Biopsy-proven NAFLD Research Articles

Obstructive Sleep Apnea Is Associated with Liver Damage and Atherosclerosis in Patients with Non-Alcoholic Fatty Liver Disease.

Background/AimsWe assessed whether obstructive sleep apnea (OSA) and nocturnal hypoxemia are associated with severity of liver fibrosis and carotid atherosclerosis in patients with biopsy-proven NAFLD and low prevalence of morbid obesity. Secondary aim was to explore the association of OSA and hypoxemia with NASH and severity of liver pathological changes.MethodsConsecutive patients (n = 126) with chronically elevated ALT and NAFLD underwent STOP-BANG questionnaire to estimate OSA risk and ultrasonographic carotid assessment. In patients accepting to perform cardiorespiratory polygraphy (PG, n = 50), OSA was defined as an apnea/hypopnea index ≥5. A carotid atherosclerotic plaque was defined as a focal thickening >1.3 mm.ResultsPrevalence of high OSA risk was similar in patients refusing or accepting PG (76% vs 68%, p = 0.17). Among those accepting PG, overall OSA prevalence was significantly higher in patients with F2-F4 fibrosis compared to those without (72% vs 44%; p = 0.04). Significant fibrosis was independently associated with mean nocturnal oxygen saturation (SaO2)<95% (OR 3.21, 95%C.I. 1.02–7.34; p = 0.04). Prevalence of OSA tended to be higher in patients with, than in those without, carotid plaques (64% vs 40%; p = 0.08). Carotid plaques were independently associated with %time at SaO2<90% >1 (OR 6.30, 95%C.I. 1.02–12.3; p = 0.01).ConclusionsIn NAFLD patients with chronically elevated ALT at low prevalence of morbid obesity, OSA was highly prevalent and indexes of SaO2 resulted independently associated with severity of liver fibrosis and carotid atherosclerosis. These data suggest to consider sleep disordered breathing as a potential additional therapeutic target in severe NAFLD patients.

PWE-120 High rate of false positives for advanced fibrosis when simple non-invasive fibrosis tests are used in older patients (≥ 65 years) with nafld

<h3>Introduction</h3> Simple non-invasive fibrosis scores, such as the FIB-4 and NAFLD fibrosis scores (NFS), are now widely used to identify/exclude advanced fibrosis in patients with NAFLD. Use of these scores in primary care provides a simple objective method to identify patients who need further investigation for advanced fibrosis. Few older patients (≥ 65 years) were included in previous studies, but older patients represent a large proportion of patients with NAFLD in the community. The aim of this study was to assess the performance of simple non-invasive fibrosis tests in older patients (≥65 years old) with biopsy-proven NAFLD. <h3>Method</h3> Patients with biopsy proven NAFLD were recruited from 4 specialist fatty liver clinics. The diagnostic performance of the AST/ALT ratio, FIB-4 and NFS were assessed using liver biopsy as the standard. <h3>Results</h3> From a total of 634 patients, 76 were ≥65 years (mean age 69 ± 3 years, 35% male, 58% diabetic). 60 (79%) of the older patients had steatohepatitis and 30 (39%) had advanced fibrosis (stage 3–4). In older patients the AUROCs for a diagnosis of advanced fibrosis were 0.73, 0.81 and 0.81 for the AST/ALT ratio, FIB-4 and NFS respectively, which was similar to younger patients (0.69, 0.80 and 0.82 respectively). However the specificity for advanced fibrosis using the FIB-4 and NFS was much lower in the older patients than younger patients (FIB-4 35% vs 79%; NFS 20% vs 66%), leading to a high false positive rate. New cut-offs for older patients were derived to improve the specificity to 70% and did not adversely affect the sensitivity (FIB-4 2.0, sensitivity 77%; NFS 0.12, sensitivity 80%). <h3>Conclusion</h3> The diagnostic accuracy for advanced fibrosis using the NFS and FIB-4 score was similar in older and younger patients. However, the specificity for advanced fibrosis was low in older patients (≥ 65 years), resulting in a high false positive rate, which may result in patients undergoing unnecessary further investigations. Adoption of our new cut-offs could reduce the proportion of older patients being referred from primary care for further investigation. <h3>Disclosure of interest</h3> None Declared.

