Immune-mediated Drug-induced Liver Injury Research Articles

Immuno-inflammatory in vitro hepatotoxicity models to assess side effects of biologicals exemplified by aldesleukin.

Hepatotoxicity induced by immunotherapeutics is an appearing cause for immune-mediated drug-induced liver injury. Such immuno-toxic mechanisms are difficult to assess using current preclinical models and the incidence is too low to detect in clinical trials. As hepatotoxicity is a frequent reason for post-authorisation drug withdrawal, there is an urgent need for immuno-inflammatory in vitro models to assess the hepatotoxic potential of immuno-modulatory drug candidates. We developed several immuno-inflammatory hepatotoxicity test systems based on recombinant human interleukin-2 (aldesleukin). Co-culture models of primary human CD8+ T cells or NK cells with the hepatocyte cell line HepaRG were established and validated with primary human hepatocytes (PHHs). Subsequently, the HepaRG model was refined by increasing complexity by inclusion of monocyte-derived macrophages (MdMs). The main readouts were cytotoxicity, inflammatory mediator release, surface marker expression and specific hepatocyte functions. We identified CD8+ T cells as possible mediators of aldesleukin-mediated hepatotoxicity, with MdMs being implicated in increased aldesleukin-induced inflammatory effects. In co-cultures of CD8+ T cells with MdMs and HepaRG cells, cytotoxicity was induced at intermediate/high aldesleukin concentrations and perforin was upregulated. A pro-inflammatory milieu was created measured by interleukin-6 (IL-6), c-reactive protein (CRP), interferon gamma (IFN-γ), and monocyte chemoattractant protein-1 (MCP-1) increase. NK cells responded to aldesleukin, however, only minor aldesleukin-induced cytotoxic effects were measured in co-cultures. Results obtained with HepaRG cells and with PHHs were comparable, especially regarding cytotoxicity, but high inter-donor variations limited meaningfulness of the PHH model. The in vitro test systems developed contribute to the understanding of potential key mechanisms in aldesleukin-mediated hepatotoxicity. In addition, they may aid assessment of immune-mediated hepatotoxicity during the development of novel immunotherapeutics.

A blinded in vitro analysis of the intrinsic immunogenicity of hepatotoxic drugs: implications for preclinical risk assessment.

In vitro preclinical drug-induced liver injury (DILI) risk assessment relies largely on the use of hepatocytes to measure drug-specific changes in cell function or viability. Unfortunately, this does not provide indications toward the immunogenicity of drugs and/or the likelihood of idiosyncratic reactions in the clinic. This is because the molecular initiating event in immune DILI is an interaction of the drug-derived antigen with MHC proteins and the T-cell receptor. This study utilized immune cells from drug-naïve donors, recently established immune cell coculture systems and blinded compounds with and without DILI liabilities to determine whether these new methods offer an improvement over established assessment methods for the prediction of immune-mediated DILI. Ten blinded test compounds (6 with known DILI liabilities; 4 with lower DILI liabilities) and 5 training compounds, with known T-cell-mediated immune reactions in patients, were investigated. Naïve T-cells were activated with 4/5 of the training compounds (nitroso sulfamethoxazole, vancomycin, Bandrowski's base, and carbamazepine) and clones derived from the priming assays were activated with drug in a dose-dependent manner. The test compounds with DILI liabilities did not stimulate T-cell proliferative responses during dendritic cell-T-cell coculture; however, CD4+ clones displaying reactivity were detected toward 2 compounds (ciprofloxacin and erythromycin) with known liabilities. Drug-responsive T-cells were not detected with the compounds with lower DILI liabilities. This study provides compelling evidence that assessment of intrinsic drug immunogenicity, although complex, can provide valuable information regarding immune liabilities of some compounds prior to clinical studies or when immune reactions are observed in patients.

Discontinuation of immunosuppression in patients with immune-mediated drug-induced liver injury or idiopathic autoimmune hepatitis: A case-control study.

