NIHR Birmingham Liver Biomedical Research Unit

Over the last 10-15 years, our understanding of the composition and functions of the human gut microbiota has increased exponentially. To a large extent, this has been due to new 'omic' technologies that have facilitated large-scale analysis of the genetic and metabolic profile of this microbial community, revealing it to be comparable in influence to a new organ in the body and offering the possibility of a new route for therapeutic intervention. Moreover, it might be more accurate to think of it like an immune system: a collection of cells that work in unison with the host and that can promote health but sometimes initiate disease. This review gives an update on the current knowledge in the area of gut disorders, in particular metabolic syndrome and obesity-related disease, liver disease, IBD and colorectal cancer. The potential of manipulating the gut microbiota in these disorders is assessed, with an examination of the latest and most relevant evidence relating to antibiotics, probiotics, prebiotics, polyphenols and faecal microbiota transplantation.

BACKGROUND: Primary biliary cholangitis (formerly called primary biliary cirrhosis) can progress to cirrhosis and death despite ursodiol therapy. Alkaline phosphatase and bilirubin levels correlate with the risk of liver transplantation or death. Obeticholic acid, a farnesoid X receptor agonist, has shown potential benefit in patients with this disease. METHODS: In this 12-month, double-blind, placebo-controlled, phase 3 trial, we randomly assigned 217 patients who had an inadequate response to ursodiol or who found the side effects of ursodiol unacceptable to receive obeticholic acid at a dose of 10 mg (the 10-mg group), obeticholic acid at a dose of 5 mg with adjustment to 10 mg if applicable (the 5-10-mg group), or placebo. The primary end point was an alkaline phosphatase level of less than 1.67 times the upper limit of the normal range, with a reduction of at least 15% from baseline, and a normal total bilirubin level. RESULTS: Of 216 patients who underwent randomization and received at least one dose of obeticholic acid or placebo, 93% received ursodiol as background therapy. The primary end point occurred in more patients in the 5-10-mg group (46%) and the 10-mg group (47%) than in the placebo group (10%; P<0.001 for both comparisons). Patients in the 5-10-mg group and those in the 10-mg group had greater decreases than those in the placebo group in the alkaline phosphatase level (least-squares mean, -113 and -130 U per liter, respectively, vs. -14 U per liter; P<0.001 for both comparisons) and total bilirubin level (-0.02 and -0.05 mg per deciliter [-0.3 and -0.9 μmol per liter], respectively, vs. 0.12 mg per deciliter [2.0 μmol per liter]; P<0.001 for both comparisons). Changes in noninvasive measures of liver fibrosis did not differ significantly between either treatment group and the placebo group at 12 months. Pruritus was more common with obeticholic acid than with placebo (56% of patients in the 5-10-mg group and 68% of those in the 10-mg group vs. 38% in the placebo group). The rate of serious adverse events was 16% in the 5-10-mg group, 11% in the 10-mg group, and 4% in the placebo group. CONCLUSIONS: Obeticholic acid administered with ursodiol or as monotherapy for 12 months in patients with primary biliary cholangitis resulted in decreases from baseline in alkaline phosphatase and total bilirubin levels that differed significantly from the changes observed with placebo. There were more serious adverse events with obeticholic acid. (Funded by Intercept Pharmaceuticals; POISE ClinicalTrials.gov number, NCT01473524; Current Controlled Trials number, ISRCTN89514817.).

