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Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease affecting all ages, characterised by elevated transaminase and immunoglobulin G levels, positive autoantibodies, interface hepatitis on histology and good response to immunosuppressive treatment. If untreated, it has a poor prognosis. The aim of this review is to analyse AIH therapeutic interventions with reference to our knowledge of the pathogenesis of AIH. Standard treatment, based on steroids and azathioprine, leads to disease remission in 80-90% of patients. Alternative first-line treatment with budesonide is effective in adults, but less so in the juvenile form of AIH; first-line treatment with ciclosporin does not provide convincing advantages compared to standard treatment. Second-line treatments are needed for patients not responding or intolerant to first-line standard management. Mycophenolate mofetil is the most widely used second-line drug, and has good efficacy particularly for patients intolerant to azathioprine, but is teratogenic. Only few and heterogeneous data on calcineurin inhibitors and m-TOR inhibitors are available. Biologicals, including anti-tumour necrosis factor- α and anti-CD20 monoclonal antibodies, have given ambivalent results and may have severe side-effects. Clinical trials with new therapeutic options aiming at targeting B lymphocytes and proinflammatory cytokines, or expanding regulatory T cells to restore tolerance are ongoing.

Circulating autoantibodies are a key diagnostic tool in autoimmune hepatitis (AIH), being positive in 95% of the cases if tested according to dedicated guidelines issued by the International Autoimmune Hepatitis Group. They also allow the distinction between type 1 AIH, characterized by positive anti-nuclear and/or anti-smooth muscle antibody, and type 2 AIH, characterized by positive anti-liver kidney microsomal type 1 and/or anti-liver cytosol type 1 antibody. Anti-soluble liver antigen is the only AIH-specific autoantibody, and is found in 20–30% of both type 1 and type 2 AIH. Anti-neutrophil cytoplasmic antibody is frequently positive in type 1 AIH, being associated also with inflammatory bowel disease and with primary/autoimmune sclerosing cholangitis. The reference method for autoantibody testing remains indirect immunofluorescence on triple tissue (rodent liver, kidney and stomach), allowing both the detection of the majority of liver-relevant reactivities, including those autoantibodies whose molecular target antigens are unknown. Of note, the current knowledge of the clinical significance of autoantibodies relies on studies based on this technique. However, immunofluorescence requires trained laboratory personnel, is observer-dependent, and lacks standardization, leading to ongoing attempts at replacing this method with automated assays, the sensitivity, and specificity of which, however, require further studies before they can be used as a reliable alternative to immunofluorescence; currently, they may be used as complementary to immunofluorescence.

Autoimmune hepatitis (AIH) is a T-cell mediated, inflammatory liver disease affecting all ages and characterized by female preponderance, elevated serum transaminase and immunoglobulin G levels, positive circulating autoantibodies, and presence of interface hepatitis at liver histology. AIH type 1, affecting both adults and children, is defined by positive anti-nuclear and/or anti-smooth muscle antibodies, while type 2 AIH, affecting mostly children, is defined by positive anti-liver-kidney microsomal type 1 and/or anti-liver cytosol type 1 antibody. While the autoantigens of type 2 AIH are well defined, being the cytochrome P4502D6 (CYP2D6) and the formiminotransferase cyclodeaminase (FTCD), in type 1 AIH they remain to be identified. AIH-1 predisposition is conferred by possession of the MHC class II HLA DRB1*03 at all ages, while DRB1*04 predisposes to late onset disease; AIH-2 is associated with possession of DRB1*07 and DRB1*03. The majority of patients responds well to standard immunosuppressive treatment, based on steroid and azathioprine; second- and third-line drugs should be considered in case of intolerance or insufficient response. This review offers a comprehensive overview of pathophysiological and clinical aspects of AIH.

Background and aims: genetic predisposition to autoimmune hepatitis (AIH) in adults is associated with possession of human leukocyte antigen (HLA) class I (A*01, B*08) and class II (DRB1*03, -04, -07, or -13) alleles, depending on geographic region. Juvenile autoimmune liver disease (AILD) comprises AIH-1, AIH-2, and autoimmune sclerosing cholangitis (ASC), which are phenotypically different from their adult counterparts. We aimed to define the relationship between HLA profile and disease course, severity, and outcome in juvenile AILD. Approach and results: we studied 236 children of European ancestry (152 female [64%], median age 11.15 years, range 0.8-17), including 100 with AIH-1, 59 with AIH-2, and 77 with ASC. The follow-up period was from 1977 to June 2019 (median 14.5 years). Class I and II HLA genotyping was performed using PCR/sequence-specific primers. HLA B*08, -DRB1*03, and the A1-B8-DR3 haplotype impart predisposition to all three forms of AILD. Homozygosity for DRB1*03 represented the strongest risk factor (8.8). HLA DRB1*04, which independently confers susceptibility to AIH in adults, was infrequent in AIH-1 and ASC, suggesting protection; and DRB1*15 (DR15) was protective against all forms of AILD. Distinct HLA class II alleles predispose to the different subgroups of juvenile AILD: DRB1*03 to AIH-1, DRB1*13 to ASC, and DRB1*07 to AIH-2. Possession of homozygous DRB1*03 or of DRB1*13 is associated with fibrosis at disease onset, and possession of these two genes in addition to DRB1*07 is associated with a more severe disease in all three subgroups. Conclusions: unique HLA profiles are seen in each subgroup of juvenile AILD. HLA genotype might be useful in predicting responsiveness to immunosuppressive treatment and course., Fifth Medical Center of PLA General Hospital; Roger Dobson Funds; King's College Hospital Charity; Medical Research Council Clinician Scientist Fellowship; Medical Research Council PhD Studentship; Alex Mowat PhD Studentship; King's College Hospital Charity; National Institute for Health Research University College London Hospital/University College London Biomedical Research Centre

A diet rich in saturated fat and carbohydrates causes low-grade chronic inflammation in several organs, including the liver, ultimately driving nonalcoholic steatohepatitis. In this setting, environment-driven lipotoxicity and glucotoxicity induce liver damage, which promotes dendritic cell activation and generates a major histocompatibility complex class II (MHC-II) immunopeptidome enriched with peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and the stress responses. Here, we demonstrated that lipotoxicity and glucotoxicity, as driven by a high-fat and high-fructose (HFHF) diet, promoted MHC-II presentation of nested T and B cell epitopes from protein disulfide isomerase family A member 3 (PDIA3), which is involved in immunogenic cell death. Increased MHC-II presentation of PDIA3 peptides was associated with antigen-specific proliferation of hepatic CD4 + immune infiltrates and isotype switch of anti-PDIA3 antibodies from IgM to IgG3, indicative of cellular and humoral PDIA3 autoreactivity. Passive transfer of PDIA3-specific T cells or PDIA3-specific antibodies also exacerbated hepatocyte death, as determined by increased hepatic transaminases detected in the sera of mice subjected to an HFHF but not control diet. Increased humoral responses to PDIA3 were also observed in patients with chronic inflammatory liver conditions, including autoimmune hepatitis, primary biliary cholangitis, and type 2 diabetes. Together, our data indicated that metabolic insults caused by an HFHF diet elicited liver damage and promoted pathogenic immune autoreactivity driven by T and B cell PDIA3 epitopes.

Genetic susceptibility to autoimmune liver diseases is conferred mainly by polymorphisms of genes encoding for the human leukocyte antigens (HLA). The strongest predisposition to autoimmune hepatitis type 1 (AIH-1) is linked to the allele DRB1*03:01, possession of which is associated with earlier disease onset and more severe course. In populations where this allele is very rare, such as in Asia, and in DRB1*03-negative patients, risk of AIH-1 is conferred by DRB1*04, which is associated with later disease onset and milder phenotype. AIH type 2 (AIH-2) is associated with DRB1*07. The pediatric condition referred to as autoimmune sclerosing cholangitis (ASC), is associated with the DRB1*13 in populations of Northern European ancestry. DRB1*1501 is protective from AIH-1, AIH-2 and ASC in Northern European populations. Possession of the DRB1*08 allele is associated with an increased risk of primary biliary cholangitis (PBC) across different populations. DRB1*03:01 and B*08:01 confer susceptibility to primary sclerosing cholangitis (PSC), as well as DRB1*13 and DRB1*15 in Europe. The hepatic blood supply is largely derived from the splanchnic circulation, suggesting a pathophysiological role of the gut microbiome. AIH appears to be associated with dysbiosis, increased gut permeability, and translocation of intestinal microbial products into the circulation; molecular mimicry between microbial and host antigens may trigger an autoaggressive response in genetically-predisposed individuals. In PBC an altered enteric microbiome may affect intestinal motility, immunological function and bile secretion. Patients with PSC have a gut microbial profile different from health as well as from patients with inflammatory bowel disease without PSC.