Drug-induced liver injury (DILI) may present with autoimmune features and require immunosuppressive therapy (IST) to reach biochemical response. Discontinuation of IST without hepatitis relapse may be more frequent in these patients as compared to patients with classical autoimmune hepatitis (AIH). We aimed to determine baseline characteristics and outcome of patients with immune-mediated drug induced liver injury (IMDILI) with particular emphasis on IST during follow-up. We performed a single-center retrospective study of consecutive patients presenting at a tertiary care center between January 2005 and December 2019 either with IMDILI or with classical AIH, for whom full baseline characteristics and a close follow-up were available over a 12-month period. Overall, 31 patients (IMDILI n= 16, mean age 59[34-74] years; AIH n= 15, mean age 47 [15-61] years) were included, showing similar biochemical, serological, and histological characteristics. Incriminating drugs in IMDILI patients were mostly represented by nonsteroidal antiinflammatory drugs and sartans. Initial corticosteroids combined with IST led to biochemical response in all patients. Compared to idiopathic AIH, more patients with IMDILI were weaned off corticosteroids at the end of follow-up (11/16 [68.7%] vs 4/15 [26.6%], P< 0.02). At 1 year of follow-up, more patients in the IMDILI group compared to the classical AIH group were off any type of IST (13/16 [81%] vs 15/15 [100%], P= 0.08). Although presenting with similar baseline biochemical and histological characteristics as idiopathic AIH, patients with IMDILI may not require long-term IST.

Hepatotoxicity of Drugs Used in Multiple Sclerosis, Diagnostic Challenge, and the Role of HLA Genotype Susceptibility.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system and the association with other autoimmune diseases is well-documented. There are many therapeutic options for the treatment of MS. Most of the available drugs cause drug-induced liver injury (DILI) to variable extents with heterogeneous clinical and biological manifestations, including liver injury with or without signs of hypersensitivity and autoimmunity. The diagnosis of DILI may be particularly difficult because MS is frequently associated with idiopathic autoimmune hepatitis. Recent advances suggest that MS and immune-mediated DILI could be promoted by genetic factors, including HLA genotype. In addition, some of these drugs may promote hepatitis B virus reactivation. This review explores the potential hepatotoxicity of drugs used to treat MS and the criteria to distinguish DILI from idiopathic autoimmune hepatitis associated with MS. The role of susceptible genes both promoting MS and causing the hepatotoxicity of the drug used for MS treatment is also discussed.

S3674 Drug-Induced Autoimmune Liver Disease (DIAILD)

Introduction: Idiosyncratic drug-induced liver injury (DILI) affects an estimated 19 per 100,000 persons yearly and is a major cause of acute liver failure. Drug-induced autoimmune liver disease (DIAILD) is a poorly defined and under-reported liver disorder, and probably an underestimated liver disease. A small number of drug-induced liver injury (DILI) cases exhibit features typical of autoimmune hepatitis (AIH). To differentiate between true AIH triggered by drugs (DI-AIH) and immune mediated DILI still remains a challenge. Case Description/Methods: We present a 71-year-old ICU nurse with past medical history of hyperlipidemia, hypothyroidism and GERD presenting for nausea, epigastric pain, and chills. Of note, 5 days prior to onset of symptoms she received her vaccine for COVID-19. Medications that she takes at home are cimetidine, Protonix, Lipitor, Synthroid, Zofran, Vitamin A, and D supplementation. Review of systems were pertinent for mild itching, nausea, and chills. Physical exam is unremarkable except for low grade fevers, scleral icterus, and jaundice. Labs are significant for T.bili 13.5, AST 1673, ALT 2372, ALP 246, INR 1.59, ANA 1:160, AFP 169, CT abdomen/pelvis with and without contrast was unremarkable except for mild constipation. Liver biopsy was obtained that revealed submassive hepatic multilobular necrosis with confluent lytic necrosis and cholestasis involving 60-70% of the cores. Portal triads showed surrounding lymphocytic infiltrate without bile duct involvement and active interface hepatitis. active interface hepatitis, . Per pathology report the histological findings were nonspecific and a diagnosis of drug toxicity is the most compatible with given history. She was started on prednisone 40 mg daily and her liver enzymes started to improve. Discussion: In our patient we attributed the acute liver injury secondary to cimetidine from immune mediated DILI. Initially we speculated that covid 19 vaccine could have attributed to her acute hepatitis. However, at the time there were no other cases reported of Covid 19 related acute liver injury;and it could have coincidentally occurred at the same time. It will be interesting to see in the future if Covid 19 vaccine presents similar to this case. Although it is a clinical challenge to differentiate AIH and DI-AIH;it is important to keep both conditions in your differential.