Nonalcoholic fatty liver disease is an independent risk factor for atherosclerosis in young adult men

IntroductionThe possible cause of accelerated atherosclerosis in NAFLD may be the relationship with the MetS and its components. Our primary goal was to evaluate the relationship between NAFLD and subclinical atherosclerosis in adult male patients between 20 and 40 years of age. Moreover, we aimed to investigate the changes in this association according to the presence or absence of MetS. MethodSixty-one male patients with biopsy-proven NAFLD and 41 healthy male volunteers were enrolled. In order to exclude any interference of confounding factors, we studied a specifically selected group with no additional cardiovascular risk. PWV, CIMT and FMD levels were measured in all patients and controls. ResultsThe levels of cf-PWV were significantly higher in SS and NASH patients compared to the control group (P < 0.001); no significant difference was found between SS and NASH patients (P > 0.05). We found significantly decreased FMD levels in patients with SS and NASH compared with control subjects (P < 0.001). Subjects with NASH had significantly greater CIMT measurements than the SS and controls (P = 0.026, P < 0.001, respectively). Although, NAFLD patients with MetS had increased cf-PWV and CIMT and reduced FMD compared to healthy subjects (P < 0.05), no significant difference existed between NAFLD with Mets and NAFLD without MetS in terms of cf-PWV, CIMT and FMD (P > 0.05) ConclusionThe present study showed that the presence of NAFLD leads to increased risk of endothelial dysfunction and atherosclerosis in adult male patients, independent of MetS.

Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease.

BackgroundChronic liver injury can lead to the development of liver fibrosis and cirrhosis but only in a minority of patients. Currently, it is not clear which factors determine progression to fibrosis. We investigated whether DNA\\methylation profile as determined by pyrosequencing can distinguish patients with mild from those with advanced/severe fibrosis in non-alcoholic liver disease (NAFLD) and alcoholic liver disease (ALD). To this end, paraffin-embedded liver biopsies were collected from patients with biopsy-proven NAFLD or ALD, as well as paraffin-embedded normal liver resections, genomic DNA isolated, bisulfite converted and pyrosequencing assays used to quantify DNA methylation at specific CpGs within PPARα, PPARα, TGFβ1, Collagen 1A1 and PDGFα genes. Furthermore, we assessed the impact of age, gender and anatomical location within the liver on patterns of DNA methylation in the same panel of genes.ResultsDNA methylation at specific CpGs within genes known to affect fibrogenesis distinguishes between patients with mild from those with severe fibrosis in both NAFLD and ALD, although same CpGs are not equally represented in both etiologies. In normal liver, age, gender or anatomical location had no significant impact on DNA methylation patterns in the liver.ConclusionsDNA methylation status at specific CpGs may be useful as part of a wider set of patient data for predicting progression to liver fibrosis.

Quantification of liver, pancreas, kidney, and vertebral body MRI-PDFF in non-alcoholic fatty liver disease.

The purpose of the present study was to determine liver, pancreas, kidney, and vertebral fat deposition in NAFLD patients by proton density fat fraction (PDFF) using magnetic resonance imaging (MRI) and to evaluate the relationships among them. A total of 41 biopsy-proven NAFLD patients underwent MRI-PDFF with IDEAL-IQ. MRI protocol included T1-independent volumetric multi-echo gradient-echo imaging with T2* correction and spectral fat modeling. The MR examinations were performed on a 1.5 HDx MRI system. MRI-PDFF measurements were obtained from liver, pancreas, renal cortex and sinus, and vertebral body. Liver biopsy specimens were retrieved from the archives and evaluated by one pathologist according to NASH CRN. The median age of the patients was 47 years. The median interval between liver biopsy and MRI examination was 16 days. Mean liver, pancreas, renal cortex, renal sinus, T12 and L1 vertebral body MRI-PDFFs were 18.7%, 5.7%, 1.7%, 51%, 43.2%, and 43.5%, respectively. No correlation between either liver MRI-PDFF or histological steatosis, and other organ MRI-PDFFs was observed. A good correlation between pancreas and vertebral body MRI-PDFFs, and pancreas and renal sinus MRI-PDFFs was observed. Diabetic patients had higher average pancreas MRI-PDFF compared to non-diabetics (12.2%, vs., 4.8%; P = 0.028). Pancreas and vertebral body MRI-PDFF is well correlated in NAFLD patients and both of them are higher in diabetic patients which may explain increased bone fractures in diabetics. MRI-PDFF can be used to demonstrate fat fractions of different organs and tissues and to understand fat metabolism.

HLA Class II Alleles Are Associated With Histological Inflammatory Features of Steatohepatitis (NASH): Presidential Poster