Funding for the development of this Practice Guideline and Guidance was provided by the American Association for the Study of Liver Diseases. Potential conflict of interest: Dr. Mack consults for Albireo. Dr. Kerkar advises High Tide and received royalties from Elsevier. Dr. Manns consults for, is on the speakers’ bureau for, and received grants from Falk. He consults for and received grants from Novartis. Dr. Vierling advises and received grants from CymaBay, Enanta, Genkyotex, Intercept, Lilly, Novartis, and TaiwanJ. He advises Arena, BioIncept, Blade, and GlaxoSmithKline and received grants from Allergan and NGM. What’s New Since 2010 Guidelines? Histological features of NAFLD are present in 17%‐30% of adult patients with AIH, and concurrent NAFLD may influence response to therapy. Diagnostic scoring systems should be used only to support clinical judgment in challenging cases of AIH and to define AIH cohorts for clinical studies. Immune checkpoint inhibitors have been associated with immune‐mediated liver injury and are frequently steroid‐responsive, but the liver injury lacks autoantibodies and typical histological features of AIH. Elastography may be used to assess the stages of hepatic fibrosis noninvasively. Testing for TPMT activity prior to AZA treatment is encouraged in all patients. Budesonide and AZA or predniso(lo)ne and AZA are recommended as first‐line AIH treatments in children and adults who do not have cirrhosis, acute severe hepatitis, or ALF. AZA can be continued throughout pregnancy, whereas the use of MMF is contraindicated in pregnancy. Liver tissue examination prior to drug withdrawal in individuals with ≥2 years of biochemical remission is preferred but not mandatory in adults and required in children. MMF or TAC can be used as second‐line treatment in children and adults with AIH who have failed to respond to first‐line therapy. Patients with acute severe AIH should receive predniso(lo)ne followed by LT if no improvement within 2 weeks, whereas patients with AIH and ALF should be evaluated directly for LT. Glucocorticoids can be discontinued after LT and patients monitored for recurrence of AIH. Purpose and Scope The objectives of this document are to provide guidance in the diagnosis and management of autoimmune hepatitis (AIH) based on current evidence and expert opinion and to present guidelines to clinically relevant questions based on systematic reviews of the literature and the quality of evidence.1 This practice guideline/guidance constitutes an update of the guidelines on AIH published in 2010 by the American Association for the Study of Liver Diseases (AASLD).2 It updates the epidemiology, diagnosis, management, and outcomes of AIH in adults and children. The document is divided into “guideline recommendations” and “guidance statements.” Guideline recommendations were based on evidence derived from systematic reviews of the medical literature and supported, if appropriate, by meta‐analyses. The systematic reviews and meta‐analyses were conducted independently by the Mayo Clinic Evidence‐Based Practice Center. Findings were analyzed and interpreted by a multidisciplinary panel of experts, including both content and methodology experts, who rated the quality of evidence and determined the strength of each recommendation. The quality of clinical evidence was determined by its source (e.g., randomized controlled trial or observational study), and the strength of the recommendation was determined by assessing the quality of evidence, balance of benefits and harms, patient values and preferences, and use of resources and costs. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system was used to categorize each recommendation as strong or conditional (Table 1).3 Details of the methodology, systematic reviews, and meta‐analyses are published separately. The guideline recommendations focus on pertinent management issues for which sufficient evidence was available to render a recommendation. They address glucocorticoid and azathioprine therapy as first‐line management, second‐line medications after failure of first‐line therapy, and maintenance management after liver transplantation (LT; see Supporting Table S1 for patient/intervention/comparison/outcome questions related to systematic reviews). Table 1 - GRADE Assessment of Clinical Studies Study Design Rating Quality Strength Determinants Strength and Implications of Recommendation Randomized controlled trial High Quality of evidence Strong Moderate Balance of benefits and harms Most people would want course Most people should take course Can be adapted as policy in most cases Low Patient values and preferences Observational Very low Resources and costs Conditional Feasibility Many people would select course Requires decision aids and shared decision‐making Debatable policy choice Acessibility Equity Quality downgrades: selection bias, inconsistency, imprecision, indirectness, publication bias. Quality upgrades: large effect, very large effect, dose–response gradient, confounders produce no effect. “Guidance statements” were developed by consensus of an expert panel based on formal review and analysis of the published literature on the topic. The quality (level) of evidence and the strength of each guidance statement were not formally rated for the guidance statements. “Guidance statements” were used to address topics for which a sufficient number of randomized controlled trials were not available to justify a systematic review and meta‐analysis. The “guidance statements” and “guideline recommendations” were also reviewed by members of the AIH Association, a 501(c)(3) nonprofit organization, in order to incorporate patient and public perspectives. “Guidance statements” and “guideline recommendations” are intended to provide health care practitioners with updated information and rigorously assessed, evidence‐based recommendations. They are intended to aid, not supersede, clinical judgment. For ease of reading this AIH guidance/guidelines document, a glossary of definitions is provided in Table 2. Table 2 - Definitions of AIH and Its Treatment Outcomes Condition Definition AIH Characteristic histologic abnormalities (lymphoplasmacytic interface hepatitis), elevated AST, ALT, and total IgG and the presence of one or more characteristic autoantibodies Inactive cirrhosis Absence of inflammatory infiltrates in both portal tracts and fibrous bands in cirrhosis Acute severe AIH Jaundice, INR > 1.5 < 2, no encephalopathy; no previously recognized liver disease370 ALF INR ≥ 2; hepatic encephalopathy within 26 weeks of onset of illness; no previously recognized liver disease100 Biochemical remission Normalization of AST, ALT, and Histological remission Absence of in liver tissue after treatment Treatment failure or histological with therapy response of and histological are to for remission of activity after of remission and drug withdrawal Treatment to maintenance therapy to with cirrhosis in biochemical remission may have of AIH is an immune‐mediated inflammatory liver of which all both and all Patients may be be or present with acute liver failure and the diagnosis be in all patients with acute or liver including patients with after LT. AIH not have a and the diagnosis the presence of a of typical features which can patients with the and can in liver to hepatic cirrhosis, from liver or LT are Treatment with been but management may be associated with and AIH all and within all and its to by and have a of AIH more present with cirrhosis, and have of and a of recurrence after LT to in adults and children the onset of AIH and but the may have been by onset have been in and New The of AIH on the and the in adults from to 2 cases of New are from to the been a in in and the The of AIH in adults from to The in children from and to with autoimmune the in AIH within the which the by most and from within and associated with AIH are the of AIH also but the for of AIH are for for AIH been associated with of or and of and the of or of may the and of and and inflammatory and AIH is a and is AIH associated with an in the autoimmune on which been associated with autoimmune 1 in the and as or can as for of to in to and to hepatic as the of and of and to in the of produce are by and to on and of to of of the of AIH. a of the of AIH, in which a in to hepatic and the are of to hepatic hepatic or to as or the present to on and and by to is a which of required for and of (e.g., and both and the of by of a of including of of into of development and and The is the failure of and and to hepatic of to can from into The is the of portal inflammatory infiltrates of of and of in of which and and portal in cirrhosis in the of therapy. of the to into and in the presence of of and of by by to and tissue can the and of all and of may a in of into also of AIH. Low of and of of with by in the in can of and and may into infiltrates of and including and and can within the portal and of into of into and hepatitis of and in patients with AIH. and of of and by of and autoantibodies may of Diagnostic and The diagnosis of AIH is based on histological abnormalities hepatitis), characteristic clinical and and and IgG and the presence of one or more characteristic AIH lacks a and the diagnosis characteristic features and the of may (e.g., hepatitis, liver are of AIH, based on the autoantibodies are 1 is by and 2 is by to liver 1 in the of and The characteristic clinical features of are in Table to of AIH cases are for and the presence of characteristic features of AIH AIH is autoantibodies may be as in Table and 2. of AIH into in management and aids in outcomes in but may be in Table - Characteristic of 1 and 2 AIH 1 AIH 2 AIH and adults of Acute onset or Acute liver failure in Acute in Acute severe in features may be with in in children with in adults after drug withdrawal Table - in the of AIH Diagnostic 1 1 2 1 after with 1 1 1 2 2 of 1 autoimmune Diagnostic for the of AIH after of and and should be in adults and to should be if and are and should be in all patients The of the liver support the diagnosis of AIH or an AIH with or The of and can to tissue and for one of autoantibodies support the diagnosis of AIH or including tissue and the for the diagnosis of AIH (Table are in of American adults with AIH are present in and are present in of patients with AIH have or as an and have can also as an in hepatitis hepatitis liver and liver and can as an in hepatitis and liver and are concurrent in of liver of AIH, and the for AIH from to if autoantibodies are are in the of and and this after for and have a low for AIH in American adults and after the of and is in patients. are in of and children with and of and are in adults and children and treatment but are not of activity or treatment to liver are present in of patients with 1 AIH, and have for the (Table have been the of AIH in of and have been associated with severe and after drug are frequently present in patients with 1 AIH but in and liver may be the only autoantibodies are a of and are present in of patients with AIH and (Table to is an is present in of patients with AIH and of patients with to and been associated with severe acute AIH, treatment and to liver 1 are present in of patients with and in children with severe liver (Table are present in of patients with 2 and may be in and have not been rigorously in the should be and with the clinical may be on of the and in with the Histological Findings The diagnosis of AIH be liver and histological hepatitis is the histological of AIH, by in and hepatitis in is also in and with in patients with and is the of one into with both to is present in of patients with AIH, and are present in of the histological is for AIH, but the of interface hepatitis with portal or into the and are typical of Histological features characteristic of AIH. inflammatory of the portal and interface hepatitis of the portal and in a portal inflammatory and of a and and of and are of of of is present in of adults in the as as in of in of adults with or The histological examination is to or concurrent the of inflammatory and the of may be present in patients with but the clinical of this Histological of are present in 17%‐30% of patients with and liver tissue examination may patients with AIH and who are of and The histological features of AIH with ALF in the and of is present in inflammatory in hepatic in and in of patients with ALF have or all of Diagnostic The scoring system of the was by an panel in in and in Table The scoring system for AIH to the scoring system whereas the scoring system and clinical judgment as the The scoring system is for patients with or whereas the scoring system is most for typical of patients with the scoring system should be the system a low a of to the of the a of and a of were associated with AIH. The scoring system can be to children and in adults as of the for the in the with the or may the of the scoring system for children by the of to the and scoring systems of by of in the of concurrent or liver failure to as and on by by Diagnostic scoring systems can in a diagnosis of AIH in challenging but are most in cohorts of patients with AIH for clinical Guidance The diagnosis of AIH histological and is by the elevated elevated IgG of and may AIH. should of and in adults and and in if to the challenging cases should be reviewed by or to an liver prior to therapy. Clinical Most patients with AIH present after the development of or is the in of and may be of or is with the and a are from of liver or of autoimmune inflammatory AIH is in of patients improvement may have histological to of frequently within and a is of patients with more severe The of should not Acute and ALF AIH with an acute onset in of ALF associated with hepatic encephalopathy in of American and definitions in Table or a or liver injury on previously AIH liver be are or in of patients with acute severe AIH, and the IgG is in Histological is a hepatitis, and interface hepatitis support the diagnosis of acute and features in the presence of cirrhosis and hepatic can be in AIH with within the liver in of patients with acute severe AIH and may be and are in of American and patients as hepatitis and as AIH by the scoring of AIH have been in and by including clinical judgment and glucocorticoid and may be in the course of the or the of and may the diagnosis to Guidance The diagnosis of AIH be in all patients with acute or liver including patients with liver and Immune Diseases autoimmune are present in of patients with and have been recognized with in patients with 1 and 2 been the most concurrent autoimmune in 1 AIH whereas 1 autoimmune and autoimmune have been most in 2 is associated with or no but in the of may the liver but in the of the concurrent may the liver of children with AIH may of the of the and autoimmune most frequently in and the by Patients years have autoimmune and more adults years whereas adults more have and autoimmune concurrent autoimmune is more in patients with or a of autoimmune in The of in patients with AIH is in the children with AIH, was present in and features associated with can be with AIH, and concurrent may to the of liver in patients with AIH and who of remission after withdrawal of AIH children Guidance AIH patients should be for and AIH patients should be for autoimmune and autoimmune based on and medical or AIH and or are clinical and not clinical is to individuals who may not respond to treatment for The patients with features of AIH and of the for should be the of or presence of and on histological for AIH in the of to the presence of interface are and IgG or presence of of the and the AIH scoring systems the were more the may not all patients with the who have The the for the diagnosis of have not been independently and is to the and of the They have also the scoring systems for AIH were not developed or for the diagnosis of the and should not be used for this to are present in of patients with AIH in the of histological features of injury or patients respond to glucocorticoid therapy, and do not into Liver tissue examination is required to the and the presence of in patients with AIH is to this for the diagnosis of as autoimmune in the presence of typical features of AIH, of and evidence of by or or evidence of based on fibrosis on is present in of adults with AIH, and of patients with AIH and concurrent have of is present in of children with AIH, and to with Patients with of histological features with or and may have or The diagnosis of should be in all patients with AIH and or to glucocorticoid Guidance Patients with AIH, with and a should be to have Patients with AIH, histological features of injury or and concurrent should be evaluated for by to have the The can in the but the may patients with who have severe the the scoring systems for AIH should be used for assessing liver injury can and an or drug been in of patients with features of and have been most and have been (Table including hepatitis, have been with the use of as the checkpoint The liver associated with the checkpoint inhibitors have with glucocorticoid therapy, but have the and histological features characteristic of cases have been to glucocorticoid therapy and associated with The liver associated with the checkpoint inhibitors should not be with AIH. Table - with Liver AIH Association Association Association from to The clinical of injury is in Table The from drug to onset from weeks to but and can have The clinical should all to and Table - of and AIH Clinical AIH but also Acute onset to with drug or autoimmune in drug Glucocorticoids with after drug withdrawal to cirrhosis transplantation The histological of interface hepatitis with portal and infiltrates of hepatitis, and are to of AIH, for the of fibrosis or cirrhosis in most may be and fibrosis is The diagnosis is by an acute features of published literature on the from drug to liver and of fibrosis or cirrhosis Liver tissue examination is if the diagnosis is severe or the of glucocorticoid therapy is Treatment withdrawal of the with and of clinical and (Table within 1 with and total the of or for LT in of of for the of glucocorticoid to glucocorticoid management are failure of the to after of the or of or the biochemical after glucocorticoid withdrawal the diagnosis of a liver whereas of abnormalities is with should be as AIH with based on the total and a of and of for this is a of The of injury been (Table The have been as or and drug do have a of and for LT in The of the Liver is a for liver It is a of the Liver of the of and and Diseases and the of of the of of Guidance liver injury be in the diagnosis of AIH. The be and to therapy for injury should be or activity are severe (e.g., or if and to or after of the after glucocorticoid withdrawal AIH and the for therapy. Assessment Assessment of by for hepatic the the the and the liver fibrosis have as the in in AIH and in assessing the or of hepatic and of and treatment Assessment of by Liver Elastography or with the histological of fibrosis in but its in fibrosis is within the of liver by is by both and the with histological of of of therapy to hepatic can cirrhosis and stages of fibrosis from severe stages The values fibrosis stages as the of were for ≥ 2, for ≥ and for ≥ in liver with biochemical of and after of Elastography The of with fibrosis and to for hepatic fibrosis in in liver of can have for portal and AIH, the and of for hepatic fibrosis are the fibrosis scoring systems and the ALT, for the diagnosis of cirrhosis in one liver the of fibrosis by the of fibrosis was liver in patients with AIH was in patients This was each fibrosis from to and in in liver by in and patients with AIH have not been but the liver by can be by therapy, by liver or hepatic and have not been in AIH. liver by in and of of are interpreted as in liver The of for cirrhosis and by of have been to in fibrosis ≥2 and by also with the of and may as a to assess of portal can hepatic fibrosis in patients with hepatic severe and hepatic Guidance for hepatic fibrosis are in AIH and should not be can fibrosis or cirrhosis in patients with AIH with but should be for after treatment of AIH in order to the of hepatic Evaluation The of the of patients with AIH are to and an Assessment of of activity patients with or TPMT activity who are for severe with AZA or or TPMT activity in only of the but the of severe may its use an analysis of and for TPMT activity not the of AZA or as and and TPMT activity not the of in Guidance patients with AIH for or TPMT activity prior to treatment with should be reviewed and prior to the of are not recommended in on of whereas and are to are in patients but not low as to Patients with hepatitis and hepatitis should prior to treatment if to and been in most patients with autoimmune liver and the of been and have developed in all patients for and in of patients for with to Guidance should be to all patients with AIH to the guidelines of the for and Patients and should therapy. and of of Patients on are for of and guidelines have been developed of patients for hepatitis and to hepatitis on the and the and of therapy, the of treatment can be as and low on the a treatment or with the of therapy can be therapy, with or treatment and for after treatment after treatment with been recommended for individuals to of with of therapy been recommended for patients low The of in patients with AIH who are with of or in with AZA is the in patients to individuals with who are of by patients but a of patients with AIH who would be for glucocorticoid patients with for but of and in a previously in patients with and have been associated with a low of as glucocorticoid therapy for weeks for autoimmune with the and of and to of for weeks have been associated with a of of Patients with AIH for and to the of and individuals for glucocorticoid therapy can be prior to The of management is to clinical and biochemical remission on glucocorticoid in with and for is in patients. of and of been by the therapy or the of or may the of and is in patients. the of therapy in patients should be Guidance Patients with AIH who are the of should therapy with or in with AZA to and the for therapy. Patients with evidence of who are with or are for and should be for therapy. by of and should be in patients with for and years in adult patients with for The most are or use of of and years for and years for and been recommended for patients on glucocorticoid in of patients with and severe in justify of the in all patients diagnosis and as and are used in children. Clinical trials support the use of is can and as a for The is by a of for and 2 may be or by glucocorticoid therapy, and its presence should be prior to the of therapy. The of the are low and or of the justify the The presence of or treatment of the glucocorticoid and and Guidance should be in all adult patients with AIH who have for and should be years of glucocorticoid of should be determined diagnosis and with and should be provided on glucocorticoid therapy and as clinically in patients with therapy is for patients with AIH and Assessment for all features of should be prior to and therapy, and its presence may treatment and should be prior to treatment to patients not only the of the but also the benefits of remission and the associated with or are patients with and are more in patients with AIH in the of is in and severe in of and with to the and of the and its and can to Low on quality of have been associated with glucocorticoid may be glucocorticoid The of and may on treatment of and in the quality of should be monitored throughout management of AIH as may justify in the of or or can be by as the the of the Patient and the Guidance Potential to should be and the of treatment and monitored of and in the quality of should be monitored throughout management of AIH, and can be by The of AIH and its medications on health should be if on and outcomes of in AIH are derived from and AIH is whereas are not but in with AIH The is in with The and of is for the but to for with are associated with and may be a but of in of but are no associated with AIH. The the or within of is is to a in AIH. in patients who are not on therapy or who have not been in remission the prior to Patients with AIH who are or within the should be continued on treatment to the of and hepatic are more and the low of may in to the of patients with cirrhosis, in can to an of and of with is as and have in (Table The of been in Table - of in the Patient with AIH in but with abnormalities AZA MMF TAC in Glucocorticoids from an of and the