The classification and diagnosis of systemic autoimmune diseases are frequently based on a collection of criteria composed of clinical, laboratory, imaging, and pathology elements that are strongly associated with the respective disease. Autoantibodies are a distinctive hallmark and have a prominent position in the classification criteria of many autoimmune diseases. The indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA), historically known as the antinuclear antibody test, is a method capable of detecting a wide spectrum of autoantibodies. A positive HEp-2 IFA test is part of the classification criteria for systemic lupus erythematosus (SLE) and juvenile idiopathic arthritis (JIA), as well as the diagnostic criteria for autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC). A positive HEp-2 IFA test can appear as different morphological patterns that are indicative of the most probable autoantibody specificities in the sample. Only some of the HEp-2 IFA patterns are associated with the specific autoantibodies relevant to SLE, JIA, AIH, and PBC, whereas some other patterns occur mainly in non-related conditions and even in apparently healthy individuals. This paper provides a critical review on the subject and proposes that the classification and diagnostic criteria for SLE, JIA, AIH, and PBC could be improved by a modification on the HEp-2 IFA (ANA) criterion in that the staining patterns accepted for each of these diseases should be restricted according to the respective relevant autoantibody specificities., Highlights • Autoantibodies play a prominent role in the classification or diagnostic criteria of many autoimmune diseases. • ANA test is part of the classification criteria for SLE and JIA, as well as the diagnostic criteria for AIH. • Different HEp-2 IFA patterns indicate different autoantibodies and only some are associated with a specific disease. • ANA classification/diagnostic criteria should reflect the HEp-2 IFA patterns associated to the relevant autoantibodies.

Background and aims Chronic hepatitis C virus (HCV) infection is associated with a wide range of immunopathological manifestations, which are significantly improved by successful interferon-based treatment. There is paucity of data on the impact of interferon-free HCV clearance on immunopathological manifestations, which might be expected to disappear more frequently as compared to what reported in interferon-induced HCV-clearance. We have investigated liver autoimmune serology before and after interferon-free clearance of HCV by treatment with direct acting antiviral agents (DAA). Method Patients within the Swiss Hepatitis C Cohort Study who underwent successful (SVR 12) HCV treatment with DAA were tested for autoimmune liver serology according to dedicated guidelines before and at least 6 months after end of treatment. Results A total of 235 patients were included; 62% males; median age 56 years; 27% with cirrhosis. Median time between end of DAA treatment and post-treatment serum sampling was 17 months. At least one autoantibody before treatment was found in 175 (74%) patients ; 32 (14%) were positive for 2 autoantibodies; no patient was positive for anti-SLA, anti-LC1 or typical AMA before or after DAA. ANA disappeared in 34%, SMA in 52% and anti-LKM1 in one of two patients after successful treatment, but, unexpectedly, one or more autoantibodies appeared in 27% of pre-treatment negative subjects. Conclusion HCV clearance by DAA is associated with autoantibody disappearance in more than one third of the patients who were positive before treatment. However, the majority of the patients remain autoantibody-positive and 27% of those who were negative before treatment developed autoantibodies after DAA-induced HCV clearance. These data confirm that HCV infection is associated with autoimmunity and show that the autoimmune imprint persists after viral clearance by DAA, suggesting that long-term follow-up may be warranted.

De novo autoimmune hepatitis (AIH) is an unusual cause of graft dysfunction after liver transplantation. This entity was originally described in 1996 in children transplanted for conditions other than AIH, who developed biochemical and histological features similar to AIH and responded to the therapy of classical AIH with steroids and azathioprine. In the last two decades, there have been reports of occurrence of de novo AIH in pediatric and adult liver transplant recipients, in the latter often being given different nomenclature including 'plasma cell hepatitis'. Typical causes of graft dysfunction in liver transplant recipients include rejection, infection, vascular and biliary complications as well as recurrence of disease that was the indication for liver transplantation like hepatitis C and primary biliary cholangitis. While acute cellular rejection and chronic rejection are well recognized complications post liver transplantation, in the last 5 years, antibody mediated rejection has become increasingly important in liver transplantation. In 2016, in the course of developing guidelines for the diagnosis of antibody mediated rejection, it was suggested that both de novo AIH and 'plasma cell hepatitis' be categorized as 'plasma cell rejection'. This review explores the literature on de novo AIH in pediatrics and adults, sheds light on the substantive differences between these two entities and suggests that they be kept distinct from each other as the two are not the same. This difference in the cause of graft dysfunction in pediatric and adult liver transplant recipients is important as the management of the two conditions is not the same.

Autoimmune serology is key to the diagnosis and management of autoimmune liver diseases. Its correct use in clinical practice requires a basic knowledge of the laboratory techniques used for autoantibody detection. Indirect immunofluorescence (IIF) on triple rodent tissue is still the gold standard screening procedure for liver-relevant autoantibodies, while HEp2 cells and human ethanol-fixed neutrophils are used as substrates to characterize nuclear reactivities and to detect anti-neutrophil cytoplasm antibody, respectively. Assays based on purified or recombinant antigens are increasingly used, having the main advantage of being observer-independent and the disadvantage of detecting only autoantibodies whose antigenic target has been identified. The AIH-specific anti-soluble liver antigen antibody cannot be detected by IIF and a molecular-based assay should be used at the screening level. Since autoantibodies may be present in the context of viral hepatitides and other inflammatory liver diseases it is important to exclude these conditions before diagnosing autoimmune liver disease. Anti-nuclear antibody (ANA), most often with a homogeneous IIF pattern on HEp2 cells, characterizes type 1 autoimmune hepatitis (AIH), and is found in association with anti-smooth muscle antibody in about half of the cases. Two IIF ANA patterns are specific for primary biliary cholangitis, namely the rim-like/membranous pattern, and the multiple nuclear dots pattern. Anti-liver kidney microsomal antibody type 1 is the serological hallmark of type 2 AIH, often in association with anti-liver cytosol type 1 antibody. Atypical perinuclear anti-neutrophil antibody, referred to as perinuclear anti-neutrophil nuclear antibody, is frequently detected in primary sclerosing cholangitis, in AIH type 1 and in inflammatory bowel diseases. The anti-asiaglycoprotein receptor antibody is liver-specific but not disease-specific, and reliable commercial assays for its detection are lacking. Anti-mitochondrial antibody is the hallmark of primary biliary cholangitis (PBC), being disease-specific and present in about 95% of the PBC patients. Its incidental detection presages the future development of PBC.

Regulatory T-cells (Tregs) are key players in the maintenance of immune homeostasis by preventing immune responses to self-antigens. Defects in Treg frequency and/or function result in overwhelming CD4 and CD8 T cell immune responses participating in the autoimmune attack. Perpetuation of autoimmune damage is also favored by Treg predisposition to acquire effector cell features upon exposure to a proinflammatory challenge. Treg impairment plays a permissive role in the initiation and perpetuation of autoimmune liver diseases, namely autoimmune hepatitis, primary biliary cholangitis and primary sclerosing cholangitis. In this Review, we outline studies reporting the role of Treg impairment in the pathogenesis of these conditions and discuss methods to restore Treg number and function either by generation/expansion in the test tube or through in vivo expansion upon administration of low dose IL-2. Challenges and caveats of these potential therapeutic strategies are also reviewed and discussed.

Detection of autoantibodies in the patient’s serum is a key diagnostic tool in autoimmune liver diseases, provided that the clinician is familiar with the laboratory methods and interprets correctly the results. Indirect immunofluorescence on triple rodent tissue should be used at a screening level, since it allows the simultaneous detection of the majority of liver-relevant autoantibodies, that is, anti-nuclear, anti-smooth muscle, anti-liver-kidney, anti-liver cytosol, and anti-mitochondrial antibody. Type 1 autoimmune hepatitis is characterized by anti-nuclear and/or anti-smooth muscle antibodies, whereas type 2 autoimmune hepatitis is characterized by anti-liver-kidney microsomal and/or anti-liver cytosolic type 1 antibodies. Anti-soluble liver antigen testing by a molecular-based assay should be included in the diagnostic work-up of liver disease of unknown origin, being highly specific for autoimmune hepatitis. Anti-mitochondrial antibody is the serological hallmark of primary biliary cholangitis and has high disease specificity. Rim-like and multiple nuclear dots anti-nuclear antibodies are also specific for primary biliary cholangitis and are of particular diagnostic value in anti-mitochondrial antibody negative patients. Atypical anti-neutrophil cytoplasmic antibody is detected by indirect immunofluorescence on human neutrophils and is associated with type 1 autoimmune hepatitis, primary/autoimmune sclerosing cholangitis, and inflammatory bowel disease.

