specimens of these patients were then used for the RNA extraction. cDNA probes were hybridized with customized microarray gene chips containing 5,220 relevant genes. Gene expression profiles were compared between groups using significance analysis of microarrays algorithm.ResultsIn comparison to all Caucasian patients, African-American patients had over-expression of EPB41L1, IGF2, FAH, ACSL4, FUT4, CYP3A (q values < 10−4). In comparison to Caucasian NAFLD patients, African-American NAFLD patients showed over-expression of EPB41L1 and ACSL4 genes. Finally, in comparison to Caucasian NASH patients, African-American NASH patients showed over-expression of GSTM 2, GSTM4 and GSTM5 as well as FH and ASCL4 genes. Some genes highlighted by this analysis, particularly cytochrome CYP3A and glutathione transferases GSTM2, 4, 5, were previously implicated in the pathogenesis of NASH.ConclusionAfrican-American patients with biopsy-proven obesity-related NAFLD and NASH have a specific hepatic gene expression pattern that may explain their differences from Caucasian patients with NAFLD in developing progressive liver disease.

Gene expression profile associated with superimposed non-alcoholic fatty liver disease and hepatic fibrosis in patients with chronic hepatitis C

Background: Hepatic steatosis occurs in 40–70% of patients chronically infected with hepatitis C virus [chronic hepatitis C (CH‐C)]. Hepatic steatosis in CH‐C is associated with progressive liver disease and a low response rate to antiviral therapy.

Expression Level of IL20, mTOR, IL13RA1, and SEMA4C Are Increased in the Visceral Adipose Tissue of Patients With Non-Alcoholic Steathepatitis While There is a Decrease in the Expression Levels of Their Regulatory Mirnas

fully understood. We hypothesized that probiotics could strengthen barrier function in an animal model of alcoholic liver disease (ALD) by activating hypoxia-inducible factor (HIF) leading to an increase in barrier-protecting factors, such as intestinal trefoil factor (ITF), vascular endothelial growth factor (VEGF), and CD73, and thus prevent endotoxemia and subsequently ALD. Male C57/6J mice were fed Lieber DiCarli diet containing 5% ethanol for 8 weeks. In last two weeks, 1 x 109 cfu/daily live Lactobacillus GG (LGG) was added to the diet. Serum endotoxin and alanine aminotransferase were measured. Intestinal and liver tissues were analyzed for ITF, VEGF and CD73. In addition, liver steatosis was assessed by histology. The mice treated with LGG had significantly less hepatic steatosis, serum ALT and lipopolysaccharide levels, and less intestinal barrier damage than alcohol-fed mice. ITF, VEGF and CD73 were deceased in the intestine of mice treated with alcohol, and LGG attenuated this decrease. In Vitro studies revealed that treatment with 5% alcohol decreased transepithelial resistance and increased in FITC-dextran permeability in Caco-2 cells. Supplementation of LGG cultural supernatant (LGGs) attenuated these deleterious effects in a timedependent manner. LGGs also prevented alcohol-induced reduction in ITF, VEGF, and CD73 which are known HIF-1 targets. These results indicate that LGG attenuating the alcohol-induced liver injury is likely, at least in part, by activating HIF signaling leading to intestinal mucosal barrier protection.