Immune-Mediated Drug-Induced Liver Injury: Immunogenetics and Experimental Models.

Drug-induced liver injury (DILI) is a challenging clinical event in medicine, particularly because of its ability to present with a variety of phenotypes including that of autoimmune hepatitis or other immune mediated liver injuries. Limited diagnostic and therapeutic tools are available, mostly because its pathogenesis has remained poorly understood for decades. The recent scientific and technological advancements in genomics and immunology are paving the way for a better understanding of the molecular aspects of DILI. This review provides an updated overview of the genetic predisposition and immunological mechanisms behind the pathogenesis of DILI and presents the state-of-the-art experimental models to study DILI at the pre-clinical level.

Recent Advances in Models of Immune-Mediated Drug-Induced Liver Injury

Hepatic inflammation is a key feature of a variety of liver diseases including drug-induced liver injury (DILI), orchestrated by the innate immune response (Kupffer cells, monocytes, neutrophils, dendritic cells) and the adaptive immune system (T cells and natural killer T cells). In contrast to acute DILI, prediction of immune-mediated DILI (im-DILI) has been more challenging due to complex disease pathogenesis, lack of reliable models and limited knowledge of underlying mechanisms. This review summarizes in vivo and in vitro systems that have been used to model im-DILI. In particular, the review focuses on state-of-the-art in vitro human-based multicellular models which have been developed to supplement the use of in vivo models due to interspecies variation and increasing ethical concerns regarding animal use. Advantages of the co-cultures in maintaining hepatocyte functions and importantly, introducing heterotypic cell-cell interactions to mimic inflammatory hepatic microenvironment are discussed. Challenges regarding cell source and incorporation of different cells with physical cell-cell contact are outlined and potential solutions are proposed. It is likely that better understanding of the interplay of immune cells in liver models will allow for the development of more accurate systems to better predict hepatotoxicity and stratification of drugs that can cause immune-mediated effects.

Alterations in the HLA-B*57:01 Immunopeptidome by Flucloxacillin and Immunogenicity of Drug-Haptenated Peptides.

Neoantigen formation due to the interaction of drug molecules with human leukocyte antigen (HLA)-peptide complexes can lead to severe hypersensitivity reactions. Flucloxacillin (FLX), a β-lactam antibiotic for narrow-spectrum gram-positive bacterial infections, has been associated with severe immune-mediated drug-induced liver injury caused by an influx of T-lymphocytes targeting liver cells potentially recognizing drug-haptenated peptides in the context of HLA-B*57:01. To identify immunopeptidome changes that could lead to drug-driven immunogenicity, we used mass spectrometry to characterize the proteome and immunopeptidome of B-lymphoblastoid cells solely expressing HLA-B*57:01 as MHC-I molecules. Selected drug-conjugated peptides identified in these cells were synthesized and tested for their immunogenicity in HLA-B*57:01-transgenic mice. T cell responses were evaluated in vitro by immune assays. The immunopeptidome of FLX-treated cells was more diverse than that of untreated cells, enriched with peptides containing carboxy-terminal tryptophan and FLX-haptenated lysine residues on peptides. Selected FLX-modified peptides with drug on P4 and P6 induced drug-specific CD8+ T cells in vivo. FLX was also found directly linked to the HLA K146 that could interfere with KIR-3DL or peptide interactions. These studies identify a novel effect of antibiotics to alter anchor residue frequencies in HLA-presented peptides which may impact drug-induced inflammation. Covalent FLX-modified lysines on peptides mapped drug-specific immunogenicity primarily at P4 and P6 suggesting these peptide sites as drivers of off-target adverse reactions mediated by FLX. FLX modifications on HLA-B*57:01-exposed lysines may also impact interactions with KIR or TCR and subsequent NK and T cell function.