Inflammatory bowel disease (IBD) and autoimmune liver disease (AILD) are closely associated, the former often dictating progression of the latter. Antibodies to tumor necrosis factor alpha (anti-TNFα) are effective in the management of IBD, but may cause liver injury.Retrospective review of medical records of patients with juvenile AILD who received anti-TNFα for IBD to evaluate the safety and efficacy of anti-TNFα.Eleven patients (6 boys), ages 9 to 15 years (median 13 years) were identified. Ten had ulcerative colitis and 1 Crohn disease; 2 had autoimmune hepatitis type 1 and 9 autoimmune hepatitis-sclerosing cholangitis variant. All patients were started on infliximab (IFX, 5 mg/kg) and 2 required dose increase (10 mg/kg); 3 of 11 switched to adalimumab due to allergic reaction or nonresponse. Three received adalimumab after losing response or developing antibodies to IFX. Liver function tests (LFTs) improved in 5, 1 continued to have stably abnormal LFTs and 2 maintained normal LFTs. Patients on adalimumab showed stable or improved liver function compared to pretreatment status. Six of 8 treated with a full course of IFX maintained clinical remission of IBD for 6 months to 2.5 years; of the 6 patients treated with adalimumab, 1 sustained IBD clinical remission for 24 months, 2 achieved remission only after tacrolimus addition and 3 did not respond.IBD in patients with AILD can be aggressive, requiring escalation to anti-TNFα or switching to other biologics. In this series, anti-TNFα did not impair liver function and improved gut disease in most of the patients, indicating that it can be beneficial and safe.

Autoimmune phenomena and clinically apparent autoimmune diseases, including autoimmune hepatitis, are increasingly been reported not only after natural infection with the SARS-CoV-2 virus, but also after vaccination against it. We report the case of a 63-year old man without a history of autoimmunity or SARS-CoV-2 natural infection who experienced acute severe autoimmune-like hepatitis seven days after the first dose of the mRNA-1273 SARS-CoV-2 vaccine. Liver histology showed inflammatory portal infiltrate with interface hepatitis, lobular and centrilobular inflammation with centrilobular necrosis, in absence of fibrosis and steatosis. Serum immunoglobulin G was slightly elevated. Autoimmune liver serology showed an indirect immunofluorescence pattern on triple rodent tissue compatible with anti-mitochondrial antibody (AMA), but, unexpectedly, this pattern was not mirrored by positivity for primary biliary cholangitis (PBC)-specific molecular tests, indicating that this antibody is different from classical AMA. Anti-nuclear antibody (ANA) was also positive with a rim-like indirect immunofluorescence pattern on liver and HEp2 cell substrates, similar to PBC-specific ANA; however, anti-gp210 and a large panel of molecular-based assays for nuclear antigens were negative, suggesting a unique ANA in our patient. He carries the HLA DRB1*11:01 allele, which is protective against PBC. Response to prednisone treatment was satisfactory. The clinical significance of these novel specificities needs to be further evaluated in this emerging condition.

Imbalance between T regulatory (Treg) and T effector (Teff) cells is likely to contribute to induction and perpetuation of liver damage in autoimmune hepatitis (AIH) and autoimmune sclerosing cholangitis (AISC) either through inability of Tregs to restrain proliferation and effector cytokine production by responders or through conversion of Tregs into T helper type 1 (Th1) or type 17 (Th17) effector lymphocytes. We investigated the effect of Treg skewing on the phenotypic and functional properties of CD4+CD127+CD25high cells, an activated subset of Teff, in 32 patients with AIH, 20 with AISC and in 36 healthy subjects (HS). In AIH/AISC we note a substantial increase in peripheral blood derived CD4+CD127+CD25high cells that display a Th1/Th17 phenotypic profile, as reflected by heightened IFNγ and IL-17 production, as well as by high levels of T-bet and RORC expression, and which is strongly correlated with disease activity. CD4+CD127+CD25high cells are unresponsive to low dose IL-2 and in patients have marked proliferative ability, further enhanced by stimulation with IL-7. CD4+CD127+CD25high cells obtained from CD4+ cells exposed to Treg polarizing conditions display enhanced IL-10 production, upregulate CD49b and LAG-3, markers of T regulatory 1 (Tr1) cells, and effectively suppress responder cell proliferation both in health and AIH/AISC patients through a mechanism which is dependent on IFNγ and IL-17. Suppressive function of CD4+CD127+CD25high cells is maintained upon pro-inflammatory challenge in HS but not in AIH/AISC. Conclusion: Treg skewing confers activated Teff phenotypic and functional properties of Tr1 cells in health and in AIH/AISC, though suppressive function is lost in patients upon pro-inflammatory challenge. Protracted modulation of the inflammatory environment is required to attenuate the effector potential while boosting immunoregulatory properties in Teff. This article is protected by copyright. All rights reserved.

We report a novel association between the commonly used antimalarial medication atovaquone/proguanil and drug-induced autoimmune-like hepatitis. The patient developed severe liver disease fulfilling biochemical, immunologic, and histologic criteria for the diagnosis of autoimmune hepatitis after the inadvertent rechallenge with the offending drug, which had caused self-limited hepatitic symptoms a year previously. Over a period of 18 months, the patient underwent two follow-up liver biopsies showing progressive resolution of the liver inflammation and achieved complete biochemical and immunologic remission on steroids. This remission persisted for 20 months following treatment withdrawal. Conclusion: This well documented case raises awareness of the potential hepatotoxicity of atovaquone/proguanil. (Hepatology Communications 2017;1:293-298).

Autoimmune hepatitis (AIH) is a severe liver disease affecting all age groups worldwide. Novel basic and clinical aspects of AIH, addressed at a Monothematic Conference in London in September 2015, are highlighted in this review. The diagnosis of AIH relies upon detection of characteristic autoantibodies, hypergammaglobulinemia, and interface hepatitis on liver histology. The International Autoimmune Hepatitis Group (IAIHG) has devised diagnostic scoring systems to help in comparative studies and clinical practice. AIH arises in a genetically predisposed host, when yet unknown triggers - such an encounter with a pathogen - lead to a T cell-mediated immune response targeting liver autoantigens. This immune response is inadequately controlled because regulatory mechanisms are impaired. The mainstay of treatment for AIH is immunosuppression, which should be instituted as soon as the diagnosis is made. Standard treatment regimens include relatively high doses of predniso(lo)ne, which are tapered gradually as azathioprine is introduced. Recent guidelines have described newer treatment regimens and have tightened the goal of therapy to complete normalization of biochemical, serological and histological parameters. Mycophenolate mofetil, calcineurin inhibitors, mTOR inhibitors and biological agents are potential salvage therapies, but should be reserved for selected non-responsive patients and administered only in experienced centers. Liver transplantation is a life-saving option for those patients who progress to end-stage liver disease. Further dissection of cellular and molecular pathways involved in AIH pathogenesis is likely to lead to the discovery of novel, tailored and better tolerated therapies.

Background & aim The diagnosis of primary biliary cholangitis (PBC), an uncommon immune-mediated cholestatic liver disease, is based on positive circulating anti-mitochondrial (AMA) and/or PBC-specific anti-nuclear autoantibodies (ANA), coupled with elevated serum alkaline phopsphatase (ALP) levels. Timely initiation of treatment with ursodeoxycholic acid prevents progression to cirrhosis and liver failure. We aimed at investigating liver histology in patients with normal ALP level and positive AMA and/or PBC-specific ANA. Methods We searched the Swiss PBC Cohort Study database, which includes subjects with positive PBC autoimmune serology and normal ALP levels, for patients who underwent a liver biopsy. Histological slides were centrally reviewed by an expert liver pathologist, and sera were centrally re-tested for AMA and ANA. Results 30 patients were included; 90% females, median age 53 (range 27–72) years. Twenty-four (80%) had liver histology typical for (n = 2), consistent with (n = 16) or suggestive of (n = 6) PBC, including three of four AMA-negative ANA-positive patients. Among 22 ursodeoxycholic acid treated patients, 14 had elevated GGT levels before treatment; a significant decrease of the median GGT level between pre- (1.46 x ULN) and post- (0.43 x ULN) treatment (p = 0.0018) was observed. Conclusions In our series, a high proportion of AMA positive patients with normal ALP levels have PBC. For the first time we show histological diagnosis of PBC in AMA-negative/PBC-specific ANA-positive subjects and the potential role of GGT as a biomarker in PBC patients with normal baseline ALP levels. Current guidelines for the diagnosis of PBC do not cover the whole extent of PBC presentation, with important clinical implications in terms of timely treatment initiation.