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of disease ranging from hepatocellular steatosis through steatohepatitis to fibrosis and irreversible cirrhosis. The prevalence of NAFLD has risen rapidly in parallel with the dramatic rise in obesity and diabetes,1,2 and is rapidly becoming the most common cause of liver disease in Western countries.3 Indeed, NAFLD is now recognized to be the aetiology in many cases previously labelled as cryptogenic cirrhosis.4 In Western populations, estimates of NAFLD prevalence vary between 20 and 30%,5,6 rising up to 90% in morbidly obese individuals.7 The more severe, and clinically significant form of NAFLD, non-alcoholic steatohepatitis (NASH) is less common, affecting an estimated 2–3% of the general population,8 and up to 37% of the morbidly obese.7 Of particular concern, and with significant implications for future disease burden, is the increasing prevalence of NAFLD in children and young adults. Studies have reported a 3% prevalence of NAFLD in the general paediatric population, rising to 53% in obese children.9,10 NAFLD has a strong association with type 2 diabetes, with steatosis present in 70% of type 2 diabetics screened with ultrasound,11 and thus it is now recognized to represent the hepatic manifestation of the metabolic syndrome. NAFLD occurs in all ethnic groups although it appears to have a lower prevalence in African-Americans compared with Hispanic and European Americans. This difference remains even after controlling for obesity and insulin resistance (IR)5,12 and may be related to ethnic differences in lipid homeostasis.5 There are no laboratory, imaging or histological findings which can accurately distinguish between NAFLD and alcohol-induced steatosis or steatohepatitis, and the diagnosis can therefore only be made in the absence of a history of significant alcohol intake. Other specific causes of steatosis need to be considered and include metabolic disorders e.g. lipodystrophy and abetalipoproteinaemia, nutritional causes such as rapid weight loss, jejuno-ileal bypass and total parenteral nutrition, and drug-induced. Commonly implicated agents include glucocorticoids, methotrexate, amiodarone, synthetic oestrogens, tamoxifen, diltiazem and highly active anti-retroviral drugs.13–15 Steatosis also commonly occurs in association with hepatitis C, particularly genotype 3, and has an increased prevalence in women with polycystic ovary syndrome, when it is usually associated with IR.16 In the great majority of patients NAFLD develops in association with features of IR and the metabolic syndrome. The metabolic syndrome comprises a cluster of clinical and biochemical features, namely IR, glucose intolerance or diabetes, central obesity, hypertension and dyslipidaemia and is associated with significant cardiovascular morbidity and mortality.17–19 Whilst simple steatosis in the absence of significant fibrosis is considered to be a relatively benign condition,20 the presence of fibrosis predicts both disease progression and liver-related complications over a subsequent 10-year period.21 Decreased survival in this sub-group is due to predominantly cardiovascular causes, although there is a significant increase in liver-related deaths.21 NASH also carries an increased risk of hepatocellular carcinoma (HCC)21 and thus the observation of increased incidence of HCC in type 2 diabetics22 is likely to be due to their high prevalence of NASH.21 In a recent US study, NASH was found to account for at least 13% of overall cases of HCC.23 There are as yet few proven therapies available for patients with NASH, and current strategies are directed towards improving aspects of the metabolic syndrome. Ultimately when such measures fail, liver transplantation remains the only option for patients with end-stage cirrhosis. Although the pathogenesis of NAFLD/NASH is not yet fully understood, much progress has been made in recent years in elucidating the mechanisms of progression from steatosis to more advanced liver inflammation and fibrosis. In this review, we discuss the current understanding of NAFLD pathogenesis, and anticipate that such knowledge will eventually translate into the development of novel treatment strategies for this increasingly important disease. Initial theories for the pathogenesis of NASH were based on a ‘2-hit hypothesis’ (Figure 1a). The ‘first hit’, hepatic triglyceride accumulation, or steatosis, increases susceptibility of the liver to injury mediated by ‘second hits’, such as inflammatory cytokines/adipokines, mitochondrial dysfunction and oxidative stress, which in turn lead to steatohepatitis and/or fibrosis.24,25 However, there is increasing recognition of the role that free fatty acids (FFA) play in directly promoting liver injury, which has led to modification of this theory (Figure 1b). In obesity and IR there is an increased influx of FFA to the liver. These FFA either undergo β-oxidation or are esterified with glycerol to form triglycerides, leading to hepatic fat accumulation. There is now substantial evidence that FFA can directly cause toxicity by increasing oxidative stress and by activation of inflammatory pathways,26 therefore hepatic triglyceride accumulation may be a protective mechanism by preventing the toxic effects of unesterified FFA.27 Additionally, a further component, or ‘third-hit’ has been added to reflect inadequate hepatocyte proliferation (Figure 1c).28 In the healthy liver, cell death stimulates replication of mature hepatocytes which replace the dead cells and reconstitute normal tissue function.28 However oxidative stress, a central feature of NAFLD pathogenesis, inhibits the replication of mature hepatocytes which results in expansion of the hepatic progenitor cell (oval cell) population.29 These cells can differentiate into hepatocyte-like cells, and both oval cell and intermediate hepatocyte-like cell numbers are strongly correlated with fibrosis stage, suggesting that cumulative hepatocyte loss promotes both accumulation of progenitor cells and their differentiation towards hepatocytes.29 Activation of these cells has also been implicated in hepatocellular carcinogenesis.29 In chronic liver injury, the development of fibrosis/cirrhosis is dependent on the efficacy of hepatocyte regeneration, and therefore cell death with impaired proliferation of hepatocyte progenitors represents the proposed ‘third hit’ in NAFLD pathogenesis.28 (a) The traditional 2-hit hypothesis: steatosis represents the ‘first hit’, which then sensitises the liver to injury mediated by ‘second hits’, such as inflammatory cytokines, adipokines, oxidative stress and mitochondrial dysfunction, leading to steatohepatitis and fibrosis. The presence of high levels of oxidative stress reduces the ability of mature hepatocytes to proliferate, resulting in reduced endogenous liver repair. (b) Modified 2-hit hypothesis: the accumulation of FFA alone has been suggested to be sufficient to induce liver damage, without recourse for a second hit. Indeed, rather than being harmful, triglyceride accumulation in the form of steatosis may actually be protective by preventing FFA-induced inflammation and oxidative stress. (c) The 3-hit hypothesis: oxidative stress reduces the ability of mature hepatocytes to proliferate, resulting in the recruitment of other pathways of liver regeneration, such as HPCs. These cells have the capability of differentiating into both cholangiocytes and hepatocytes and contributing to liver repair. It has been suggested that an inability to mount such a ductular response, as is seen in patients transplanted for NASH who have denervated livers, may be responsible for a more progressive pattern of liver damage. Thus, impaired proliferation of hepatocyte progenitors represents the proposed ‘third hit’ in NAFLD pathogenesis.28 NAFLD is characterized by the accumulation of triglycerides, which are formed from the esterification of FFA and glycerol within the hepatocyte. FFAs arise in the liver from three distinct sources; lipolysis (the hydrolysis of FFA and glycerol from triglyceride) within adipose tissue, dietary sources, and de novo lipogenesis (DNL).30 In contrast, FFA may be utilized either through β-oxidation, re-esterification to triglycerides and storage as lipid droplets, or packaged and exported as very low density lipoprotein (VLDL). Hence hepatic fat accumulation can occur as a result of increased fat synthesis, increased fat delivery, decreased fat export, and/or decreased fat oxidation (Figure 2).30 Mechanisms of hepatic fat accumulation. To establish the relative contribution of lipid accumulation in patients with NAFLD, Donnelly et al. used a multiple-stable-isotope method, demonstrating that approximately 60% of liver triglyceride content derived from FFA influx from adipose tissue, 26% from DNL, and 15% from diet.31 This contrasts with healthy individuals in whom DNL contributes <5% of hepatic triglyceride formation.32,33 Triglyceride can also be exported from the liver in VLDL particles, which are formed by the incorporation of triglyceride into apolipoprotein B (apoB) by microsomal transfer protein (MTP).34 Aberrant alterations of MTP/apoB synthesis and secretion have been proposed as potential mechanisms underpinning the pathogenesis of NAFLD leading to a decreased capacity for lipid export.35,36 In healthy individuals, binding of insulin to its receptor leads to phosphorylation of several substrates including insulin receptor substrates (IRS)-1, -2, -3 and -4, which propagate the insulin signal.30,37 Insulin stimulation of IRS-1 and -2 leads to activation of intracellular PI3K (phosphoinositide 3-kinase) and AKT/PKB (protein kinase B) pathways, which are intimately involved in mediating the metabolic effects of insulin.30 Ultimately, AKT/PKB activation results in translocation of glucose transporter, GLUT4, containing vesicles to the plasma membrane, thus facilitating glucose uptake. In addition, the expression of key lipogenic genes is increased, with a concomitant decrease in gluconeogenic gene expression via its regulation of forkhead (FOXO) transcription factor activity. Insulin has a potent action to suppress adipose tissue lipolysis. However, in situations of IR, such as NAFLD, this suppression is impaired resulting in an increased efflux of FFA from adipose tissue.38 The hyperinsulinaemia associated with IR leads to: (i) up-regulation of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c), which is a key transcriptional regulator of genes involved in DNL,15 and (ii) Inhibition of β-oxidation of FFA thus further promoting hepatic lipid accumulation.30 Many of the abnormalities reported in NAFLD interfere with the insulin signalling cascade, and thus contribute to IR. These include FFAs, tumour necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), ceramide, jun N-terminal kinase 1 (JNK1), SOCS (suppressors of cytokine signalling) and cytochrome CYP2E1.39,40 Increased lipid metabolites such as diacylglycerol (DAG) have been implicated in a protein kinase Cε (PKCε) dependent mechanism, to interfere with insulin signalling through inhibition of insulin receptor activity and modulation of IRS-2 phosphorylation.30 Similar processes occur in skeletal muscle cells, leading to a more generalized state of IR. The presence of steatosis is tightly associated with chronic hepatic inflammation,41 an effect in part mediated by activation of the Iκκ-β/NF-κB signalling pathway. In murine models of high-fat diet (HFD)-induced steatosis, increased NF-κB activity is associated with elevated