Introduction: The genotype-phenotype interaction in chronic liver diseases is a subject of growing interest. Previous studies have linked liver fibrosis susceptibility to specific HLA alleles. In this study we aimed to examine potential associations between HLA class II alleles and histological characteristics and severity in patients with NAFLD/ NASH. Methods: DNA from biopsy-proven NAFLD patients was genotyped using polymerase chain reaction sequence-specific oligonucleotide method (PCR-SSO) for HLA class II antigens (HLA-DR1, -DR3, -DP &-DQ). Liver biopsies were assessed for hepatic steatosis, inflammation, and fibrosis. Multivariate analysis was performed to draw correlations between HLA antigen frequencies and the different variables; p≤0.05 were considered to be significant. Results: The study cohort include 85 patients with biopsy-proven NAFLD [age=44.25±10.78 years; female=68 (80.0%); white=59 (69.41%); NASH=35 (41.18%); diabetes mellitus=44 (22.4%); hyperlipidemia= 48 (56.47%) 75(38.9%), hypertension=46 (54.12%)]; diabetes=30 (35.29%); portal Inflammation=46 (54.12%), lobular Inflammation: [Kupffer cell hypertrophy=3 (3.8%%), inflammatory cells=18 (21.18%), polymorhnuclear cells=9 (10.59%)], [pericellular fibrosis=33 (38.82%), portal fibrosis=53 (62.35%); bridging fibrosis and cirrhosis=13 (15.29%)] and controls (N=55) without liver disease. DPB1*03 was found to be associated with patients with advanced steatosis [N=7 (31.82%) vs. non-steatosis N=6 (9.52%), p=0.01]. Interestingly DQA*05 was found to be associated with apoptosis and necrosis [N=25 (35.71%) vs. 1 (6.67%, p=0.02]. DRB3*01 was only found in patients with apoptotic or necrotic markers [N=16 (22.86%) vs. 0 (0.00%), p=0.03]. DQA1*03 was associated with higher risk of inflammation [n= 7 (53.85%) vs. 17 (25.76%), p=0.04] than those without inflammation. Inversely, DPA1*01 [n= 3 (33.33%) vs. 64 (84.21%), p<0.001], DPB1*04 [n= 0 (0.00%) vs. 47 (61.84%), p<0.001], DQB1*03 [n=2 (22.22%) vs. 49 (64.47%), p=0.01], and DRB4*01 [n= 2 (22.22%) vs. 47 (61.84%), p=0.04] were associated with lower risk of any type of inflammation. In multivariate analysis, DQA1*03 was independently associated with inflammation [odds ratio (OR): 6.12 (95% CI 1.39-27.73; p=0.01), while DPA1*01 was independently associated with lower risk of any type of inflammation (OR 0.08; 95% CI 0.01-0.61; p =0.01]. Conclusion: HLA-class II genes play important role in determining the disease mechanisms. DQA1*03 alleles play important role in predisposing to sever histological features of NASH among patients with NALFD. These HLA class II alleles that are risk haplotypes may help identify NAFLD patients with increased risk for developing inflammation and sever liver disease.

The development of the pediatric NAFLD fibrosis score (PNFS) to predict the presence of advanced fibrosis in children with nonalcoholic fatty liver disease.

BackgroundNoninvasive hepatic fibrosis scores that predict the presence of advanced fibrosis have been developed and validated in adult patients with NAFLD. The aims of our study were to assess the utility of commonly used adult fibrosis scores in pediatric NAFLD and to develop a pediatric specific fibrosis score that can predict advanced fibrosis.MethodsConsecutive children with biopsy-proven NAFLD were included. Fibrosis was determined by an experienced pathologist (F0–4). Advanced fibrosis was defined as fibrosis stage ≥3. The following adult fibrosis scores were calculated for each child: AST/ALT ratio, AST/platelet ratio index (APRI), NAFLD fibrosis score (NFS), and FIB-4 Index. Multivariable logistic regression analysis was performed to build a new pediatric model for predicting advanced fibrosis.ResultsOur cohort consisted of 242 children with a mean age of 12.4±3.1 years and 63% were female. 36 (15%) subjects had advanced fibrosis. APRI and FIB-4 were higher in patients with advanced fibrosis compared to those with fibrosis stage 0–2; however, AST/ALT ratio and NFS were not different between the two groups. We used our data to develop a new model to predict advanced fibrosis which included: ALT, alkaline phosphatase, platelet counts and GGT. The multivariable logistic regression model (z) was defined as follows: z = 1.1+(0.34*sqrt(ALT))+(0.002*alkaline phosphatase) – (1.1*log(platelets) – (0.02*GGT). This value was then converted into a probability distribution (p) with a value between 0 to 100 by the following formula: p = 100×exp(z)/[1+exp(z)]. The AUCROC for this model was 0.74 (95% CI: 0.66, 0.82). This was found to be significantly better than APRI, NAFLD Fibrosis Score and FIB-4 Index.ConclusionNoninvasive hepatic fibrosis scores developed in adults had poor performance in diagnosing advanced fibrosis in children with NAFLD. We developed a new pediatric NAFLD fibrosis score with improved performance characteristics.