Background & aims: T-helper-type 17 (Th17) cells are involved in autoimmune tissue damage. CD39 is an ectonucleotidase that catalyzes extracellular ATP/ADP hydrolysis, culminating in the generation of immunosuppressive adenosine. Functional CD39 expression confers immunosuppressive properties upon immune cells. As the proportion of CD39 lymphocytes is decreased in juvenile autoimmune liver disease (AILD), we have explored whether decreased CD39 expression is present on Th17 cells and whether this phenomenon is associated with heightened effector function and inflammation. Methods: Thirty-eight patients with juvenile AILD (22 autoimmune hepatitis and 16 autoimmune sclerosing cholangitis), 8 disease controls (DC) and 16 healthy subjects (HS) were studied. Peripheral blood cell phenotype was determined by flow cytometry; ability to suppress by inhibition of cell proliferation/ effector cytokine production; ectoenzymatic activity by thin layer chromatography; expression of adenosine receptor, adenosine deaminase (ADA) and phosphodiesterases (PDE) by quantitative real-time PCR or by Western Blot. Results: CD39þ Th17 (Th17CD39þ) cells from HS appear activated and contain high frequencies of lymphocytes producing regulatory cytokines. In AILD, however, Th17CD39þ cells are markedly diminished and fail to generate AMP/adenosine, thereby limiting control of both target cell proliferation and IL-17 production. When compared to HS, Th17 cells from AILD patients also show lower A2A adenosine receptor expression while displaying similar levels of PDE4A, PDE4B and ADA. Only rare Th17CD39þ cells are observed by liver immunohistochemistry. Conclusions: Th17CD39þ cells in juvenile AILD are both quantitatively decreased and qualitatively deficient. Low levels CD39 and A2A expression may contribute to the perpetuation of Th17 cell effector properties and unfettered inflammation in this disease.

Autoimmune hepatitis (AIH) is a severe life-threatening hepatopathy of unknown etiology, affecting both pediatric and adult populations, and characterised by inflammatory liver histology, circulating non-organ-specific autoantibodies, and hypergammaglobulinaemia. AIH is a very heterogeneous disease with a variety of clinical presentations, ranging from asymptomatic liver test abnormalities to acute severe hepatitis or even acute liver failure. It responds very well to immunosuppressive treatment with prednisolone with or without azathioprine. Patients who are intolerant or fail to respond to standard therapy are candidates for alternative immunosuppressive regimens, the combination of steroids with mycophenolate mofetil or calcineurin inhibitors being the most frequently reported. The pathogenesis of AIH remains not completely understood, although there is evidence that genetic predisposition, molecular mimicry and defective immunoregulatory mechanisms contribute to the autoimmune liver damage. Areas covered: A literature search was conducted using the key-words 'autoimmune hepatitis', 'immunogenetics', 'regulatory T-cells' and 'immunosuppression'. The aim of this review is to discuss recent breakthroughs in the understanding AIH pathogenesis, diagnosis and treatment. Expert commentary: Progress in the understanding of AIH pathogenesis is likely to contribute to the development of novel therapeutic strategies, such as the adoptive transfer of autologous expanded antigen-specific regulatory T-cells.

Overlap between autoimmune hepatitis (AIH) and either primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC) is not rare and has extensively been reported. We herein report the first well documented case of triple overlap. A 68-year-old male patient presented with asymptomatic PBC including normal alkaline phosphatase serum level, developed AIH five years later, associated with magnetic resonance cholangiopancreatography biliary changes typical for PSC. Despite treatment with ursodeoxycholic acid and mycophenolate mofetil, owing to prednisone and azathioprine intolerance, he continued to have interface hepatitis and developed increasing fibrosis at follow-up liver biopsy. Our case report raises awareness for this rare and difficult to diagnose and treat clinical phenotype., Highlights • We report the first well documented case of coexisting primary biliary cholangitis, autoimmune hepatitis and primary sclerosing cholangitis. • Our patient, a 68-year old male, had normal serum alkaline phosphatase level despite florid bile duct lesions at liver biopsy. • Our patient had normal serum transaminase levels despite marked interface hepatitis at follow-up liver biopsy.

Juvenile sclerosing cholangitis is a rare chronic hepatobiliary disorder characterized by inflammation of the intra- and/or extrahepatic bile ducts, bile duct dilatation, narrowing and obliteration, and, histologically, by inflammatory bile duct damage leading to periductular fibrosis. The diagnosis is based on endoscopic retrograde cholangiopancreatography or magnetic resonance cholangiopancreatography. In children, it may be associated to a variety of systemic and hepatic conditions: thus, the term "primary" sclerosing cholangitis should be reserved for the rare cases without a known cause. Small duct disease is diagnosed in the presence of histological features diagnostic of sclerosing cholangitis and normal cholangiography. Autoimmune sclerosing cholangitis (ASC) is a form of sclerosing cholangitis with strong autoimmune features overlapping with those of autoimmune hepatitis (AIH). It is a well-recognized nosological entity in paediatrics, where it accounts for the majority of sclerosing cholangitis cases. It is as prevalent as AIH in children, is equally frequent in males and females, half of the patients have concomitant inflammatory bowel disease, virtually all patients have raised immunoglobulin G levels and positive anti-nuclear and/or anti-smooth muscle antibodies. Half of the ASC patients respond well to standard immunosuppressive treatment for AIH with the addition of ursodeoxycholic acid, but the transplant rate is higher than in AIH, and post-transplant recurrence is frequent. A number of open questions remain: are ASC and AIH distinct entities or different manifestations of the same condition? What is the role of histology? Is small duct disease a specific entity? What is the relationship between ASC and adult primary sclerosing cholangitis? What is the role of inflammatory bowel disease? In addition, validated diagnostic criteria for ASC are needed.

Primary Biliary Cholangitis (PBC) is an uncommon, chronic, cholangiopathy of autoimmune origin and unknown etiology characterized by positive anti-mitochondrial autoantibodies (AMA), female preponderance and progression to cirrhosis if left untreated. The diagnosis is based on AMA- or PBC-specific anti-nuclear antibody (ANA)-positivity in the presence of a cholestatic biochemical profile, histologic confirmation being mandatory only in seronegative cases. First-line treatment is ursodeoxycholic acid (UDCA), which is effective in preventing disease progression in about two thirds of the patients. The only approved second-line treatment is obeticholic acid. This article summarizes the most relevant conclusions of a meeting held in Lugano, Switzerland, from September 23rd-25th 2018, gathering basic and clinical scientists with various background from around the world to discuss the latest advances in PBC research. The meeting was dedicated to Ian Mackay, pioneer in the field of autoimmune liver diseases. The role of liver histology needs to be reconsidered: liver pathology consistent with PBC in AMA-positive individuals without biochemical cholestasis is increasingly reported, raising the question as to whether biochemical cholestasis is a reliable disease marker for both clinical practice and trials. The urgent need for new biomarkers, including more accurate markers of cholestasis, was also widely discussed during the meeting. Moreover, new insights in interactions of bile acids with biliary epithelia in PBC provide solid evidence of a role for impaired epithelial protection against potentially toxic hydrophobic bile acids, raising the fundamental question as to whether this bile acid-induced epithelial damage is the cause or the consequence of the autoimmune attack to the biliary epithelium. Strategies are needed to identify difficult-to-treat patients at an early disease stage, when new therapeutic approaches targeting immunologic pathways, in addition to bile acid-based therapies, may be effective. In conclusion, using interdisciplinary approaches, groundbreaking advances can be expected before long in respect to our understanding of the etiopathogenesis of PBC, with the ultimate aim of improving its treatment.

The etiology of autoimmune hepatitis (AIH) is unknown, though hepatotropic viruses may be potential triggers. Hepatitis E virus (HEV) infection, an increasingly recognized cause of acute hepatitis, has been misdiagnosed as AIH due to the occurrence of autoantibodies during its acute phase. It has also been suggested that HEV infection may lead to or unmask AIH. The HEV seroprevalence has been ascertained in patients with AIH, but the prevalence of AIH-related autoantibodies in patients with HEV infection has not been systematically tested. We aimed to investigate whether acute HEV infection is associated with the presence of AIH-relevant autoantibodies, following the liver autoimmune serology guidelines of the International AIH Group. We tested 48 patients with acute HEV infection. Half of them had at least one autoantibody, 17% two autoantibodies. Anti-nuclear antibody (ANA) were detected in 16 (33%), anti-smooth muscle antibody (SMA) in 10 (21%), and anti-neutrophil cytoplasmic antibody (ANCA) in 7 (14.6%). Of note, two patients showed SMA with VG or VGT patterns and five had ANA with homogeneous appearance, both being typical of AIH type 1. Other AIH-specific autoantibodies were negative. Atypical anti-mitochondrial antibody, without evidence of primary biliary cholangitis, was positive in one patient, disappearing at follow-up. Follow-up (median 12 months) serum was available from seven autoantibody positive patients: two became negative, while five remained positive, although no patient developed AIH to date. In conclusion, autoantibodies are frequently present during acute HEV infection, indicating that HEV should always be excluded before diagnosing AIH. Importantly, a minority of patients with acute hepatitis E develops AIH-specific autoantibodies, and, though they did not progress to autoimmune liver disease in the short-term, they warrant long-term monitoring.