hepatic expression of inflammatory cytokines such as TNF-α, interleukin-6 (IL-6) and interleukin 1-beta (IL-1β), and activation of Kupffer cells.41 Liver-specific NF-κB inhibition prevents HFD-induced inflammatory gene expression, whereas HFD-induced hyperglycaemia and IR can be reproduced by selective over-expression of constitutively active Iκκ-β in hepatocytes.41 The Iκκ-β/NF-κB pathway in hepatocytes can also be activated directly by FFA, providing a further mechanism by which central obesity with consequent increased hepatic FFA supply can contribute to inflammation (Figure 3).26 Furthermore, the conversion of FFA to hepatic triglyceride may serve as a protective measure to prevent direct hepatic lipotoxicity. This is endorsed by a murine model of NAFLD, where inhibition of DGAT2, the enzyme that catalyzes the final step in triglyceride synthesis, resulted in improvement of hepatic steatosis and IR but exacerbation of injury and fibrosis.27,42 Proposed pathogenesis of NASH. The likelihood of progression to advanced NASH/cirrhosis results from a complex interplay between genetic predisposition and the mechanisms described earlier. Both serum and hepatic levels of TNF-α are elevated in patients with NASH,43,44 and levels correlate with histological severity.45 In addition to its proinflammatory effects, TNF-α promotes IR.46 Conversely, inhibition of TNF-α signalling improves IR and histological parameters of NASH.47–49 Similarly, serum IL-6 levels are also elevated in both animal and human models of IR and NAFLD,43,50,51 and levels correlate with increasing liver inflammation and fibrosis.52 The key role of hepatocyte cytokine production in the progression of steatosis to NASH is supported by studies demonstrating that cytokines can replicate all of the histological features associated with NASH, including neutrophil chemotaxis, hepatocyte apoptosis/necrosis, Mallory body formation and stellate cell activation.25 Additionally, data suggests that inflammation and NF-κB activation can promote carcinogenesis,53 and that the chronic inflammatory state associated with hepatic steatosis may also play a key role in HCC development.25 Adipose tissue is not just an inert site of energy storage, but an actively secreting endocrine organ. The functional role of adipocyte-derived cytokines (adipokines), is now increasingly recognized, with leptin and adiponectin amongst the most well described. Leptin is a 16 kDa hormone produced mainly by mature adipocytes whose actions include the regulation of energy intake and expenditure,54 regulation of the immune system,55,56 and promotion of inflammation and fibrogenesis.56,57 Higher leptin levels are observed in obese patients and those with NAFLD,54,58–60 which are commonly regarded as states of leptin resistance.58 It remains plausible that leptin may have a functional role to play in the pathogenesis of NAFLD. In contrast to leptin, secretion and circulating levels of adiponectin are inversely proportional to body fat and are reduced in patients with is and increases insulin and the of adiponectin improves as well as the biochemical and histological parameters of NAFLD in a murine the effects of TNF-α, which adiponectin The of adiponectin in NAFLD is supported by studies that serum adiponectin levels can to distinguish NASH from simple Other adipose tissue derived found in in NAFLD include TNF-α, and all of which the lipogenic effects of but their role in the pathogenesis of NAFLD remains to be (Figure The role of oxidative stress and mitochondrial dysfunction in NASH is with more advanced disease with of oxidative β-oxidation within the normal liver in the but in the of this can as a result of increased FFA rise to induce oxidative stress, with subsequent activation of inflammatory and also mitochondrial damage. mitochondrial and a in mitochondrial activity have been observed in human studies of expression and activity of the hepatic microsomal fatty enzyme cytochrome has been observed in human and animal models of NASH and represents a potent of over-expression of activity is associated with oxidative stress, IR and hepatic fat Other mechanisms implicated in NASH pathogenesis include stress and stress can be by a of including hyperinsulinaemia and and can result in activation of pathways leading to IR, and mitochondrial stress is to be important in alcohol-induced steatohepatitis and further of its role in NASH is is also for a role of in the pathogenesis of NASH. results in production of and of both of which can TNF-α production in Kupffer cells and thus induce hepatic and increased have been found more in patients with NASH when compared with This has led to the that this may the of NASH and liver fibrosis as a of bypass This is further supported by evidence that of with and can hepatic inflammation in both and from both and endogenous are a cause of NAFLD. with syndrome, who have increased circulating levels a metabolic of central obesity, IR and a significant of these patients will also hepatic The mechanisms by which promote hepatic fat accumulation include inhibition of fatty β-oxidation and promotion of hepatocyte However most patients with NAFLD have normal circulating suggesting that mechanisms are the metabolic This has led to in enzyme which play a key role in and consequent to and the type 1 to the active and thus increases levels and Inhibition of has been to lower body weight and lipid levels and glucose in animal and increase hepatic insulin in In the and are responsible for the of to its Increased hepatic activity has been in patients with and which may represent a mechanism to decrease in an to prevent development or progression of NAFLD. In animal both and inhibition of activity has been to increase susceptibility to development of IR and fatty Hence of hepatic by modulation of and the may represent a potential for preventing the development and progression of NAFLD. and its more advanced form represents the final common pathway of all chronic liver including NASH. fibrosis results in liver and hypertension with its associated complications of and as well as an increased risk of The pathogenesis of fibrosis is not within the of this and is well There are aspects of liver fibrosis and which are relatively specific to NASH and are In most of liver injury by replication of mature the presence of such as NASH or is associated with high levels of oxidative stress which the ability of these mature hepatocytes to In this other pathways of liver regeneration, hepatic progenitor cells are are cells which in the of and which on proliferation form a complex of and cholangiocytes as a ductular This was used to the of cells at the of the and the and to proliferation of progenitor cell activation and of intermediate these cells have the capability of differentiating into both cholangiocytes and hepatocytes and contributing to liver repair. inability to mount such a ductular response, as is seen in patients transplanted for NASH who have denervated livers, may be responsible for a more progressive pattern of liver damage. to in the the interplay of the ductular and fibrosis in NAFLD. Initial a association between the expansion of and the ductular in liver of NASH. The of ductular in turn strongly correlated with the of suggesting that may be responsible for a progressive mechanisms for this include the secretion of cytokines and by the ductular as well as direct of the cholangiocytes to This is in that it a for the of fibrosis in NAFLD which is a key feature of progressive disease. recent in the model of murine NAFLD has suggested that liver fibrosis the proliferation of suggesting that fibrosis not occur as a result of The of the presence of the histological findings is less on further the of an more complex between fibrosis and in which both processes can other Indeed, the of a progenitor cell with of may not only as a pathway for to from the into the but also for the survival of these Although hepatic steatosis is common in patients with obesity and IR only a progress to NASH and suggesting an important interplay between genetic predisposition and in genes related to lipid IR, oxidative stress, and may all increase susceptibility to NASH studies have which fibrosis development in other liver particularly chronic hepatitis Studies in NASH have in the and genes to be associated with advanced hepatic fibrosis in obese In addition, in the type 1 receptor are associated with an increased risk of NAFLD and studies are to more genes which will be not only as to the pathogenesis and of the but also may represent novel treatment an increasing understanding of the mechanisms of NAFLD pathogenesis, there are few therapies are directed towards improving the metabolic parameters which contribute to disease pathogenesis, such as weight loss and IR and improving In addition to current therapies utilized for patients with NAFLD include insulin e.g. and the weight loss e.g. and and of for morbidly obese transplantation remains the only treatment option for end-stage cirrhosis. The need for specific for NAFLD has this a of with particular on which can or prevent the more advanced and clinically of NASH. The presence of fibrosis predicts likelihood of liver related complications and therefore therapies which can prevent or fibrosis are important in NASH include such as C, and and Whilst of these has yet evidence of further are may represent a novel option for the progression of NAFLD. In patients such as have been to increase insulin suppress and increase in association with weight and in animal models reduced IR, of oxidative stress and hepatic fibrosis is the of hepatic which predominantly arise from activation of hepatic stellate cells that in the of the nuclear activated receptor Studies have that by such as the and leads to reduced results with these agents have been in patients with NAFLD with in both liver and However including and weight are of also to be on of has been to promote survival and liver and thus the effects of and receptor are likely to include clinical studies of such as which have been to suppress fibrosis in have to that dietary of improves histological fibrosis in of cytokines are intimately involved in the and of of which represents potential for include factor factor tissue factor factor factor and which also their effects through with studies in animal models of liver fibrosis have effects kinase such as which is for in chronic and Other potential to include and modulation of the liver and NAFLD now represents of the causes of liver disease in the Western and the rising levels of obesity, and metabolic syndrome will that it remains a cause of morbidity and Although simple steatosis carries a relatively benign a significant of patients will progress to NASH and with risk of The traditional of NAFLD pathogenesis has been several in most patients NAFLD to with lipid accumulation, or steatosis, which is in turn by obesity and IR. to steatohepatitis and fibrosis on such as FFAs, inflammatory cytokines and adipokines, oxidative stress and mitochondrial dysfunction in a complex interplay with genetic treatment strategies for NASH on improving of the metabolic syndrome, such as obesity and IR, with no agents yet However, modulation of of the mechanisms involved in NASH pathogenesis to prevent the development of fibrosis and its associated This and the significant that to be made in understanding of the pathogenesis of NASH, are to the development of novel strategies for this increasingly important is a in and is therefore from the of