40 The Development of the Pediatric NAFLD Fibrosis Score (PNFS) to Predict the Presence of Advanced Fibrosis in Children With Nonalcoholic Fatty Liver Disease

Background: Noninvasive hepatic fibrosis scores that predict the presence of advanced fibrosis have been developed and validated in adult patients with NAFLD. The aims of our study were to assess the utility of commonly used adult fibrosis scores in pediatric NAFLD and to develop a pediatric specific fibrosis score that can predict advanced fibrosis. Methods: Consecutive children with biopsy-proven NAFLD were included. Fibrosis was determined by an experienced pathologist (F0–4). Advanced fibrosis was defined as fibrosis stage $3. The following adult fibrosis scores were calculated for each child: AST/ALT ratio, AST/platelet ratio index (APRI), NAFLD fibrosis score (NFS), and FIB-4 Index. Multivariable logistic regression analysis was performed to build a new pediatric model for predicting advanced fibrosis. Results: Our cohort consisted of 242 children with a mean age of 12.463.1 years and 63% were female. 36 (15%) subjects had advanced fibrosis. APRI and FIB-4 were higher in patients with advanced fibrosis compared to those with fibrosis stage 0–2; however, AST/ALT ratio and NFS were not different between the two groups. We used our data to develop a new model to predict advanced fibrosis which included: ALT, alkaline phosphatase, platelet counts and GGT. The multivariable logistic regression model (z) was defined as follows: z=1.1+(0.34*sqrt(ALT))+(0.002*alkaline phosphatase) – (1.1*log(platelets) – (0.02*GGT). This value was then converted into a probability distribution (p) with a value between 0 to 100 by the following formula: p=1006exp(z)/[1+exp(z)]. The AUCROC for this model was 0.74 (95% CI: 0.66, 0.82). This was found to be significantly better than APRI, NAFLD Fibrosis Score and FIB-4 Index. Conclusion: Noninvasive hepatic fibrosis scores developed in adults had poor performance in diagnosing advanced fibrosis in children with NAFLD. We developed a new pediatric NAFLD fibrosis score with improved performance characteristics.

Sa1036 Hepatic Fibrosis After Small-for-Size Liver Transplantation in Rats: Possible Role of TGFβ/SMAD Signaling

this study, we explored whether concomitant medication altered the risk of hepatic fibrosis in NAFLD. Specifically, we sought to ascertain the association between RAS blocking agents (angiotensin converting enzyme blocker or angiotensin II receptor blocker) and severity of hepatic fibrosis in NAFLD patients with hypertension. Methods: This study cohort consisted of 259 hypertensive patients with biopsy proven NAFLD prospectively followed up in hepatology outpatient clinics in Cleveland, OH. Clinical data including demographics, anthropometry, medical history, medication history, biochemical and liver biopsy findings were evaluated. Patients with and without RAS blocker use were compared in relation to liver fibrosis stage. Utilizing electronic medical records, risk factors for advanced fibrosis were explored, in particular, the association between commonly used medications and advanced fibrosis. Independent risk factors for advanced fibrosis were ascertained using binary regression. Results: The mean age of the cohort was 52.2 years and mean BMI was 37.33 kg/m2. Females, diabetics and patients with advanced fibrosis comprised 64.9%, 60.2% and 35.9% of the cohort respectively. Patients treated with RAS blockers had significantly less ballooning (1.05 vs. 1.33, p=0.002) and lower fibrosis stage (1.64 vs. 2.24, p=0.001) on liver biopsy compared to patients not treated with RAS blockers. Compared to patients without advanced fibrosis, patients with advanced fibrosis were older (54.6 vs. 50.9 years, p=0.014), more often diabetic (76.3 vs. 50.9%, p=0.000), had more ballooning on histology (1.57 vs. 0.93, p=0.000), more likely to be on insulin (22.6 vs. 11.5%, p=0.018), and sulfonylureas (29.0 vs. 15.8%, p=0.011) while less likely to be on RAS blockers (49.5 vs. 70.3%, p=0.001). On multivariate analysis, grade of ballooning had an independent positive association with advanced fibrosis (OR 7.539; 95%CI 3.402-16.706, p=0.000) while usage of RAS blockers had a negative association with advanced fibrosis (OR 0.293; 95%CI 0.1090.787, p=0.015). Conclusion: Hypertensive patients with NAFLD treated with RAS blockers had less advanced hepatic fibrosis, suggesting a beneficial role for angiotensin blockers to protect against NASH fibrosis. Prospective clinical trials are warranted to explore this association in greater detail.

Mo1779 Identification of a Novel Stimulus-Dependent Phosphorylation in the NTerminal Region of Protein Kinase D1 (PKD1) in Intestinal Epithelial Cells