S2557 A Case of Immune-Mediated Liver Injury Secondary to Olaparib

INTRODUCTION: Immune mediated drug-induced liver injury (IM-DILI) presents with a hepatocellular or mixed pattern of liver injury that, for some patients, persists after drug withdrawal. Olaparib is an oral inhibitor of poly-ADP-ribose polymerase and is approved for treatment of relapsed metastatic ovarian cancer with BRCA 1/2 mutations. Mild alterations in liver tests have been reported in < 5% of treated patients. CASE DESCRIPTION/METHODS: A 56-year-old female with BRCA1 mutation and relapsed metastatic ovarian cancer was admitted to the hospital in August 2019 due to a severe acute hepatocellular liver injury with jaundice, elevated INR, and concern for impending fulminant liver failure (Table 1). Her liver tests had previously been normal prior to and until three months after she had started olaparib in May 2019. She had no alcohol history or use of herbal supplements. Physical examination was notable for jaundice, mild right upper quadrant tenderness, and no hepatic encephalopathy. Computed tomography showed mild hepatomegaly with periportal edema. Laboratory work-up for infectious, metabolic, and autoimmune etiologies of liver injury was negative. Liver biopsy revealed areas of submassive necrosis with lobular collapse, moderate to severe portal and lobular inflammation with predominantly lymphocytes, cholestasis with focal hepatocyte rosette formation, acute cholangitis with bile duct injury and ductular proliferation, and ceroid-laden histiocytes, consistent with IM-DILI. No portal fibrosis was seen. Olaparib was promptly withdrawn and prednisone (60 mg/day) was initiated. Liver tests had significantly improved within 4 weeks (Table 1 and Figure 1) and prednisone was tapered while liver enzymes normalized. DISCUSSION: We report the first case of IM-DILI secondary to olaparib use in the United States. Olaparib is largely metabolized by the liver through cytochrome P450 and it is hypothesized that reactive metabolites can bind cellular proteins, leading to the creation of neo-antigens and subsequent liver injury. This case report highlights the need for close monitoring of liver tests while on olaparib and that the diagnosis of IM-DILI and prompt initiation of corticosteroids should be considered if an acute liver injury develops.Table 1.: Liver tests and INR values after olaparib withdrawalFigure 1.: A graphical illustration of livers tests and INR after olaparib was withdrawn and prednisone was started.Figure 2.: liver biopsy shows areas of submassive necrosis with lobular collapse. Note that no viable hepatocytes are identified in this microscopic area.

Approach to the patient with acute severe autoimmune hepatitis.

SummaryAutoimmune hepatitis is associated with varied clinical presentations and natural history, as well as somewhat unpredictable treatment responses. Understanding how to stratify patients who require further escalation of therapy will help clinicians manage these patients. The presentation of acute severe autoimmune hepatitis (AS-AIH) is relatively uncommon, although its prevalence is potentially greater than currently perceived. Previous studies consist of small retrospective single-centre series and are not directly comparable due to the diversity of presentations, disease definitions and non-standardised treatment regimens. We define AS-AIH as those who present acutely with AIH and are icteric with an international normalised ratio ≥1.5 and no evidence of hepatic encephalopathy. Those with hepatic encephalopathy should be defined as having AS-AIH with acute liver failure. In this review, we provide a structured practical approach for diagnosing and managing this unique group of patients.

Drug-Induced Liver Injury: Understanding the Different Immune-Mediated Phenotypes and Clinical Management

The aim of this manuscript is to review two immune-mediated drug-induced liver injury (DILI) phenotypes, immune-allergic DILI (IA-DILI) and DILI with autoimmune hepatitis features (DILI-AIH), which are poorly defined and can lead to significant morbidity and mortality. Recent information on this topic improves the understanding of IA-DILI and DILI-AIH regarding risk factors, associated medications, clinical presentations, histopathologic findings, and management strategies. Although the pathogenesis and specific mechanisms remain elusive, clinical and pathological advancements in our understanding of IA-DILI and DILI-AIH have provided clinicians with added tools for the diagnosis and management of these diseases. Further studies are needed to improve our understanding of immune-mediated DILI.