BACKGROUND: Chronic kidney disease (CKD) is a frequent, under-recognized condition and a risk factor for renal failure and cardiovascular disease. Increasing evidence connects non-alcoholic fatty liver disease (NAFLD) to CKD. We conducted a meta-analysis to determine whether the presence and severity of NAFLD are associated with the presence and severity of CKD. METHODS AND FINDINGS: English and non-English articles from international online databases from 1980 through January 31, 2014 were searched. Observational studies assessing NAFLD by histology, imaging, or biochemistry and defining CKD as either estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m2 or proteinuria were included. Two reviewers extracted studies independently and in duplicate. Individual participant data (IPD) were solicited from all selected studies. Studies providing IPD were combined with studies providing only aggregate data with the two-stage method. Main outcomes were pooled using random-effects models. Sensitivity and subgroup analyses were used to explore sources of heterogeneity and the effect of potential confounders. The influences of age, whole-body/abdominal obesity, homeostasis model of insulin resistance (HOMA-IR), and duration of follow-up on effect estimates were assessed by meta-regression. Thirty-three studies (63,902 participants, 16 population-based and 17 hospital-based, 20 cross-sectional, and 13 longitudinal) were included. For 20 studies (61% of included studies, 11 cross-sectional and nine longitudinal, 29,282 participants), we obtained IPD. NAFLD was associated with an increased risk of prevalent (odds ratio [OR] 2.12, 95% CI 1.69-2.66) and incident (hazard ratio [HR] 1.79, 95% CI 1.65-1.95) CKD. Non-alcoholic steatohepatitis (NASH) was associated with a higher prevalence (OR 2.53, 95% CI 1.58-4.05) and incidence (HR 2.12, 95% CI 1.42-3.17) of CKD than simple steatosis. Advanced fibrosis was associated with a higher prevalence (OR 5.20, 95% CI 3.14-8.61) and incidence (HR 3.29, 95% CI 2.30-4.71) of CKD than non-advanced fibrosis. In all analyses, the magnitude and direction of effects remained unaffected by diabetes status, after adjustment for other risk factors, and in other subgroup and meta-regression analyses. In cross-sectional and longitudinal studies, the severity of NAFLD was positively associated with CKD stages. Limitations of analysis are the relatively small size of studies utilizing liver histology and the suboptimal sensitivity of ultrasound and biochemistry for NAFLD detection in population-based studies. CONCLUSION: The presence and severity of NAFLD are associated with an increased risk and severity of CKD. Please see later in the article for the Editors' Summary.