Natural killer T (NKT) cells are a distinct lineage of T cells which express both the T cell receptor (TCR) and natural killer (NK) cell markers. Invariant NKT (iNKT) cells bear an invariant TCR and recognize a small variety of glycolipid antigens presented by CD1d (nonclassical MHC-I). CD1d-restricted iNKT cells are regulators of immune responses and produce cytokines that may be proinflammatory (such as interferon-gamma (IFN-γ)) or anti-inflammatory (such as IL-4). iNKT cells also appear to play a role in B cell regulation and antibody production. Alpha-galactosylceramide (α-GalCer), a derivative of the marine sponge, is a potent stimulator of iNKT cells and has been proposed as a therapeutic iNKT cell activator. Invariant NKT cells have been implicated in the development and perpetuation of several autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus (SLE). Animal models of SLE have shown abnormalities in iNKT cells numbers and function, and an inverse correlation between the frequency of NKT cells and IgG levels has also been observed. The role of iNKT cells in autoimmune liver disease (AiLD) has not been extensively studied. This review discusses the current data with regard to iNKT cells function in AiLD, in addition to providing an overview of iNKT cells function in other autoimmune conditions and animal models. We also discuss data regarding the immunomodulatory effects of vitamin D on iNKT cells, which may serve as a potential therapeutic target, given that deficiencies in vitamin D have been reported in various autoimmune disorders.

Paediatric autoimmune liver disease is characterised by inflammatory liver histology, circulating autoantibodies and increased levels of IgG, in the absence of a known etiology. Three conditions have a likely autoimmune pathogenesis: autoimmune hepatitis (AIH), autoimmune sclerosing cholangitis (ASC), and de novo AIH after liver transplantation. Two types of paediatric AIH are recognized according to seropositivity for smooth muscle and/or antinuclear antibody (SMA/ANA, AIH-1) or liver kidney microsomal type 1 and/or anti-liver cytosol type 1 antibodies (anti-LKM-1/anti-LC-1; AIH-2).Pertinent issues addressing the diagnosis, treatment and long term follow up were formulated by a core group of ESPGHAN members. They have commissioned the first authors with execution of this project. Initially, they have performed a systematic literature search on MEDLINE, ResearchGate and Mendeley databases over the last 30 years and produced a document focusing on prospective and retrospective studies in children. The ESPGHAN core group and ESPGHAN Hepatology Committee members voted on each recommendation, using a formal voting technique.

Autoimmune hepatitis (AIH) is a rare, chronic disease that affects both adults and children, including infants. The disease is probably triggered by environmental factors in genetically predisposed individuals. The clinical presentation ranges from asymptomatic patients or patients with non-specific symptoms, such as fatigue, to fulminant liver failure, many children presenting with symptoms indistinguishable from those of acute hepatitis. Raised transaminase and immunoglobulin G (IgG) levels, in association with circulating autoantibodies, guide towards the diagnosis. The histological hallmark is interface hepatitis, which however is non-specific and may be absent. There are no bile duct changes on cholangiography. Presence of anti-nuclear antibody (ANA) and/or anti-smooth muscle antibody (SMA) is characteristic for type 1 AIH, whereas presence of anti-liver kidney microsomal type 1 (LKM1) antibody and/or anti-liver cytosol type 1 (LC1) antibody defines type 2 AIH. The latter accounts for about one third of the juvenile AIH cases, presents more acutely than type 1 AIH and is very rare in adults. Immunosuppressive therapy, based on steroids and azathioprine, is required, and in the vast majority of patients leads to clinical and biochemical remission, defined as absence of symptoms, normal transaminase and IgG levels, and negative or low-titer autoantibodies. In patients intolerant or non-responder to standard therapy, a number of second line drugs have been employed with variable results. For the rare cases who progress to end-stage liver disease, liver transplantation is life-saving, but recurrence of the disease is possible. A better understanding of the underlying pathogenic mechanisms will help to develop new, more effective and less toxic therapies, and to tailor treatment regimens to the individual patient.

Autoimmune hepatitis (AIH) in humans is a severe inflammatory liver disease characterized by interface hepatitis, the presence of circulating autoantibodies, and hyper-gammaglobulinemia. There are two types of AIH, type 1 (AIH-1) and type 2 (AIH-2), characterized by distinct autoimmune serology. Patients with AIH-1 are positive for anti–smooth muscle and/or antinuclear autoantibodies, whereas patients with AIH-2 have anti–liver kidney microsomal type 1 and/or anti–liver cytosol type 1 autoantibodies. Cytochrome P4502D6 is the antigenic target of anti–liver kidney microsomal type 1, and formiminotransferase cyclodeaminase is the antigenic target of anti–liver cytosol type 1. It is known that AIH, both types 1 and 2, is strongly linked to the human leukocyte antigen (HLA) alleles -DR3, -DR4, and -DR7. However, direct evidence of the association of HLA with AIH is lacking. We developed a novel mouse model of AIH using the HLA-DR3 transgenic mouse on the nonobese-diabetic background by immunization of HLA-DR3– and HLA-DR3+ nonobese-diabetic mice with a DNA plasmid, coding for human cytochrome P4502D6/formiminotransferase cyclodeaminase fusion protein. Immunization with cytochrome P4502D6/formiminotransferase cyclodeaminase leads to a sustained elevation of alanine aminotransferase, development of antinuclear autoantibodies and anti–liver kidney microsomal type 1/anti–liver cytosol type 1 autoantibodies, chronic immune cell infiltration, and parenchymal fibrosis on liver histology in HLA-DR3+ mice. Immunized mice also showed an enhanced T helper 1 immune response and paucity of the frequency of regulatory T cells in the liver. Moreover, HLA-DR3+ mice with exacerbated AIH showed reduced diversity and total load of gut bacteria. Conclusion: Our humanized animal model has provided a novel experimental tool to further elucidate the pathogenesis of AIH and to evaluate the efficacy and safety of immunoregulatory therapeutic interventions in vivo. (Hepatology 2015;62:1536–1550)

Autoimmune hepatitis (AIH), a liver disorder affecting both children and adults, is characterized by inflammatory liver histology, elevated transaminase levels, circulating nonorganspecific autoantibodies, and increased levels of immunoglobulin G, in the absence of a known etiology. Two types of AIH are recognized according to seropositivity: smooth muscle antibody and/or antinuclear antibody define AIH type 1 and antibodies to liver-kidney microsome type 1 and/or liver cytosol type 1 define AIH type 2. AIH type 1 affects both adults and children, while AIH type 2 is mainly a paediatric disease, though it does occasionally affects young adults. AIH should be considered during the diagnostic workup of any patient with increased liver enzyme levels. AIH is exquisitely responsive to immunosuppressive treatment with prednisolone with or without azathioprine, with symptom free long-term survival for the majority of patients. For those who do not respond to standard treatment, or who are difficult-to-treat, mycophenolate mofetil and, in the absence of a response, calcineurin inhibitors should be tried in addition to steroids. The pathogenesis of AIH is not fully understood, although there is mounting evidence that genetic susceptibility, molecular mimicry and impaired immunoregulatory networks contribute to the initiation and perpetuation of the autoimmune attack. Liver damage is thought to be mediated primarily by CD4 T-cells, although recent studies support the involvement of diverse populations, including Th17 cells. A deeper understanding of the pathogenesis of AIH is likely to contribute to the development of novel treatments, such as the adoptive transfer of autologous expanded antigenspecific regulatory T-cells, which ultimately aim at restoring tolerance to liver-derived antigens.

There are three classic liver diseases with probable autoimmune etiology: primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. The occurrence of these autoimmune conditions is determined by the breakdown of immune-regulatory mechanisms that in health are responsible for maintaining immunological tolerance against self-antigens. Among the multiple T cell subsets with suppressive function, the regulatory T cells (Tregs), defined by the expression of CD4, the IL-2 receptor α chain (CD25), and the transcription factor FOXP3, have emerged as having a central role in maintaining immune-tolerance to autoantigens. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways, and the production of anti-inflammatory cytokines. In all the three autoimmune liver diseases mentioned above, there is evidence pointing for either a reduced frequency and/or function of Tregs. Here, we review the definition, phenotypic characteristics, and mechanisms of suppression employed by Tregs and then we discuss the evidence available pointing to their impairment in patients with autoimmune liver disease.

Regulatory T cells (Tregs) are central to the maintenance of self-tolerance and tissue homeostasis. Markers commonly used to define human Tregs in the research setting include high expression of CD25, FOXP3 positivity and low expression/negativity for CD127. Many other markers have been proposed, but none unequivocally identifies bona fide Tregs. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways and the production of anti-inflammatory cytokines. Treg impairment has been reported in a number of human autoimmune conditions and includes Treg numerical and functional defects and conversion into effector cells in response to inflammation. In addition to intrinsic Treg impairment, resistance of effector T cells to Treg control has been described. Discrepancies in the literature are common, reflecting differences in the choice of study participants and the technical challenges associated with investigating this cell population. Studies differ in terms of the methodology used to define and isolate putative regulatory cells and to assess their suppressive function. In this review we outline studies describing Treg frequency and suppressive function in systemic and organ specific autoimmune diseases, with a specific focus on the challenges faced when investigating Tregs in these conditions.