Challenges and difficulties in pathological diagnosis of autoimmune hepatitis.

Autoimmune hepatitis (AIH) with acute presentation is widely recognized as a distinct clinical entity, and its clinicopathology has been extensively studied. In most cases, AIH with acute presentation is merely acute exacerbation of classical chronic AIH, but pure acute-onset AIH without previous symptoms of chronic liver disease is also encountered. Rapid diagnosis and initiation of immunosuppressive treatment are necessary for both acute exacerbation and acute-onset to prevent fatal liver failure. The diagnostic criteria commonly used for classical AIH are generally applicable to acute exacerbation, but acute-onset AIH may present with additional pathological features. These features include an acute hepatitis phase characterized by centrilobular necrosis. However, centrilobular necrosis is also a feature of drug-induced liver injury, and there are no known histological characteristics exclusive to acute-onset AIH. Moreover, the possibilities of drug-induced AIH and immune-mediated drug-induced liver injury make diagnosis even more difficult. At present, liver biopsy is mandatory for the diagnosis of AIH with acute presentation, but careful consideration of all clinicopathological signs is necessary for differential diagnosis.

Immunogenomics reveal molecular circuits of diclofenac induced liver injury in mice.

Diclofenac is a non-steroidal anti-inflammatory drug and its use can be associated with severe adverse reactions, notably myocardial infarction, stroke and drug-induced liver injury (DILI). In pursue of immune-mediated DILI mechanisms an immunogenomic study was carried out. Diclofenac treatment of mice at 30 mg/kg for 3 days caused significant serum ALT and AST elevations, hepatomegaly and degenerative changes including hepatic glycogen depletion, hydropic swelling, cholesterolosis and eosinophilic hepatocytes with one animal presenting subsegmental infarction due to portal vein thrombosis. Furthermore, portal/periportal induction of the rate limiting enzyme in ammonia detoxification, i.e. carbamoyl phosphate synthetase 1 was observed. The performed microarray studies informed on > 600 differential expressed genes of which 35, 37 and 50 coded for inflammation, 51, 44 and 61 for immune and 116, 129 and 169 for stress response, respectively after single and repeated dosing for 3 and 14 days. Bioinformatic analysis defined molecular circuits of hepatic inflammation with the growth hormone (Ghr)− and leptin receptor, the protein-tyrosine-phosphatase, selectin and the suppressor-of-cytokine-signaling (Socs) to function as key nodes in gene regulatory networks. Western blotting confirmed induction of fibronectin and M-CSF to hallmark tissue repair and differentiation of monocytes and macrophages. Transcript expression of the macrophage receptor with collagenous structure increased > 7-fold and immunohistochemistry of CD68 evidenced activation of tissue-resident macrophages. Importantly, diclofenac treatment prompted strong expression of phosphorylated Stat3 amongst individual animals and the associated 8- and 4-fold Soc3 and Il-6 induction reinforced Ghr degradation as evidenced by immunoblotting. Moreover, immunohistochemistry confirmed regulation of master regulatory proteins of diclofenac treated mice to suggest complex pro-and anti-inflammatory reactions in immune-mediated hepatic injury. The findings encourage translational research.