INTRODUCTION: With the rise of obesity, the role of brown adipose tissue (BAT) capable of eliminating lipids via thermogenesis, is garnering interest. Novel research has shown TGFB's influence in the browning of white adipose.AIM: To ascertain the expression levels of Brown Fat specific genes and the interaction of these genes with core regulatory genes such as TGFB, T-cell transcription factors, and cell cycle related genes such as KCNRG in the visceral adipose tissue (VAT) of morbidly obese NAFLD patients.METHODS: RNAs were extracted from frozen visceral fat samples of 84 patients with liver biopsy proven NAFLD using Bio-Rad's Aurum Total RNA Fatty and Fibrous Tissue Kit. RNA from each sample was converted to cDNA using SABiosciences' RT2 First Strand Kit. Custom primers were designed for all genes and were validated. These primers and the cDNA samples were added to 10 μL q-PCR reactions in a 96 well plate format. Co-expression patterns were analyzed using Spearman's rank-sum correlation. RESULTS: Visceral fat samples were obtained from 84 biopsy-proven NAFLD patients undergoing Bariatric surgery (age 42.62 ± 11.55 years, BMI 47.23 ± 9.99, 39.3% NASH, 67.9% female, AST 27.20 ± 20.25U/L, ALT 35.85 ± 29.18U/L). BAT specific genes UCP1 and PRDM16 both positively correlate with expression of inflammatory regulator TGFB1 (r = 0.35, p-value < 0.001 and r = 0.43, p-value < 7.6e05 respectively) in VAT and UCP1 also correlates with the receptor TGFBR1 (r = 0.28, pvalue < 0.009). The data also show a positive correlation between UCP1and the cell cycle gene KCNRG v2 (r = 0.25, p-value < 0.02), as well as the T-cell transcription factors T-bet (r=0.23, p-value<0.04), CD3e (r=0.26, p-value<0.02), and GATA3 (r=0.22, p-value<0.04). PRDM16 also correlates with KCNRGv2 (r = 0.29, p-value < 0.01) and CD3e (r = 0.36, pvalue < 0.001). In additions UCP1 and PRDM16 show a positive correlation (r = 0.6032, p-value < 4.0e-9).CONCLUSIONS: The correlation between the expression of BAT specific genes UCP1 and PRDM16 with T-bet, CD3e, GATA3, TGFBR1, TGFB1, and KCNRGV2 in VAT could suggest a novel role for the activation of brown adipocytes. Further studies would be needed on brown adipocytes to confirm specific receptor interactions and their significance.

Serum immunoglobulin levels predict fibrosis in patients with non-alcoholic fatty liver disease

A third of the population are estimated to have NAFLD of varying severity. Serum immunoglobulins are frequently elevated in patients with chronic liver disease, but little is known about serum immunoglobulin levels in patients with NAFLD. Aim of this study was to evaluate serum immunoglobulin levels (IgA, IgG, and IgM) in a large cohort of patients with biopsy-proven NAFLD and determine if immunoglobulin levels are associated with clinical or histological features. Patients seen in a tertiary fatty liver clinic between 1999 and 2009 were included. Liver biopsies were assessed using the Kleiner score. Immunoglobulin levels and other blood tests were taken at time of biopsy. 285 patients (110 simple steatosis and 175 NASH) had serum immunoglobulins measured within 6months of liver biopsy. 130 (46%) patients had elevated (>1× upper limit of normal) serum IgA levels, 28 (10%) patients had elevated IgG and 22 (8%) raised IgM. Serum IgA levels were elevated more frequently in patients with NASH compared with subjects with simple steatosis (55% vs. 31%, p<0.001). Overall, 55 (19%) patients had advanced liver fibrosis (Kleiner stage 3-4). There was a significant positive association between serum IgA levels and the stage of fibrosis (p<0.001). Serum IgA, age, platelets, AST/ALT ratio and BMI were all independently with advanced fibrosis following multivariate analysis. A model constructed from these independent predictors accurately predicted advanced fibrosis (AUROC 0.87). The serum IgA level was frequently elevated in patients with NAFLD and was an independent predictor of advanced fibrosis.

PNPLA3 GG Genotype and Carotid Atherosclerosis in Patients with Non-Alcoholic Fatty Liver Disease

Background and AimTo evaluate if the presence of carotid atherosclerosis in patients with NAFLD, could be related to gene variants influencing hepatic fat accumulation and the severity of liver damage.MethodsWe recorded anthropometric, metabolic and histological data(Kleiner score) of 162 consecutive, biopsy-proven Sicilian NAFLD patients. Intima-media thickness(IMT), IMT thickening(IMT≥1 mm) and carotid plaques(focal thickening of >1.3 mm at the level of common carotid artery) were evaluated using ultrasonography. IL28B rs12979860 C>T, PNPLA3 rs738409 C>G, GCKR rs780094 C>T, LYPLAL1 rs12137855 C>T, and NCAN rs2228603 C>T single nucleotide polymorphisms were also assessed. The results were validated in a cohort of 267 subjects with clinical or histological diagnosis of NAFLD from Northern Italy, 63 of whom had follow-up examinations.ResultsCarotid plaques, IMT thickening and mean maximum IMT were similar in the two cohorts, whereas the prevalence of diabetes, obesity, NASH, and PNPLA3 GG polymorphism(21%vs.13%, p = 0.02) were significantly higher in the Sicilian cohort. In this cohort, the prevalence of carotid plaques and IMT thickening was higher in PNPLA3 GG compared to CC/CG genotype(53%vs.32%, p = 0.02; 62%vs.28%, p<0.001, respectively). These associations were confirmed at multivariate analyses (OR2.94;95%C.I. 1.12–7.71, p = 0.02, and OR4.11;95%C.I. 1.69–9.96, p = 0.002, respectively), although have been observed only in patients <50years. Also in the validation cohort, PNPLA3 GG genotype was independently associated with IMT thickening in younger patients only (OR: 6.00,95%C.I. 1.36–29, p = 0.01), and to IMT progression (p = 0.05) in patients with follow-up examinations.ConclusionPNPLA3 GG genotype is associated with higher severity of carotid atherosclerosis in younger patients with NAFLD. Mechanisms underlying this association, and its clinical relevance need further investigations.