In paediatrics, there are 2 liver disorders in which liver damage most likely stems from an autoimmune attack: ‘classical' autoimmune hepatitis (AIH) and the AIH/sclerosing cholangitis overlap syndrome (also known as autoimmune sclerosing cholangitis, ASC). The presentation of childhood autoimmune liver disease (AILD) is non-specific and can mimic most other liver disorders. AIH is exquisitely responsive to immunosuppressive treatment, which should be instituted promptly to prevent rapid deterioration and promote remission and long-term survival. Difficult-to-treat or non-responsive patients should be treated with mycophenolate mofetil; if this fails then calcineurin inhibitors can be tried. Persistent failure to respond or lack of adherence to treatment result in end-stage liver disease. These patients, and those with fulminant liver failure at diagnosis, will require liver transplantation. ASC responds to the same immunosuppressive treatment used for AIH when treatment is initiated early. Abnormal liver function tests often resolve within a few months of treatment, although medium- to long-term prognosis is worse than that of AIH because bile duct disease continues to progress despite treatment in approximately 50% of patients. Ursodeoxycholic acid is usually added to conventional treatment regimen in ASC, but whether this actually helps arrest the progression of bile duct disease remains to be established. The pathogenesis of paediatric-onset AILD is not fully understood, although there is mounting evidence that genetic susceptibility, molecular mimicry and impaired immunoregulatory networks contribute to the initiation and perpetuation of the autoimmune attack. Liver damage is thought to be mediated primarily by CD4pos T-cells. While Th1 effector cells are associated with hepatocyte damage in both AIH and ASC, Th17 immune responses predominate in the latter where they correlate with biochemical indices of cholestasis, indicating that IL-17 is involved in the bile duct damage characteristic of this condition. Since a substantial difference between these 2 pathologies is the frequent association of ASC with inflammatory bowel disease, it can be speculated that lymphocytes of intestinal origin are present in patients with a diagnosis of ASC rather than in those with AIH. Animal models faithfully representing the human conditions are needed to unravel the contribution of innate and adaptive, effector and regulatory immune responses. A deeper understanding of the pathogenesis of AILD is likely to contribute to the development of novel treatments, such as the adoptive transfer of autologous expanded antigen-specific regulatory T-cells, which ultimately aim at restoring tolerance to liver-derived antigens.

Serology is key to the diagnosis of autoimmune hepatitis (AIH). Clinicians need to be aware of which tests to request, how to interpret the laboratory reports, and be familiar with the laboratory methodology. If correctly tested, >95% of AIH patients show some serological reactivity. Indirect immunofluorescence on triple rodent tissue is recommended as first screening step, since it allows the detection of all liver-relevant autoantibodies, except for anti-soluble liver antigen (SLA) antibody, which needs to be detected by molecular based assays. The threshold of immunofluorescence positivity is a titer equal or exceeding 1/40, but for patients younger than 18years even lower titers are clinically significant. Anti-nuclear antibody (ANA) and/or anti-smooth muscle (SMA) antibody characterize type 1 AIH. ANA in AIH typically shows a homogeneous staining pattern on HEp2 cells, without any specific target antigen. Anti-SMA displays different staining patterns on indirect immunofluorescence: the vascular/glomerular (VG) and the vascular/glomerular/tubular (VGT) patterns are considered specific for AIH, whilst the V pattern can be found in a variety of diseases. Type 2 AIH, which is rare and affects mostly children/adolescents, is characterized by anti-liver kidney microsomal 1 and/or anti-liver cytosol 1 antibodies. The presence of anti-neutrophil cytoplasmic antibody (ANCA), particularly atypical p-ANCA (pANNA), points to the diagnosis of AIH, especially in absence of other autoantibodies. Since it is associated with sclerosing cholangitis and inflammatory bowel disease, these conditions have to be ruled out. The only antibody specific for AIH is anti-SLA, which is associated with a more severe disease course.

Imbalance between effector and regulatory T-cells (Treg) underlies the loss of immune-tolerance to self-antigens in autoimmune disease. In autoimmune hepatitis type 2 (AIH-2), effector CD4 T-cell immune responses to cytochrome P450IID6 (CYP2D6) are permitted by numerically and functionally impaired Treg. Restoration of CYP2D6-specific Treg in AIH-2 would enable control over effectors sharing the same antigen specificity, leading to re-establishment of immune-tolerance. We have previously developed a protocol for generating antigen-specific Treg through co-culture with semi-mature dendritic cells presenting CYP2D6 peptides. In this study, we aimed to explore phenotypic and functional features of patient Treg compared to health, to test Treg stability under pro-inflammatory conditions, and to investigate the potential benefit of supplementation with all-trans-retinoic acid (RA) or rapamycin (RP), agents proven to enhance Treg function. We show that antigen-specific Treg from patients have comparable phenotypic and functional features to those from healthy controls, suppressing both proliferation and pro-inflammatory cytokine production by effector cells. Treg exposure to inflammatory challenge results in decreased suppressive function and up-regulation of Th1/Th2/Th17 transcription factors both in health and AIH-2. The increase of Th1 and Th17 transcription factors is limited by addition of RA in controls and Th1 expression is decreased by RP in patients. Importantly, inflammation-induced decrease in Treg function is also abrogated by RA/RP in health and RA in patients. Our data provide important information for the optimization of protocols aiming at generating antigen-specific Treg for treatment of autoimmune disease and for understanding their biology upon pro-inflammatory challenge and RP/RA supplementation.

Objective: Familial clustering of juvenile autoimmune liver disease (AILD), including autoimmune hepatitis and autoimmune sclerosing cholangitis (ASC), is rare, despite a high prevalence of autoimmune disorders in AILD families. Methods: To investigate this discrepancy, we measured autoantibodies diagnostic for AILD, anti-nuclear, anti-smooth muscle, anti-liver kidney microsomal type 1, anti-liver cytosol type 1, and anti-soluble liver antigen antibodies, and human leukocyte antigen profiles in 31 patients and 65 of their first-degree relatives (FDR). The autoantibody profile was compared with that of 42 healthy subjects (HS). Results: Autoantibodies were detected in 71% (22/31) patients. Antinuclear antibody or anti-smooth muscle antibody were present in 4/65 FDR (6.2%). HS were negative for all autoantibodies. The frequencies of homozygous HLA DRB1� 0301 (DR3) genes and haplotype A1-B8-DR3 were higher in the patients (25% and 43%) than in FDR (9% and 27%) and HS (0% and 16%). The frequencies of disease-protective genes DR4 and/or DR15 were lower in the patients (25%) than in FDR (42%) and HS (42%). Only 1 family contained 2 patients with AILD, 1 with ASC and 1 with primary sclerosing cholangitis. Both patients possessed A1-B8-DR3 genes, the ASC being homozygous and the primary sclerosing cholangitis heterozygous. Six FDR had nonhepatic autoimmune disorders, none being autoantibody positive. Conclusions: Homozygosity for DR3 plays a major role in the predisposition to juvenile AILD. Diagnostic autoantibodies for AILD are rare among patients’ FDR and not linked to clinical manifestation of AILD.

Acetaminophen-induced acute liver failure (AALF) is characterized both by activation of innate immune responses and susceptibility to sepsis. Circulating monocytes and hepatic macrophages are central mediators of inflammatory responses and tissue repair processes during human AALF. Secretory leukocyte protease inhibitor (SLPI) modulates monocyte/macrophage function through inhibition of nuclear factor kappa B (NF-κB) signaling. The aims of this study were to establish the role of SLPI in AALF. Circulating levels of SLPI, monocyte cluster of differentiation 163 (CD163), human leukocyte antigen-DR (HLA-DR), and lipopolysaccharide (LPS)-stimulated levels of NF-κBp65, tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 were determined in patients with AALF, chronic liver disease, and healthy controls. Immunohistochemistry and multispectral imaging of AALF explant tissue determined the cellular sources of SLPI and hepatic macrophage phenotype. The phenotype and function of monocytes and macrophages was determined following culture with recombinant human (rh)-SLPI, liver homogenates, and plasma derived from AALF patients in the presence and absence of antihuman (α)SLPI. Hepatic and circulatory concentrations of SLPI were elevated in AALF and immunohistochemistry revealed SLPI expression in biliary epithelial cells and within hepatic macrophages (h-mψ) in areas of necrosis. H-mψ and circulating monocytes in AALF exhibited an anti-inflammatory phenotype and functional characteristics; typified by reductions in NF-κBp65, TNF-α, and IL-6 and preserved IL-10 secretion following LPS challenge. Culture of healthy monocytes with AALF liver homogenates, plasma, or rhSLPI induced monocytes with strikingly similar anti-inflammatory characteristics which were reversed by inhibiting the activity of SLPI. Conclusion: SLPI is a pivotal mediator of anti-inflammatory responses in AALF through modulation of monocyte/macrophage function, which may account for the susceptibility to sepsis in AALF. (Hepatology 2014;59:1564-1576)

