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 serum AST, ALT, and IgG* levels Histological remission Absence of inflammation in liver tissue after treatment Treatment failure Worsening laboratory or histological findings despite compliance with standard therapy Incomplete response Improvement of laboratory and histological findings that are insufficient to satisfy criteria for remission Relapse Exacerbation of disease activity after induction of remission and drug withdrawal (or nonadherence) Treatment intolerance Inability to continue maintenance therapy due to drug‐related side effects *Patients with cirrhosis in biochemical remission may have persistent elevation of IgG. AIH is an immune‐mediated inflammatory liver disease of uncertain cause which affects all ages, both genders, and all ethnicities. Patients may be asymptomatic, be chronically ill, or present with acute liver failure (ALF); and the diagnosis must be considered in all patients with acute or chronic liver inflammation, including patients with graft dysfunction after LT. AIH does not have a signature diagnostic feature, and the diagnosis requires the presence of a constellation of typical features which can vary between patients with the same disease and can occur in other liver diseases. Progression to advanced hepatic fibrosis, cirrhosis, death from liver failure, or LT are possible outcomes. Treatment with immunosuppressive agents has been life‐saving, but management regimens may be long‐term, associated with serious side effects, and variably effective. Background Epidemiology AIH occurs at all ages and within all ethnic groups, and its manifestations appear to vary by race and ethnicity. Alaskan Natives have a high frequency of icteric AIH at presentation, Hispanics more commonly present with cirrhosis, and African Americans have accelerated progression of disease and a higher rate of recurrence after LT compared to other races.5 Female predominance occurs in adults (71%‐95% women)7 and children (60%‐76% girls).13 Early epidemiological reports suggested that the onset of AIH had age peaks at 10‐30 and 40‐60 years, but the findings may have been influenced by referral bias.17 Older peak ages at onset (>60 years) have been reported in Denmark11 and New Zealand.10 The estimated incidence of AIH varies worldwide depending on the region and the age at onset. Incidence rates in adults range from 0.67 (southern Israel) to 2 cases per 100,000 person‐years (Canterbury region of New Zealand).10 Pediatric incidences are lower, ranging from 0.23 (Canada)16 to 0.4 per 100,000 person‐years (United States).15 Over the past few decades there has been a near 50% increase in incidence in Spain, Denmark, Sweden, and the Netherlands.11 The prevalence of AIH in adults ranges from 4 (Singapore) to 42.9 (Alaska natives) per 100,000 persons.17 The prevalence in children ranges from 2.4 (non‐native Canadian children)26 and 3 per 100,000 persons (United States)15 to 9.9 per 100,000 persons (native Canadian children).17 Genetic Predispositions In common with other autoimmune diseases, the primary genetic associations in AIH involve major histocompatibility complex loci. Human leukocyte antigen (HLA) associations cluster within the conserved 8.1 ancestral haplotype which defines the alleles carried by most Caucasians27 and results from linkage disequilibrium within HLA class I, II, and III loci: HLA‐A1, Cw7, B8, TNFAB*a2b3, TNFN*S, C2*C, Bf*s, C4A*Q0, C4B*1, DRB1*03:01, DRB1*04:01, DRB1*13:01, DRB3*01:01, DQA1*05:01, DQB1*02:01.28HLA‐DRB1*03:01 haplotypes associated with AIH are the result of additional genetic recombinations. AIH also has non‐HLA genetic associations, but the odds ratios (ORs) for risk of AIH are far lower than those for HLA alleles. Susceptibility for AIH has been associated with genetic polymorphisms encoding cytotoxic T lymphocyte antigen‐4 (CTLA‐4),33 tumor necrosis factor‐alpha (TNF‐α),34 Fas (cluster of differentiation 95 [CD95] or apoptosis antigen‐1),36 vitamin D receptor,38 signal transducer and activator of transcription 4,40 transforming growth factor‐beta 1,41 macrophage migration inhibitory factor,42 SH2B adapter protein 3,43 caspase recruitment domain family member 10,43 and the interleukin‐23 (IL‐23) receptor.44 Dysfunctional products of genetic variants or deficient levels of gene product may disrupt homeostatic mechanisms that affect the proliferation and survival of autoreactive T and B cells, regulate cytokine production, and modulate inflammatory and immune responses. AIH is a complex genetic disease that requires interplay among genetic, epigenetic, immunologic, and environmental factors. A rare exception is AIH associated with an autosomal recessive mutation in the autoimmune regulator gene on chromosome 21q22.3, which has been associated with autoimmune polyglandular syndrome type 1 (APS‐1).45 Environmental exposures play greater roles than genetics in shaping the immune repertoire, and specific environmental factors, such as viral infections or xenobiotic exposures, can act as environmental triggers for loss of self‐tolerance to autoantigens in persons genetically susceptible to AIH.46 Pathogenesis Autoreactive CD4 and CD8 T cells break self‐tolerance to hepatic autoantigens as the result of environmental triggers and inability of autoantigen‐specific natural T regulatory cells (nTregs) and inducible T regulatory cells (iTregs) to prevent autoreactivity48 (Fig. 1). Concurrently, in the absence of effective B regulatory cell (Breg) inhibition, autoreactive B cells produce autoantibodies.51 Peptide autoantigens are presented by class II and class I HLA alleles to autoreactive T‐cell receptors on CD4 T helper (Th) cells and CD8 cytotoxic