Profiles of Immune Cells and Reproduction of Immune-mediated Drug-induced Liver Injury

Introduction: Immune-mediated drug-induced liver injury (DILI) is induced by various drugs that can evoke immune responses. However, the mechanisms of immune-mediated DILI have not been clarified yet. The aim of the study is to determine the immune profiles in the peripheral blood of DILI subjects and the patterns of liver injury, and to investigate whether CYP3A4-mediated active metabolites of drugs can participate in inducing the immune responses. Methods: 1. We analyzed the proportions of immune cells in peripheral blood mononuclear cells (PBMCs) of DILI using BD FACS canto II, comparing 18 healthy controls with 6 acute hepatitis subjects (type A 2cases, type B 4cases). PBMCs were stained with the following 4 sets of antibodies. 1. DC:CD1c, CD14, CD19, CD86, CD141, CD303, CD304, PD-L1; 2.Tcell: CD3, CD4, CD8a, CD25, CD127, PD-1; 3.NK/NKT:CD3, CD16, CD56, NKG2D, 4. Myeloid-derived suppressor cells (MDSC); CD11b, CD14, CD16, CD33, PD-L1, HLA-DR. 2. PBMCs from immune-mediated DILI patients or healthy subjects were cultured with CYP3A4 expressing HepG2 cells and amiodarone (AMD) using a transwell system. We collected PBMCs in the upper chamber and analyzed the proportions of the various kinds of immune cells at 48 hours after co-incubation by BD FACS canto II. To assess the effects of the metabolites of AMD, we measured the concentrations of AMD and N-desethyamiodarone, a major metabolite of AMD, using LS/MS. Moreover, PBMCs from DILI subjects treated with asunaprevir were analyzed the same way since asunaprevirmediated DILI could be induced by immune-dysfunction. Results: 1. There was significantly elevated proportion of MDSCs in the PBMCs of DILI and acute hepatitis(type A, type B), compared with those of healthy controls(P<0.0001). The frequency of regulatory T cells (Tregs) in the PBMCs of DILI was higher than in Tregs of acute hepatitis (type A, type B) and healthy controls (P=0.0862). Comparisons of immune profiles between the dominant injury of hepatocytes and the dominant injury of bile ducts did not reveal any significant differences. 2. The in vitro co-culture system could partially reproduce the immune profiles in some DILI patients. Conclusion: These results demonstrated that immune-mediated DILI was associated with specific immune subsets. The profiles of immune cells in peripheral blood appear to be prognostic. We might reproduce immune-mediated drug-induced liver injury with the in vitro co-culture system.Figure 1Figure 2

Relationships Between Pharmacovigilance, Molecular, Structural, and Pathway Data: Revealing Mechanisms for Immune-Mediated Drug-Induced Liver Injury.

Immune-mediated drug-induced liver injury (IMDILI) can be devastating, irreversible, and fatal in the absence of successful transplantation surgery. We present a novel approach that combines the methods of pharmacoepidemiology with in silico molecular modeling to identify specific features in toxic ligands that are associated with clinical features of IMDILI. Specifically, from pharmacovigilance data multivariate logistic regression identified 18 drugs associated with IMDILI (P < 0.00015). Eleven of these drugs, along with their known and proposed metabolites, constituted a training set used to develop a four-point pharmacophore model (sensitivity 75%; specificity 85%). Subsequently, this information was combined with information from immune-pathway reviews and genetic-association studies and complemented with ligand-protein docking simulations to support a hypothesis implicating two putative targets within separate, possibly interacting, immune-system pathways: the major histocompatibility complex within the adaptive immune system and Toll-like receptors (TLRs), in particular TLR-7, which represent pattern recognition receptors of the innate immune system.

Hepatotoxicants induce cytokine imbalance in response to innate immune system

In recent years, attention has been paid to innate immune systems as mechanisms to initiate or promote drug-induced liver injury (DILI). Kupffer cells are hepatic resident macrophages and might be involved in the pathogenesis of DILI by release of pro- and anti-inflammatory mediators such as cytokines, chemokines, reactive oxygen species, and/or nitric oxides. The purpose of this study was to investigate alterations in mediator levels induced by hepatotoxic compounds in isolated Kupffer cells and discuss the relation between balance of each cytokine or chemokine and potential of innate immune-mediated DILI. Primary cultured rat Kupffer cells were treated with hepatotoxic (acetaminophen, troglitazone, trovafloxacin) or non-hepatotoxic (pioglitazone, levofloxacin) compounds with or without lipopolysaccharide (LPS). After 24 hr treatment, cell supernatants were collected and various levels of mediators released by Kupffer cells were examined. Although hepatotoxicants had no effect on the LPS-induced tumor necrosis factor-alpha (TNF-α) secretion, they enhanced the release of pro-inflammatory cytokine interleukin-1 beta (IL-1β) and suppressed the anti-inflammatory cytokines interleukin-6 (IL-6) and interleukin-10 (IL-10) induced by LPS. These cytokine shifts were not associated with switching the phenotypes of M1 and M2 macrophages in Kupffer cells. In conclusion, the present study suggested that the levels of some specific cytokines are affected by DILI-related drugs with LPS stimulation, and imbalance between pro- and anti-inflammatory cytokines, induced by the up-regulation of IL-1β and the down-regulation of IL-6 or IL-10, plays a key role in innate immune-mediated DILI.