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its alpha subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1alpha is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1alpha expression varies among different organs. In liver and kidney, HIF-1alpha reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1alpha is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1beta is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1alpha. Overall, HIF-1alpha expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.

BACKGROUND: Portal vein thrombosis (PVT) has been seen as an obstacle to liver transplantation (LTx). Recent data suggest that favorable results may be achieved in this group of patients but only limited information from small size series is available. The present study was conducted in an effort to review the surgical options in patients with PVT and to assess the impact of PVT on LTx outcome. Risk factors for PVT and the value of screening tools are also analyzed. METHODS: Adult LTx performed from 1987 through 1996 were reviewed. PVT was retrospectively graded according to the operative findings: grade 1: <50% PVT +/- minimal obstruction of the superior mesenteric vein (SMV); grade 2: grade 1 but >50% PVT; grade 3: complete PV and proximal SMV thrombosis; grade 4: complete PV and entire SMV thrombosis. RESULTS: Of 779 LTx, 63 had operatively confirmed PVT (8.1%): 24 had grade 1, 23 grade 2, 6 grade 3, and 10 grade 4 PVT. Being male, treatment for portal hypertension, Child-Pugh class C, and alcoholic liver disease were associated with PVT. Sensitivity of ultrasound (US) in detecting PVT increased with PVT grade and was 100% in grades 3-4. In patients with US-diagnosed PVT, an angiogram was performed and ruled out a false positive US diagnosis in 13%. In contrast with US, angiograms differentiated grade 1 from grade 2, and grade 3 from grade 4 PVT. Grade 1 and 2 PVT were managed by low dissection and/or a thrombectomy; in grade 3 the distal SMV was directly used as an inflow vessel, usually through an interposition donor iliac vein; in grade 4 a splanchnic tributary was used or a thrombectomy was attempted. Transfusion requirements in PVT patients (10 U) were higher than in non-PVT patients (5 U) (P<0.01). In-hospital mortality for PVT patients was 30% versus 12.4% in controls (P<0.01). Patients with PVT had more postoperative complications, renal failure, primary nonfunction, and PV rethrombosis. The overall actuarial 5-year patient survival rate in PVT patients (65.6%) was lower than in controls (76.3%; P=0.04). Patients with grade 1 PVT, however, had a 5-year survival rate (86%) identical to that of controls, whereas patients with grades 2, 3, and 4 PVT had reduced survival rates. The 5-year patient survival rate improved from the 1st to the 2nd era in non-PVT patients (from 72% to 83%; P<0.01), in grade 1 PVT (from 53% to 100%; P<0.01), and in grades 2 to 4 PVT (from 38% to 62%; P=0.11). CONCLUSIONS: The value of US diagnosis in patients with PVT depends on the PVT grade, and false negative diagnoses occur only in incomplete forms of PVT (grades 1-2). The degree of PVT dictates the surgical strategy to be used, thrombectomy/low dissection in grade 1-2, mesoportal jump graft in grade 3, and a splanchnic tributary in grade 4. Taken altogether, PVT patients undergo more difficult surgery, have more postoperative complications, have higher in-hospital mortality rates, and have reduced 5-year survival rates. Analysis by PVT grade, however, reveals that grade 1 PVT patients do as well as controls; only grades 2 to 4 PVT patients have poorer outcomes. With increased experience, results of LTx in PVT patients have improved and, even in severe forms of PVT, a 5-year survival rate >60% can now be achieved.