T lymphocytes (CTLs), respectively. Binding of different autoantigens to B‐cell receptors initiates secretion of specific autoantibodies.Figure 1: Current concepts of the immunopathogenesis of AIH. Current knowledge supports a multistep working model of the immunopathogenesis of AIH, in which a break in self‐tolerance to hepatocyte autoantigens initiates immunological responses causing progressive hepatic necroinflammation and fibrogenesis.50 In the first step, thymic autoantigen‐specific nTregs are incapable of preventing immune responses to hepatic autoantigens during hepatic or systemic immune responses to environmental triggers, such as viral infections or xenobiotics. In the second step, professional antigen‐presenting cells (APCs) present autoantigenic peptides to autoreactive α/β T cell receptors (TCRs) on naive CD4+ Th cells, and CD8+ T cells and APCs activate MAIT cells by presenting bacterially processed vitamin B antigens to MAIT cell TCRs.54 Costimulation is a crucial third step, which induces expression of T‐cell genes required for proliferation, differentiation, and maturation of autoantigen‐specific CD4+ Th subsets (e.g., Th1, Th2, Th3, Th9, Th17, iTregs, Tr1, Tfh cells) and both CD8+ CTLs and CD8+ Tregs. In the fourth step, secretion of specific cytokines by subsets of CD4+ Th cells produces a variety of immunological sequelae, including CD4+ Th2 cytokine stimulation of B‐cell autoantibody production, CD4+ Tfh‐cell activation of B cells into antibody‐secreting plasma cells, Treg stimulation of Breg development through IL‐35 mechanisms and cytokine‐activated macrophages, and CD4+ Th17 cell–mediated pathogenic cytotoxicity. The fifth step is the cumulative failure of CD4+ and CD8+ Tregs and Bregs to control autoantigen‐specific effector mechanisms causing hepatic injury.53 Moreover, exposure of CD4+ iTregs to specific cytokines can transform them from regulatory cells into pathogenic CD4+ Th17 cells.52 The sixth step is the generation of complex portal inflammatory infiltrates of effector cells that cause cytotoxicity of periportal and lobular hepatocytes. Necroinflammatory destruction of hepatocytes results in activation of periportal stellate cells, which amplify local immune responses through contact‐dependent and independent mechanisms and cause progressive portal fibrosis, culminating in cirrhosis in the absence of effective immunosuppressive therapy. Abbreviations: Ag, antigen; IFN, interferon; TGF, transforming growth factor.The composition of the local cytokine milieu dictates CD4 Th cells to differentiate into Th1, Th2, Th9, Th17, iTregs, and T follicular helper (Tfh) cell subsets in the presence of costimulatory signaling.50 CD4 Th1 cells secrete cytokines that promote proliferation of autoantigen‐specific CD8 CTLs and activation of macrophages. CD4 Th2 cytokines augment immunoglobulin production by B cells, while cytokines produced by Tfh cells induce their conversion to immunoglobulin G (IgG)–secreting plasma cells. CD4 Th17 cells intensify inflammation and tissue injury. Autoantigen‐specific iTregs can down‐regulate the proliferation and functions of all CD4 Th subtypes, and inadequate numbers and/or dysfunction of CD4 iTregs may play a key role in AIH.52 Cytokine‐mediated transformation of CD4 iTregs into pathogenic CD4 Th17 cells also promotes perpetuation of AIH. Low doses of IL‐2 preferentially stimulate proliferation and function of CD4 iTregs, while high doses promote production of other pathogenic CD4 Th subsets. Mucosal invariant T (MAIT) cells that react with bacterially processed vitamin B antigens presented by major histocompatibility complex class I–related molecules congregate in the peribiliary region in AIH.54 MAIT cells can express characteristics of CD4 Th1 and Th17 cells, and they may transform CD4 iTregs into proinflammatory CD4 Th17 cells. Inflammatory infiltrates composed of CD4 Th subsets, CD8 CTLs, MAIT cells, B cells, plasma cells, and innate immune cells, including natural killer (NK) and NK T cells and activated macrophages, can accumulate within the portal tracts. Adhesion molecules and chemokines mediate transendothelial migration of immune cells into tissues.50 Extension of inflammation into periportal hepatocytes (interface hepatitis) and lobular hepatitis causes apoptosis of hepatocytes and fibrogenesis in untreated patients with AIH. Uptake and processing of immune complexes of autoantigen and immunoglobulin by antigen‐presenting cells greatly increases activation of autoantigen‐specific CD8 CTLs, and autoantibodies may enhance CD8 CTL cytotoxicity of hepatocytes. Diagnosis Diagnostic Requisites and Subtypes The diagnosis of AIH is based on histological abnormalities (interface hepatitis), characteristic clinical and laboratory findings (elevated serum aspartate aminotransferase [AST] and alanine aminotransferase [ALT] levels and increased serum IgG concentration), and the presence of one or more characteristic autoantibodies.2 AIH lacks a signature diagnostic marker, and the diagnosis requires characteristic features and the exclusion of other diseases that may resemble it (e.g., viral hepatitis, drug‐induced liver injury, Wilson's disease, hereditary hemochromatosis).56 There are two types of AIH, based on the specific autoantibodies that are present. Type 1 is characterized by antinuclear antibodies (ANA) and/or smooth muscle antibodies (SMA)/anti‐actin antibodies, and type 2 is characterized by antibodies to liver kidney microsome type 1 (anti‐LKM1), usually in the absence of ANA and SMA.57 The characteristic clinical features of these two types are presented in Table 3. In addition, up to 20% of AIH cases are negative for ANA, SMA, and LKM1 autoantibodies, despite