More on Current Evidences on Probiotics as a Novel Treatment for Non-Alcoholic Fatty Liver Disease

Dear Editor, In their recent article Kelishadi et al. (1) analyzed in a clear and concise way a number of key aspects of obesity-related NAFLD, deepening in particular the role of gut microflora and its potential therapeutic modulation by probiotics. From their paper it is apparent that most promises still derive from a relevant number of studies conducted in different NAFLD animal models. When interpreting data obtained in these models one should however remind that other factors like physical activity, social and chemical environment, psychological stress factors and genetics are also important contributors to the development of human NAFLD and to its incredible resistance to treatments as well (2). In this respect Dr. Kelishadi and colleagues' review of four available clinical trials on human population is therefore particularly welcomed. In three studies probiotic treatments appear to improve somehow NAFLD related parameters. The fourth study, published in 2008 by doctor Diehl’s group, instead warned versus probiotics treatment of NAFLD, reporting that steatosis measured by proton magnetic resonance spectroscopy increased in three out of four patients treated with VSL#3 for 4 months (3). In addition to these meagre evidences, in recent times, Malguarnera et al. showed in histologically monitored NAFLD patients that Bifidobacterium longum + fructo-oligosaccharides (FOS) treatment and lifestyle modification, when compared to lifestyle modification alone, significantly reduced a number of serum inflammatory, metabolic, toxinic and cytolitic parameters, along with improved histological steatosis, and NASH activity index (4). Even more recently, a further open label randomized controlled trial in 20 obese adult patients with biopsy proven NAFLD appeared fairly promising. Patients received a six month formula containing five strains of bacteria (4 lactobacilli + Bifidobacterium bifidum) + FOS. Treatment resulted in reduction of liver fat (P = 0.034) and AST level (absolute change P = 0.021; percentage of baseline value P = 0.008) (5). In all cases, it should be emphasized that the hitherto available study designs are not homogeneous and data results cannot be easily compared. Study population of two examined series by dr Kelishadi and colleagues for example included different chronic liver diseases in addition to NAFLD (6, 7); probiotics preparations often contained different bacteria strains, and sometimes confounding prebiotics (FOS) and/or vitamins cocktails (4-6); study duration was quite different, and it is still not clear if longer study may be affected by a number of biases like concurrent unpredictable major life style modifications, and at which extent. Last, but not least, although the Authors reminded the seriousness of epidemiologic data of pediatric NAFLD, they do not refer further to pediatric therapeutic studies targeting gut microbiota. In this regard we think therefore it might be valuable to the discussion completeness also the inclusion of our 2011 study published in Journal of Pediatric Gastroenterology and Nutrition (8). In that double-blind, placebo-controlled pilot study the effects of a short-term (8 weeks) probiotic treatment in 20 children with obesity-related liver disease were evaluated. Patients received Lactobacillus Rhamnosus strain GG (12 billion CFU/day): this probiotic strain was chosen because of the proven safety and efficacy profile in previous pediatric studies. Consistently with available adult data, in our population a multivariate analysis after probiotic treatment revealed a significant decrease in alanine aminotransferase (average variation vs. placebo P = 0.03), with normalization in most (80%) cases, irrespective of changes in BMI z score and visceral fat. We measured anti peptidoglycan-polysaccharide (PGPS) antibodies, an indirect indicator of intestinal bacterial overgrowth. PGPS is a ubiquitous bacterial antigen found in both gram-positive and gram-negative bacteria wall, and anti PGPS production is a sign of bacteria or bacterial membrane translocation through intestine barrier. Interestingly PGPS antibodies were significantly reduced after probiotic treatment (average variation vs. placebo P = 0.03). Serum levels of tumor necrosis factor-alpha, and US bright liver parameters remained stable during the study frame. Summing up, taking into account also the above additional evidences, we agree with Dr. Kelishadi and colleagues that available animal and human studies altogether may authorize to further support the view of a reasonable therapeutic role of probiotics in adult and also pediatric NAFLD patients not compliant to lifestyle changes, also because of minimal adverse effects and low cost (9). For better clarifying these aspects, future studies still need to be planned on well characterized larger populations, possibly with single probiotic agents, followed by a wash out period, and/or with gut microbiota characterization. The latter, much interestingly, has been defined only very recently both in adult (10)and in pediatric NAFLD (11).