Chronic hepatitis B is caused by persistent infection with the hepatitis B virus (HBV), a unique DNA virus that replicates through an RNA intermediate produced from a stable covalently closed circular DNA molecule. Viral persistence appears to be due to inadequate innate and adaptive immune responses. Chronic infection has a variable course after several decades resulting in cirrhosis in up to one-third of patients and liver cancer in a proportion of those with cirrhosis. Sensitive assays for HBV DNA levels in serum have been developed that provide important insights into pathogenesis and natural history. Therapy of hepatitis B is evolving. Peginterferon induces long-term remissions in disease in one-third of patients with typical hepatitis B e antigen (HBeAg) positive chronic hepatitis B, but a lesser proportion of those without HBeAg. Several oral nucleoside analogues with activity against HBV have been shown to be effective in suppressing viral levels and improving biochemical and histological features of disease in a high proportion of patients with and without HBeAg, at least in the short term. What is uncertain is which agent or combination of agents is most effective, how long therapy should last, and which criteria should be used to start, continue, switch or stop therapy. Long-term therapy with nucleoside analogues may be the most appropriate approach to treatment, but the expense and lack of data on long-term safety and efficacy make recommendations difficult. Clearly, many basic and clinical research challenges remain in defining optimal means of management of chronic hepatitis B.

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease, and is strongly associated with the metabolic syndrome. In the last decade, it has become apparent that the clinical burden of NAFLD is not restricted to liver-related morbidity or mortality, and the majority of deaths in NAFLD patients are related to cardiovascular disease (CVD) and cancer. These findings have fuelled concerns that NAFLD may be a new, and added risk factor for extrahepatic diseases such as CVD, chronic kidney disease (CKD), colorectal cancer, endocrinopathies (including type 2 diabetes mellitus [T2DM] and thyroid dysfunction), and osteoporosis. In this review we critically appraise key studies on NAFLD-associated extrahepatic disease. There was marked heterogeneity between studies in study design (cross-sectional versus prospective; sample size; presence/absence of well-defined controls), population (ethnic diversity; community-based versus hospital-based cohorts), and method of NAFLD diagnosis (liver enzymes versus imaging versus biopsy). Taking this into account, the cumulative evidence to date suggests that individuals with NAFLD (specifically, nonalcoholic steatohepatitis) harbor an increased and independent risk of developing CVD, T2DM, CKD, and colorectal neoplasms. We propose future studies are necessary to better understand these risks, and suggest an example of a screening strategy.

BACKGROUND & AIMS: We evaluated the efficacy and safety of obeticholic acid (OCA, α-ethylchenodeoxycholic acid) in a randomized controlled trial of patients with primary biliary cirrhosis who had an inadequate response to ursodeoxycholic acid therapy. METHODS: We performed a double-blind study of 165 patients with primary biliary cirrhosis (95% women) and levels of alkaline phosphatase (ALP) 1.5- to 10-fold the upper limit of normal. Patients were randomly assigned to groups given 10 mg, 25 mg, or 50 mg doses of OCA or placebo, once daily for 3 months. Patients maintained their existing dose of ursodeoxycholic acid throughout the study. The primary outcome was change in level of ALP from baseline (day 0) until the end of the study (day 85 or early termination). We also performed an open-label extension of the trial in which 78 patients were enrolled and 61 completed the first year. RESULTS: OCA was superior to placebo in achieving the primary end point. Subjects given OCA had statistically significant relative reductions in mean ALP from baseline to the end of the study (P < .0001 all OCA groups vs placebo). Levels of ALP decreased 21%-25% on average from baseline in the OCA groups and 3% in the placebo group. Sixty-nine percent (68 of 99) of patients given OCA had at least a 20% reduction in ALP compared with 8% (3 of 37) of patients given placebo (P < .0003). Among secondary end points, levels of γ-glutamyl transpeptidase decreased 48%-63%, on average, among subjects given OCA, vs a 7% decrease in the group given placebo; levels of alanine aminotransferase decreased 21%-35% on average among subjects given OCA vs none of the patients given placebo. Pruritus was the principal adverse event; incidence values in the OCA 10 mg, 25 mg, and 50 mg groups were 47% (not significantly different), 87% (P < .0003), and 80% (P < .006), respectively, vs 50% in the placebo group. In the extension study, levels of ALP continued to decrease to a mean level of 202 ± 11 U/L after 12 months vs 285 ± 15 U/L at baseline. CONCLUSIONS: Daily doses of OCA, ranging from 10 to 50 mg, significantly reduced levels of ALP, γ-glutamyl transpeptidase, and alanine aminotransferase, compared with placebo, in patients with primary biliary cirrhosis who had inadequate responses to ursodeoxycholic acid. The incidence and severity of pruritus were lowest among patients who received 10 mg/d OCA. Biochemical responses to OCA were maintained in a 12-month open-label extension trial. ClinicalTrials.gov ID: NCT00550862.

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in developed countries, and accumulating evidence suggests it as the hepatic manifestation of the metabolic syndrome (MS). Although the published prevalence of hepatocellular carcinoma (HCC) is low in NAFLD/NASH patients, most of these data have been derived from areas endemic for viral hepatitis. We recruited 162 adults with HCC between February 2007 and March 2008, investigated the underlying etiologies and determined the prevalence of the MS and related features within each group. Patients with NAFLD/NASH-associated HCC exhibited a higher prevalence of metabolic features (Type 2 diabetes mellitus, hypertension, dyslipidemia, coronary artery disease) compared to non-NAFLD/NASH-HCC. Intriguingly, a significant number (41.7%; p < 0.005) of individuals with NAFLD/NASH-HCC had no evidence of cirrhosis. Patients with alcohol-induced liver disease also displayed many features (14/19, 73.7%) of the MS, although, in contrast to NAFLD/NASH-HCC, alcohol-associated HCC was highly associated with cirrhosis (95.0%; p = 0.064). NAFLD/NASH as the hepatic entity of the MS may itself pose a risk factor for HCC, even in the absence of cirrhosis. The MS may also promote development of HCC among those with alcoholic liver disease. Increased awareness of liver manifestations in the MS may instigate early interventions against developing HCC.

There is a limited access to liver transplantation, however, many organs are discarded based on subjective assessment only. Here we report the VITTAL clinical trial (ClinicalTrials.gov number NCT02740608) outcomes, using normothermic machine perfusion (NMP) to objectively assess livers discarded by all UK centres meeting specific high-risk criteria. Thirty-one livers were enroled and assessed by viability criteria based on the lactate clearance to levels ≤2.5 mmol/L within 4 h. The viability was achieved by 22 (71%) organs, that were transplanted after a median preservation time of 18 h, with 100% 90-day survival. During the median follow up of 542 days, 4 (18%) patients developed biliary strictures requiring re-transplantation. This trial demonstrates that viability testing with NMP is feasible and in this study enabled successful transplantation of 71% of discarded livers, with 100% 90-day patient and graft survival; it does not seem to prevent non-anastomotic biliary strictures in livers donated after circulatory death with prolonged warm ischaemia.

BACKGROUND & AIMS: Insulin resistance and lipotoxicity are pathognomonic in non-alcoholic steatohepatitis (NASH). Glucagon-like peptide-1 (GLP-1) analogues are licensed for type 2 diabetes, but no prospective experimental data exists in NASH. This study determined the effect of a long-acting GLP-1 analogue, liraglutide, on organ-specific insulin sensitivity, hepatic lipid handling and adipose dysfunction in biopsy-proven NASH. METHODS: Fourteen patients were randomised to 1.8mg liraglutide or placebo for 12-weeks of the mechanistic component of a double-blind, randomised, placebo-controlled trial (ClinicalTrials.gov-NCT01237119). Patients underwent paired hyperinsulinaemic euglycaemic clamps, stable isotope tracers, adipose microdialysis and serum adipocytokine/metabolic profiling. In vitro isotope experiments on lipid flux were performed on primary human hepatocytes. RESULTS: Liraglutide reduced BMI (-1.9 vs. +0.04kg/m(2); p<0.001), HbA1c (-0.3 vs. +0.3%; p<0.01), cholesterol-LDL (-0.7 vs. +0.05mmol/L; p<0.01), ALT (-54 vs. -4.0IU/L; p<0.01) and serum leptin, adiponectin, and CCL-2 (all p<0.05). Liraglutide increased hepatic insulin sensitivity (-9.36 vs. -2.54% suppression of hepatic endogenous glucose production with low-dose insulin; p<0.05). Liraglutide increased adipose tissue insulin sensitivity enhancing the ability of insulin to suppress lipolysis both globally (-24.9 vs. +54.8pmol/L insulin required to ½ maximally suppress serum non-esterified fatty acids; p<0.05), and specifically within subcutaneous adipose tissue (p<0.05). In addition, liraglutide decreased hepatic de novo lipogenesis in vivo (-1.26 vs. +1.30%; p<0.05); a finding endorsed by the effect of GLP-1 receptor agonist on primary human hepatocytes (24.6% decrease in lipogenesis vs. untreated controls; p<0.01). CONCLUSIONS: Liraglutide reduces metabolic dysfunction, insulin resistance and lipotoxicity in the key metabolic organs in the pathogenesis of NASH. Liraglutide may offer the potential for a disease-modifying intervention in NASH.