Autoimmune hepatitis (AIH) is an important cause of severe liver disease and is associated with both quantitative and qualitative regulatory T-cell (Treg) impairments. Tregs express CD39, an ectonucleotidase responsible for extracellular nucleotide hydrolysis, culminating in the production of immunosuppressive adenosine. Here, we describe multiple CD39pos Treg defects that potentially contribute to the impaired immunoregulation that is characteristic of AIH. We have examined the frequency and phenotype of CD39pos Tregs by flow cytometry and measured their ectonucleotidase activity. The capacity of CD4posCD25high, CD4posCD25highCD39pos, and CD4posCD25highCD39neg subsets to suppress both proliferation of effector T cells and interleukin (IL)-17 production was evaluated. In AIH, CD39pos Tregs are decreased in frequency, exhibit limited adenosine triphosphate/adenosine diphosphate hydrolysis activity, and fail to suppress IL-17 production by effector CD4 T cells. Moreover, these CD39pos Tregs display a more proinflammatory profile in AIH, which is characterized by elevated CD127 positivity, and a greater propensity to produce interferon-gamma or IL-17 upon challenge with proinflammatory stimuli. Conclusions: In AIH, CD39pos Tregs are decreased in number, fail to adequately hydrolyze proinflammatory nucleotides and do not efficiently suppress IL-17 production by effector CD4 T cells. CD39pos Tregs show plasticity and are unstable upon proinflammatory challenge, suggesting that defective immunoregulation in AIH might result not only from reduced Treg number and function, but also from increased conversion of Tregs into effector cells. (Hepatology 2014;59:1007–1015)

The accurate diagnosis and classification of autoimmune hepatitis (AIH) rely upon the detection of characteristic autoantibodies. Positivity for anti-nuclear (ANA) and/or anti-smooth muscle (SMA) autoantibodies defines AIH type 1 (AIH-1), whereas anti-liver kidney microsomal type 1 (anti-LKM1) and/or anti-liver cytosol type 1 (anti-LC1) define AIH type 2 (AIH-2). ANA and SMA, and less commonly anti-LKM1, have also been detected in de-novo autoimmune hepatitis developing after liver transplantation, a condition that may affect patients transplanted for non-autoimmune liver disease. The diagnostic autoantibodies associated with AIH-1 are also detected in the paediatric AIH/sclerosing cholangitis overlap syndrome, referred to as autoimmune sclerosing cholangitis (ASC). ASC, like adult primary sclerosing cholangitis, is often associated with atypical perinuclear anti-neutrophil cytoplasmic autoantibodies (p-ANCA), although p-ANCA are also detected in other autoimmune liver diseases. These associations highlight the necessity for simple and prompt diagnostic autoantibody testing, and the requirement for the accurate interpretation of the results of the tests in the clinical context. Fine-mapping of antigenic autoantibody targets has facilitated the development of rapid molecular assays that have the potential to revolutionise the field if properly standardised and when used in combination with classical immunofluorescence. Despite their diagnostic significance, the pathogenic role of the various autoantibodies and the mechanisms by which they can potentially inflict damage onto the liver cell remain a topic for further research.

Autoimmune liver disorders in childhood include autoimmune hepatitis (AIH) and AIH/sclerosing cholangitis overlap syndrome (henceforth referred to as autoimmune sclerosing cholangitis, ASC). These inflammatory liver disorders are characterised histologically by interface hepatitis, biochemically by elevated transaminase levels, and serologically by autoantibodies and increased levels of immunoglobulin G. AIH is particularly aggressive in children and progresses rapidly unless immunosuppressive treatment is started promptly. With appropriate treatment, 80% of patients achieve remission and long-term survival. For non-responders and difficult-to-treat patients, novel and more effective therapeutic approaches are sought. ASC responds to the same treatment used for AIH in regards to parenchymal inflammation, but bile duct disease progresses in about 50% of cases, leading to a worse prognosis and a higher liver transplantation requirement; moreover, it has a high recurrence rate after transplant. Progression of liver disease and recurrence after transplant are more common in patients with associated poorly controlled inflammatory bowel disease. Though the mechanisms underlying the pathogenesis of liver autoimmunity are not fully understood, genetic and environmental factors are likely to be involved. A deeper understanding of the pathogenesis of these conditions will contribute to the development of novel treatments, aimed ultimately at the restoration of tolerance to liver-derived antigens.

Liver transplantation (LT) is an effective treatment for patients with end-stage autoimmune liver diseases such as primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. Indications for LT for these diseases do not differ substantially from those used for other acute or chronic liver diseases. Despite the good outcomes reported, the recurrence of autoimmune liver disease is relatively common in the allograft. In addition, it has become apparent that autoimmunity and autoimmune liver disease can arise de novo after transplantation for nonautoimmune liver disorders. An awareness of the existence of recurrent autoimmune liver diseases and de novo autoimmune hepatitis after LT has important clinical implications because their management differs from the standard antirejection treatment and is similar to the management of classic autoimmune liver diseases in the native liver.

Patients with alcohol-related liver disease (ALD) have antibodies directed to alcohol dehydrogenase (ADH), anti-ADH titers being associated with disease severity and active alcohol consumption. ADH-specific T-cell responses have not been characterized. We aimed to define anti-ADH cellular immune responses and their association with active alcohol consumption and disease severity. Using cultures of peripheral blood mononuclear cells (PBMCs) from 25 patients with alcohol-related cirrhosis (ARC; 12 were actively drinking or abstinent for6 months, and 13 were abstinent for6 months) and hepatic mononuclear cells (HMCs) from 14 patients with ARC who were undergoing transplantation, we investigated T-cell reactivity to 25 overlapping peptides representing the full human ADH protein (beta 1 subunit). ADH-specific peripheral T-cell responses were assessed by the quantification of T-cell proliferation and cytokine production and were correlated with the clinical course. In active alcohol consumers, proliferative T-cell responses targeted ADH31-95 and other discontinuous sequences in the ADH peptide, whereas only one sequence was targeted in abstinents. ADH peptides induced the production of interferon-γ (IFN-γ), interleukin-4 (IL-4), and IL-17. IL-4 production was lower in active drinkers versus abstinents, and IL-17 production was higher. Peptides inducing IFN-γ production outnumbered those inducing T-cell proliferation. The intensity of the predominantly T helper 1 (Th 1) responses directly correlated with disease severity. Similar to PBMCs in abstinents, ADH peptides induced weak T-cell proliferation and a similar level of IL-4 production in HMCs but less vigorous Th 1 and T helper 17 responses.This suggests that Th 1 responses to ADH in ARC are induced by alcohol consumption. A Th 1/T helper 2 imbalance characterizes T-cell responses in active drinkers with ARC, whereas IL-4 production prevails in abstinents. This identifies new targets for immunoregulatory therapies in ALD patients for halting detrimental effector T-cell responses, which may encourage liver fibrogenesis and progression to end-stage liver disease.

Autoimmune hepatitis (AIH), a severe hepatopathy characterized by hypergammaglobulinaemia, autoantibodies and interface hepatitis, is occasionally associated with systemic autoimmune manifestations [systemic lupus erythematosus (SLE); mixed connective tissue disease (MCTD)]. In both AIH and SLE/MCTD numerical and/or functional impairment of regulatory T-cells (T-regs) is believed to favour autoimmunity. To investigate whether immune-tolerance breakdown profiles differ in patients with AIH and SLE/MCTD, isolated AIH or systemic autoimmunity, we studied phenotypic and functional features of T-regs in 10 patients with AIH-SLE/MCTD, 22 with AIH, 12 with SLE and 20 healthy subjects. Compared to health, CD4(pos)CD25(pos) cells were decreased in number and expressed high levels of the CD127 activation marker in all three disease groups; in AIH-SLE/MCTD and in SLE they displayed low levels of FOXP3. In AIH-SLE/MCTD, they also contained a high proportion of IFNγ positive cells, indicating a Th1 profile. Similarly, in AIH-SLE/MCTD, CD4(pos)CD25(pos)CD25(high) T-regs were reduced in number and contained an increased proportion of activated CD127(pos) and IFNγ(pos) cells. Such skewing towards a Th1 profile was also present at effector level, as a high frequency of IFNγ-producing cells was observed within AIH-SLE/MCTD CD4(pos)CD25(neg) responder cells. Impairment in suppressor function both of CD4(pos)CD25(pos) cells and CD4(pos)CD25(pos)CD127(neg) T-regs was observed in all three autoimmune conditions, but while addition of CD4(pos)CD25(pos)CD127(neg) T-regs decreased CD4(pos)CD25(neg) responder cell proliferation in healthy subjects and partially in AIH patients, it had no effect in AIH-SLE/MCTD and SLE patients. In conclusion, in AIH-SLE/MCTD T-regs display a distinctive phenotypic and functional signature, characterized by marked activation, elevated IFNγ production and by a profound impairment of suppressive function, suggesting that multiple autoimmune manifestations may derive from a complex defect of immune-regulation.