PWE-139 Further Validation of Terminal Peptide of Procollagen Iii (PIIINP) for the Detection and Assessment of Nonalcoholic Steatohepatitis in Patients with Nonalcoholic Fatty Liver Disease

IntroductionPIIINP has recently been shown to discriminate between simple steatosis (SS) and NASH both in patients without advanced fibrosis and in patients with all degrees of fibrosis1. In this study...

Validation study of systems for noninvasive diagnosis of fibrosis in nonalcoholic fatty liver disease in Latin population

Background. The incidence of liver cirrhosis is significantly high in Latin population. The high prevalence of nonalcoholic fatty liver disease NAFLD is likely partially responsible for these figures. Liver biopsy is not a practical diagnostic option in this scenario. The validation of noninvasive markers of fibrosis is important in populations with a high prevalence of NAFLD.Aim. To compare the diagnostic value of noninvasive assessment systems to detect fibrosis in a cohort of Latin patients with biopsy-proven NAFLD.Material and methods. Patients with biopsy-proven NAFLD were included. Noninvasive evaluations included calculations of NAFLD fibrosis, FIB–4, BARD scores, APRI, and AST/ALT ratio. The sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver-operating characteristic curve (AUROC) were calculated.Results. A total of 228 patients (mean age, 48.6 ± 12.7 years) were included. Fifty-one percent were women; 48% were overweight and 23% were obese. The severity of fibrosis was classified as G0, 56.6%; G1, 25%; G2, 6.6%; G3, 7%; and G4, 4.8%. The AUROC values for advanced fibrosis were 0.72 for the NAFLD fibrosis score, 0.74 for FIB–4 score, 0.67 for AST/ALT ratio, 0.66 for APRI score, and 0.65 for BARD score. In 54% of patients with undetermined FIB–4 score and in 60% of patients with undetermined NAFLD fibrosis score, fibrosis was observed in the liver biopsy.Conclusions. The NAFLD fibrosis, FIB–4, and APRI scores can be used for the noninvasive diagnosis of fibrosis. However, 25% of patients evaluated by these methods have an indeterminate degree of fibrosis.

707 MRI Identifies Unsuspected Liver Dysfunction in Patients After Fontan Procedure

Since the development of the original NAFLD activity score (NAS), it became evident that pediatric NAFLD has a distinct histopathological pattern including the presence of portal inflammation (PI). The aim of this study was to develop and validate a new grading score for Pediatric NAFLD that takes into account the presence of PI and the weight of each histological feature. Methods: Our training set consisted of 203 pediatric patients with biopsy-proven NAFLD. The diagnosis of nonalcoholic steatohepatitis (NASH) versus not NASH was based on Brunt criteria. The individual histological features of NAFLD were scored as follows: steatosis (0-3), lobular inflammation (0-3), ballooning (0-2), and portal inflammation (0-2; 0= no PI, 1= mild PI, 2= more than mild). Logistic regression analysis was performed to apply weight to each histological feature (to improve correlation with NASH). The new score was called the Pediatric NAFLD Histological Score or PNHS. A p value < 0.05 was considered statistically significant. The validation set consisted of 100 children with biopsy-proven NAFLD. Results: The mean age of the initial cohort was 12.4 ± 3.4 years, the median BMI percentile was 97% (94%-98%), and significant fibrosis (fibrosis stage ≥ 2) was present in 26 patients (12.8%). NASH was diagnosed in 135 patients with a mean NAS of 4.5 ± 1.4. The mean PNHS in the NASH group was 89 ± 20.5 compared to 21.9 ± 24.5 in not NASH group, p< 0.001. PNHS correlated with the presence of NASH according to pathologist diagnosis better than NAS, p= 0.011. The area under the ROC curve (AUC) for diagnosis of NASH was 0.96 for PNHS. Similar findings were noted in the validation set with AUC of 0.94. Conclusion: PNHS may be used for histological grading of pediatric NAFLD with better correlation with the presence of NASH than NAS. The utility of this score in other populations and in therapeutic trials should be further investigated.