the presence of other characteristic features of AIH (seronegative AIH). If seronegative AIH is suspected, other autoantibodies may be sought, as indicated in Table 4 and Fig. 2. Classification of AIH into types assists in management and aids in predicting outcomes in children, but it may be less informative in adults.58 Table 3 - Characteristic Features of Type 1 and Type 2 AIH Features Type 1 AIH Type 2 AIH Frequency US adults, 96%61 US children, 9%‐12%14 UK children, 38%13 Age at presentation Peripubertal and adults Usually under 14 years153 Mode of presentation Chronic symptoms common Acute onset (~40%) Ascites or GI bleeding rare Acute liver failure possible555 Asymptomatic in 25%‐34% Relapse frequent108 Acute in 25%‐75% Acute severe in 2%‐6% Laboratory features Hypergammaglobulinemia IgA levels may be reduced153 Autoantibodies ANA Anti‐LKM1 SMA, anti‐actin [Anti‐LC1, Anti‐LKM3] SLA Concurrent immune diseases Autoimmune thyroiditis Autoimmune thyroiditis Rheumatic diseases Diabetes mellitus IBD Vitiligo Autoimmune overlap with PSC (ASC in children) Common in children Rare Atypical pANCA‐positive Atypical pANCA‐negative Overlap with PBC Seen in adults (not children) Not reported Cirrhosis at presentation Rare after drug withdrawal usually Abbreviations: serum immunoglobulin Table 4 - Autoantibodies in the Diagnosis of AIH Diagnostic ANA Type 1 Type 1 LKM1 Type 2 SLA Type 1 after with Type 1 Type 1 Type 1 family Type 2 Type 2 of overlap Type 1 Abbreviations: autoimmune Diagnostic for the of AIH after exclusion of and diseases. ANA and should be in adults and antibodies to LKM1 should be if ANA and are ANA, SMA, and LKM1 should be in all patients at presentation The findings of the liver support the diagnosis of AIH or that an overlap AIH with or The absence of ANA, SMA, and LKM1 additional that can antibodies to tissue and for one of these autoantibodies support the diagnosis of AIH or other including disease Abbreviations: tissue ANA, SMA, and the for the diagnosis of AIH (Table ANA are in of American adults with AIH at presentation, are present in and are present in of patients with AIH have ANA, SMA, or as an at and have ANA can also occur as an in primary chronic hepatitis chronic hepatitis B liver disease and chronic liver disease and can occur as an in PSC chronic hepatitis and chronic liver disease ANA and are concurrent in of liver diseases of AIH, and the diagnostic for AIH from to if two autoantibodies are at Anti‐LKM1 are commonly in the absence of ANA and SMA, and this has their after first for ANA and (Fig. have a low for AIH in American adults and their after first the absence of ANA and is in these patients. Anti‐LKM1 are in of and Canadian children with and of ANA, SMA, and are usually at in adults and children disease and treatment but they are not of disease activity or treatment to liver antigen are present in of patients with type 1 AIH, and they have high for the (Table have been the of AIH in of and they have been associated with severe disease and after drug Atypical antibodies are frequently present in patients with type 1 AIH but they diagnostic in overlap and liver may be the only autoantibodies are a of SMA, and they are present in of patients with AIH and (Table to is an that is present in of patients with AIH and of patients with to anti‐actin and has been associated with severe acute AIH, treatment and to liver type 1 are present in of patients with and they occur in children with severe liver (Table are present in of patients with type 2 and may be in seronegative and have not been rigorously in the should be and with the clinical may be depending on results of the and in with the diagnostic (Fig. Histological Findings The diagnosis of AIH be liver and histological hepatitis is the histological of AIH, by plasma cell in and lobular hepatitis in necrosis is also in and it occurs with frequency in patients with and is the of one cell into with both cells to is present in of patients with AIH, and hepatocyte are present in (Fig. of the histological findings is specific for AIH, but the findings of interface hepatitis with portal or cells into the and are considered typical of Histological features characteristic of AIH. inflammatory of the portal and interface hepatitis of the portal and cell predominance in a portal inflammatory and of a and A hepatocyte and of hepatocytes and are of of of is present in of adults at presentation, in the as as in of Cirrhosis in of adults with necrosis or The histological examination at presentation is to or concurrent the of inflammatory and the of plasma cells may be present in patients with but the clinical of this Histological findings of are present in 17%‐30% of patients with and liver tissue examination may patients with AIH and who are at increased risk of and The histological features of AIH with ALF in the and of is present in plasma inflammatory in hepatic necrosis in and in of patients with ALF have two or all of these Diagnostic The diagnostic scoring system of the Autoimmune was by an panel in in and in Table The scoring system has greater for AIH compared to the scoring system whereas the scoring system has and clinical judgment as the The diagnostic scoring system is for patients with complex or whereas the scoring system is most for typical of patients with the scoring system should be considered the system a low In children, a of to the of the criteria a of and a of In that were associated with seronegative AIH. The diagnostic scoring system can be to children and lower autoantibody than in adults as diagnostic of the serum for the serum in the with the serum or may the of the scoring system for children by the of to the and scoring systems of by