The role played by chemokines in regulating the selective recruitment of lymphocytes to different tissue compartments in disease is poorly characterized. In hepatitis C infection, inflammation confined to portal areas is associated with a less aggressive course, whereas T cell infiltration of the liver parenchyma is associated with progressive liver injury and cirrhosis. We propose a mechanism to explain how lymphocytes are recruited to hepatic lobules during bursts of necroinflammatory activity in chronic hepatitis C infection. We report here that lymphocytes infiltrating hepatitis C-infected liver express high levels of the chemokine receptors CCR5 and CXCR3. However, whereas the CCR5 ligands macrophage inflammatory protein-1alpha and -1beta were largely confined to vessels within portal tracts, the CXCR3 ligands IFN-inducible protein-10 and monokine-induced by IFN-gamma were selectively up-regulated on sinusoidal endothelium. In vitro, human hepatic sinusoidal endothelial cells secreted IFN-inducible protein-10 and monokine-induced by IFN-gamma in response to stimulation with IFN-gamma in combination with either IL-1 or TNF-alpha. This suggests that intrahepatic Th1 cytokines drive the increased expression of IFN-inducible protein-10 and monokine-induced by IFN-gamma and thereby promote the continuing recruitment of CXCR3-expressing T cells into the hepatic lobule in chronic hepatitis C infection.

Currently, no vaccine exists for hepatitis C virus (HCV), a major pathogen thought to infect 170 million people globally. Many studies suggest that host T cell responses are critical for spontaneous resolution of disease, and preclinical studies have indicated a requirement for T cells in protection against challenge. We aimed to elicit HCV-specific T cells with the potential for protection using a recombinant adenoviral vector strategy in a phase 1 study of healthy human volunteers. Two adenoviral vectors expressing NS proteins from HCV genotype 1B were constructed based on rare serotypes [human adenovirus 6 (Ad6) and chimpanzee adenovirus 3 (ChAd3)]. Both vectors primed T cell responses against HCV proteins; these T cell responses targeted multiple proteins and were capable of recognizing heterologous strains (genotypes 1A and 3A). HCV-specific T cells consisted of both CD4+ and CD8+ T cell subsets; secreted interleukin-2, interferon-γ, and tumor necrosis factor-α; and could be sustained for at least a year after boosting with the heterologous adenoviral vector. Studies using major histocompatibility complex peptide tetramers revealed long-lived central and effector memory pools that retained polyfunctionality and proliferative capacity. These data indicate that an adenoviral vector strategy can induce sustained T cell responses of a magnitude and quality associated with protective immunity and open the way for studies of prophylactic and therapeutic vaccines for HCV.

Primary sclerosing cholangitis (PSC), a chronic inflammatory liver disease characterized by progressive bile duct destruction, develops as an extra-intestinal complication of inflammatory bowel disease (IBD) (Chapman, R.W. 1991. Gut. 32:1433-1435). However, the liver and bowel inflammation are rarely concomitant, and PSC can develop in patients whose colons have been removed previously. We hypothesized that PSC is mediated by long-lived memory T cells originally activated in the gut, but able to mediate extra-intestinal inflammation in the absence of active IBD (Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Adams. 2002. Lancet. 359:150-157). In support of this, we show that liver-infiltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemokine CCL25 that activates alpha4beta7 binding to mucosal addressin cell adhesion molecule 1 on the hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the pathogenesis of extra-intestinal complications of IBD.

BACKGROUND: The T-cell receptor (TCR), located on the surface of T cells, is responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an inflammatory response. Analysing the TCR repertoire may help to gain a better understanding of the immune system features and of the aetiology and progression of diseases, in particular those with unknown antigenic triggers. The extreme diversity of the TCR repertoire represents a major analytical challenge; this has led to the development of specialized methods which aim to characterize the TCR repertoire in-depth. Currently, next generation sequencing based technologies are most widely employed for the high-throughput analysis of the immune cell repertoire. RESULTS: Here, we report on the latest methodological advancements in the field by describing and comparing the available tools; from the choice of the starting material and library preparation method, to the sequencing technologies and data analysis. Finally, we provide a practical example and our own experience by reporting some exemplary results from a small internal benchmark study, where current approaches from the literature and the market are employed and compared. CONCLUSIONS: Several valid methods for clonotype identification and TCR repertoire analysis exist, however, a gold standard method for the field has not yet been identified. Depending on the purpose of the scientific study, some approaches may be more suitable than others. Finally, due to possible method specific biases, scientists must be careful when comparing results obtained using different methods.

UNLABELLED: The biochemical response to ursodeoxycholic acid (UDCA)--so-called "treatment response"--strongly predicts long-term outcome in primary biliary cholangitis (PBC). Several long-term prognostic models based solely on the treatment response have been developed that are widely used to risk stratify PBC patients and guide their management. However, they do not take other prognostic variables into account, such as the stage of the liver disease. We sought to improve existing long-term prognostic models of PBC using data from the UK-PBC Research Cohort. We performed Cox's proportional hazards regression analysis of diverse explanatory variables in a derivation cohort of 1,916 UDCA-treated participants. We used nonautomatic backward selection to derive the best-fitting Cox model, from which we derived a multivariable fractional polynomial model. We combined linear predictors and baseline survivor functions in equations to score the risk of a liver transplant or liver-related death occurring within 5, 10, or 15 years. We validated these risk scores in an independent cohort of 1,249 UDCA-treated participants. The best-fitting model consisted of the baseline albumin and platelet count, as well as the bilirubin, transaminases, and alkaline phosphatase, after 12 months of UDCA. In the validation cohort, the 5-, 10-, and 15-year risk scores were highly accurate (areas under the curve: >0.90). CONCLUSIONS: The prognosis of PBC patients can be accurately evaluated using the UK-PBC risk scores. They may be used to identify high-risk patients for closer monitoring and second-line therapies, as well as low-risk patients who could potentially be followed up in primary care.

Antiviral T cell responses in hepatotropic viral infections such as hepatitis B virus (HBV) are profoundly diminished and prone to apoptotic deletion. In this study, we investigate whether the large population of activated NK cells in the human liver contributes to this process. We show that in vitro removal of NK cells augments circulating CD8(+) T cell responses directed against HBV, but not against well-controlled viruses, in patients with chronic hepatitis B (CHB). We find that NK cells can rapidly eliminate HBV-specific T cells in a contact-dependent manner. CD8(+) T cells in the liver microcirculation are visualized making intimate contact with NK cells, which are the main intrahepatic lymphocytes expressing TNF-related apoptosis-inducing ligand (TRAIL) in CHB. High-level expression of the TRAIL death receptor TRAIL-R2 is found to be a hallmark of T cells exposed to the milieu of the HBV-infected liver in patients with active disease. Up-regulation of TRAIL-R2 renders T cells susceptible to caspase-8-mediated apoptosis, from which they can be partially rescued by blockade of this death receptor pathway. Our findings demonstrate that NK cells can negatively regulate antiviral immunity in chronic HBV infection and illustrate a novel mechanism of T cell tolerance in the human liver.