705 Prevalence of Suspected NAFLD is Increasing Among US Adolescents

Since the development of the original NAFLD activity score (NAS), it became evident that pediatric NAFLD has a distinct histopathological pattern including the presence of portal inflammation (PI). The aim of this study was to develop and validate a new grading score for Pediatric NAFLD that takes into account the presence of PI and the weight of each histological feature. Methods: Our training set consisted of 203 pediatric patients with biopsy-proven NAFLD. The diagnosis of nonalcoholic steatohepatitis (NASH) versus not NASH was based on Brunt criteria. The individual histological features of NAFLD were scored as follows: steatosis (0-3), lobular inflammation (0-3), ballooning (0-2), and portal inflammation (0-2; 0= no PI, 1= mild PI, 2= more than mild). Logistic regression analysis was performed to apply weight to each histological feature (to improve correlation with NASH). The new score was called the Pediatric NAFLD Histological Score or PNHS. A p value < 0.05 was considered statistically significant. The validation set consisted of 100 children with biopsy-proven NAFLD. Results: The mean age of the initial cohort was 12.4 ± 3.4 years, the median BMI percentile was 97% (94%-98%), and significant fibrosis (fibrosis stage ≥ 2) was present in 26 patients (12.8%). NASH was diagnosed in 135 patients with a mean NAS of 4.5 ± 1.4. The mean PNHS in the NASH group was 89 ± 20.5 compared to 21.9 ± 24.5 in not NASH group, p< 0.001. PNHS correlated with the presence of NASH according to pathologist diagnosis better than NAS, p= 0.011. The area under the ROC curve (AUC) for diagnosis of NASH was 0.96 for PNHS. Similar findings were noted in the validation set with AUC of 0.94. Conclusion: PNHS may be used for histological grading of pediatric NAFLD with better correlation with the presence of NASH than NAS. The utility of this score in other populations and in therapeutic trials should be further investigated.

Involvement of a periodontal pathogen, Porphyromonas gingivalis on the pathogenesis of non-alcoholic fatty liver disease

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that is closely associated with multiple factors such as obesity, hyperlipidemia and type 2 diabetes mellitus. However, other risk factors for the development of NAFLD are unclear. With the association between periodontal disease and the development of systemic diseases receiving increasing attention recently, we conducted this study to investigate the relationship between NAFLD and infection with Porphyromonas gingivalis (P. gingivalis), a major causative agent of periodontitis.MethodsThe detection frequencies of periodontal bacteria in oral samples collected from 150 biopsy-proven NAFLD patients (102 with non-alcoholic steatohepatitis (NASH) and 48 with non-alcoholic fatty liver (NAFL) patients) and 60 non-NAFLD control subjects were determined. Detection of P. gingivalis and other periodontopathic bacteria were detected by PCR assay. In addition, effect of P. gingivalis-infection on mouse NAFLD model was investigated. To clarify the exact contribution of P. gingivalis-induced periodontitis, non-surgical periodontal treatments were also undertaken for 3 months in 10 NAFLD patients with periodontitis.ResultsThe detection frequency of P. gingivalis in NAFLD patients was significantly higher than that in the non-NAFLD control subjects (46.7% vs. 21.7%, odds ratio: 3.16). In addition, the detection frequency of P. gingivalis in NASH patients was markedly higher than that in the non-NAFLD subjects (52.0%, odds ratio: 3.91). Most of the P. gingivalis fimbria detected in the NAFLD patients was of invasive genotypes, especially type II (50.0%). Infection of type II P. gingivalis on NAFLD model of mice accelerated the NAFLD progression. The non-surgical periodontal treatments on NAFLD patients carried out for 3 months ameliorated the liver function parameters, such as the serum levels of AST and ALT.ConclusionsInfection with high-virulence P. gingivalis might be an additional risk factor for the development/progression of NAFLD/NASH.

Elevated asymmetric dimethylarginine in plasma: An early marker for endothelial dysfunction in non-alcoholic fatty liver disease?

AimsNon-alcoholic fatty liver disease (NAFLD) is associated with cardiovascular disease. Asymmetric dimethylarginine (ADMA) is a novel marker of endothelial dysfunction and atherosclerosis. We aimed to investigate circulating ADMA concentrations in biopsy proven NAFLD and also to search its association with carotid atherosclerosis. MethodsSixty-seven nondiabetic and normotensive patients with NAFLD and 35 healthy controls were enrolled. Plasma ADMA was measured along with glucose, lipids and insulin levels. Insulin resistance (IR) was assessed by homeostasis model assessment-estimated insulin resistance (HOMA-IR) method. Carotid atherosclerosis was evaluated by carotid artery intima-media thickness (CIMT) using carotid ultrasonography. ResultsADMA levels and CIMT measurements were significantly higher in NAFLD group than the controls. However, the difference regarding the CIMT disappeared when the findings were adjusted according to the metabolic parameters and insulin sensitivity. In contrast, the difference for ADMA remained significant between two groups. No significant association was found between ADMA, CIMT and histopathological findings. ConclusionsPlasma ADMA levels are increased in subjects with NAFLD. This increase seems to be independent from traditional cardiovascular risk factors, insulin resistance and liver histology. Circulating ADMA may be an earlier marker of vascular damage with respect to CIMT in subjects with NAFLD.

OP11 Are simple non-invasive scoring systems for fibrosis reliable in patients with NAFLD and normal ALT levels?

IntroductionWith a quarter of the UK population estimated to have some degree of NAFLD, there is a need for a robust and economical population based screen to identify individuals with...