Hepatitis B virus (HBV) is a noncytopathic virus, and the recognition of infected hepatocytes by HBV-specific CD8 cells has been assumed to be the central mechanism causing both liver damage and virus control. To understand the role of cytotoxic T cells in the pathogenesis of HBV infection, we used functional assays that require T cell expansion in vitro and human histocompatibility leukocyte antigen (HLA)-peptide tetramers that allow direct ex vivo quantification of circulating and liver-infiltrating HBV-specific CD8 cells. Two groups of patients with persistent HBV infection were studied: one without liver inflammation and HBV replication, the other with liver inflammation and a high level of HBV replication. Contrary to expectation, a high frequency of intrahepatic HBV-specific CD8 cells was found in the absence of hepatic immunopathology. In contrast, virus-specific T cells were more diluted among liver infiltrates in viremic patients, but their absolute number was similar because of the massive cellular infiltration. Furthermore, inhibition of HBV replication was associated with the presence of a circulating reservoir of CD8+ cells able to expand after specific virus recognition that was not detectable in highly viremic patients with liver inflammation. These results show that in the presence of an effective HBV-specific CD8 response, inhibition of virus replication can be independent of liver damage. When the HBV-specific CD8 response is unable to control virus replication, it may contribute to liver pathology not only directly but by causing the recruitment of nonvirus-specific T cells.

Autoimmune hepatitis (AIH) is a progressive inflammatory hepatopathy and an important cause of end-stage liver disease. Its aetiology remains unknown, though both genetic and environmental factors are involved in its development. The major mechanism of autoimmune liver damage involves immune reactions against host liver antigens. Numerical and functional defects of regulatory T-cells play a permissive role enabling autoimmune liver injury to occur and persist. The most typical features of AIH are female preponderance, hypergammaglobulinaemia, seropositivity for circulating autoantibodies and a picture of interface hepatitis on histology. Two types of AIH are distinguished according to serological profile: AIH type 1 patients are positive for anti-nuclear and/or anti-smooth muscle antibodies, whereas AIH type 2 patients are defined by the positivity for anti-liver kidney microsomal type 1 antibody and/or for anti-liver cytosol type 1 antibody. Clinical manifestations are variable, and AIH onset is often ill-defined, frequently mimicking acute hepatitis; its course may be fluctuating. AIH responds to immunosuppressive treatment in the majority of cases. Steroids with or without azathioprine should be instituted promptly upon diagnosis. Remission is achieved in some 80% of patients. For the remaining 20% of patients, alternative immunosuppressive agents such as mycophenolate mofetil and calcineurin inhibitors are an option. Liver transplantation should be considered for those patients who progress to cirrhosis and develop complications of end-stage liver disease, as well as for those presenting with acute liver failure; outcomes are excellent, although the disease may recur in the allograft.

Approximately 10% of children with autoimmune hepatitis (AIH) and 30% of those with sclerosing cholangitis (SC) require liver transplantation (LT). LT is indicated in patients who present with fulminant hepatic failure (ie, with encephalopathy) and in those who develop end-stage liver disease despite treatment. After LT, recurrent AIH is reported in approximately 30% of patients and recurrent SC in up to 50%. Diagnosis of recurrence is based on biochemical abnormalities, seropositivity for autoantibodies, interface hepatitis on histology, steroid dependence, and, for SC, presence of cholangiopathy. Recurrence of SC after LT is often associated with poorly controlled inflammatory bowel disease (IBD). Recurrence may even appear years after LT; therefore, steroid-based immunosuppression should be maintained at a higher dose than that used for patients transplanted for nonautoimmune liver diseases. Although the impact of recurrent disease on graft function is controversial, it seems that in pediatric LT recipients recurrence of AIH or SC is associated with compromised graft survival. Exacerbation of preexistent IBD may be observed after LT for SC or AIH, and IBD appears to have a more aggressive course than before LT. In addition, IBD can develop de novo following LT. Liver Transplantation 22 1275-1283 2016 AASLD.

Changes in monocytes and their subsets (CD14(hi)/CD16(neg), CD14(hi)/CD16(pos) and CD14(lo)/CD16(pos)) have been described in several diseases. The combination of CD14, CD16 and HLA-DR has been suggested to discriminate monocytes from the CD16(pos) /HLA-DR(neg) NK-cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA-DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66b(pos) neutrophils, CD56(pos) NKcells, CD19(pos) B-cells, and CD3(pos) T-cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA-DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA-DR expression]. Bland-Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14(hi)/CD16(pos) monocytes was higher (7% vs 4%) whilst the percentage of CD14(lo)/CD16(pos) was lower (1.9% vs. 7%) (P ≤ 0.001); HLA-DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P0.05). The relative expression by monocyte subsets of HLA-DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody-fluorochrome "backbone" targeting HLA-DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients0.8). Monocytes and their subsets can be reliably identified using an antibody-fluorochrome "backbone" of HLA-DR, CD14, and CD16. CD16(pos) monocytes continue to constitutively express HLA-DR even in conditions where HLA-DR is pathologically reduced on CD14(hi)/CD16(neg) monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico-pathological diseases may allow the development of novel biomarkers or therapeutic strategies.

In autoimmune hepatitis (AIH), liver-damaging CD4 T cell responses are associated with defective CD4(pos) CD25(pos) regulatory T cells (T-regs). Galectin-9 (Gal9), a β-galactosidase-binding protein expressed by T-regs, is key to their function, inhibiting T helper 1 immune responses by binding T cell immunoglobulin and mucin domain 3 (Tim-3) on CD4 effector cells. We investigated whether impaired immunoregulation in AIH results from reduced expression of Gal9 in T-regs and/or Tim-3 on CD4 effector cells. Circulating Gal9(pos) CD4(pos) CD25(pos) and Tim-3(pos) CD4(pos) CD25(neg) T cell phenotype was assessed by flow cytometry in 75 AIH patients. To evaluate whether Tim-3 expression renders CD4(pos) CD25(neg) T cells amenable to T-reg control, purified CD4(pos) CD25(neg) Tim-3(pos) (Tim-3(pos)) and CD4(pos) CD25(neg) Tim-3(neg) (Tim-3(neg)) cells were cocultured with T-regs. To determine whether Gal9 expression is essential to function, T-regs were treated with small interfering RNA (siRNA) to repress Gal-9 translation; T-reg suppressor function was assessed by proliferation. In AIH, Tim-3(pos) cells within CD4(pos) CD25(neg) cells and their T-bet(pos) and RORC(pos) subsets were fewer and contained higher numbers of interferon-γ (IFNγ)(pos) and interleukin (IL)-17(pos) cells than healthy subjects (HS). In AIH and HS, Tim-3(pos) cells proliferated less vigorously and were more susceptible to T-reg control than Tim-3(neg) cells. In AIH, Gal9(pos) T-regs were fewer and contained less FOXP3(pos), IL-10(pos), and transforming growth factor β(pos) and more IFNγ(pos) and IL-17(pos) cells than HS. siRNA treatment of Gal-9(pos) T-regs drastically reduced T-reg ability to suppress CD4(pos) CD25(neg) and Tim-3(pos) cell proliferation in AIH and HS. Tim-3(pos) cell percentage correlated inversely with aminotransferase and CD25(neg) T-bet(pos) cell values. Conclusion Reduced levels of Tim-3 on CD4(pos) CD25(neg) effector cells and of Gal9 in T-regs contribute to impaired immunoregulation in AIH by rendering effector cells less prone to T-reg control and T-regs less capable of suppressing.

Liver transplantation is an effective treatment for patients with end-stage acute and chronic autoimmune hepatitis. However, despite the good outcomes reported, disease recurrence is relatively common in the allograft. In addition, autoimmunity and autoimmune liver disease can arise de novo after transplantation for non-autoimmune liver disorders. Little is known about the mechanisms by which autoimmune diseases develop after liver transplantation, but genetic factors, molecular mimicry, impaired regulatory T-cell responses, and exposures to new alloantigens might be involved. Regardless of the pathogenic mechanisms, it is important to remain aware of the existence of recurrent and de novo autoimmune hepatitis after liver transplantation; these disorders are similar to classic autoimmune hepatitis and are therefore not treated with standard antirejection strategies.