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, and is rapidly becoming the most common cause of liver disease in Western countries. Indeed, NAFLD is now recognized to be the aetiology in many cases previously labelled as cryptogenic cirrhosis.

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: 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.

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.

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.

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.

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.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist.

Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by clinical homogeneity among patients, an overwhelming female predominance, production of a multilineage immune response to mitochondrial autoantigens, inflammation of small bile ducts, and in some patients the development of fibrosis and cirrhosis. The targets in this disease are small bile ducts, and the prototypic serologic response includes antimitochondrial antibodies (AMAs). Several key observations have greatly advanced our understanding of PBC. First, the multilineage immune response, including AMAs, is directed at the E2 component of the 2-oxo-dehydrogenase pathway, particularly PDC-E2. Second, such autoantibodies may be identified years before the clinical diagnosis of disease. Third, the autoreactive T cell precursor frequency for both CD4 and CD8 cells is significantly higher in liver and regional lymph node than in blood, so the multilineage antimitochondrial response may be required for the development of this disease. Fourth, the apotope of biliary cells contains intact PDC-E2; this apotope, in a setting that includes granulocyte macrophage colony-stimulating factor-stimulated macrophages and AMAs, produces an intense proinflammatory response. Fifth, several mouse models of PBC highlight the importance of loss of tolerance to PDC-E2 as well as a critical role for the interleukin (IL)-12 signaling pathway. Finally, genome-wide association studies suggest an important role for the IL-12 pathway in disease susceptibility. Taken together, these findings have resulted in a better understanding of the mechanism for selective biliary cell destruction and have also suggested unique pathways for therapeutic intervention.