Drug-induced autoimmune liver disease: A diagnostic dilemma of an increasingly reported disease.

The aetiology of autoimmune hepatitis (AIH) is uncertain but the disease can be triggered in susceptible patients by external factors such as viruses or drugs. AIH usually develops in individuals with a genetic background mainly consisting of some risk alleles of the major histocompatibility complex (HLA). Many drugs have been linked to AIH phenotypes, which sometimes persist after drug discontinuation, suggesting that they awaken latent autoimmunity. At least three clinical scenarios have been proposed that refers to drug- induced autoimmune liver disease (DIAILD): AIH with drug-induced liver injury (DILI); drug induced-AIH (DI-AIH); and immune mediated DILI (IM-DILI). In addition, there are instances showing mixed features of DI-AIH and IM-DILI, as well as DILI cases with positive autoantibodies. Histologically distinguishing DILI from AIH remains a challenge. Even more challenging is the differentiation of AIH from DI-AIH mainly relying in histological features; however, a detailed standardised histologic evaluation of large cohorts of AIH and DI-AIH patients would probably render more subtle features that could be of help in the differential diagnosis between both entities. Growing information on the relationship of drugs and AIH is being available, being drugs like statins and biologic agents more frequently involved in cases of DIAILD. In addition, there is some evidence on the fact that patients diagnosed with DIAILD may have had a previous episode of hepatotoxicity. Further collaborative studies in DIAILD will strengthen the knowledge and understanding of this intriguing and complex disorder which might represent different phenotypes across the spectrum of disease.

Immunological aspects of drug hypersensitivity—from molecule to man

ImmuNologICal aSpeCTS of drug hyperSeNSITIVITy—from moleCule To maN K. Park Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom Summary: Drug-induced liver injury (DILI) accounted for > 50% of cases of acute liver failure and 15% of patients undergoing liver transplantation between 1990 and 2002. One of the biggest fears is unpredictable idiosyncratic drug-induced liver injury (IDILI), which is a major contributor to the failure of a drug to process through development and is a major cause of withdrawal and black box warnings. Due to the low concordance of the hepatotoxicity of drugs in animals and humans, a fundamental understanding of the mechanistic basis from novel human-relevant in vitro models and biomarkers is critical for the development of effective strategies to prevent and predict DILI. Hepatocellular activation of drugs has been widely implicated in immune-mediated DILI with concomitant activation of the adaptive immune system. Consistent with this hypothesis, a number of HLA alleles that are associated with DILI have been identified. Studies with patient PBMC and PBMC from drug-naive donors carrying HLA risk alleles have shown that drugs form specific associations with HLA risk alleles generating T-cell antigens and hence provide the immunogenetic basis for the reaction. The identification of HLA alleles as predisposing factors for IDILI suggests that the adaptive immune system also participates in reactions targeting the liver. Furthermore, the release of damage-associated molecular pattern molecules (DAMPs), such as high mobility group box-1, may serve to activate cells of the innate immune system. Certain mediators produced by innate immune cells may cause liver injury, but other factors are actually important to promote tissue repair and regeneration. However, to fully define cause and effect (bioactivation vs liver failure), an integrated approach based on clinical studies, in vitro experiments, and animal models will be required, underpinned by appropriate bioanalysis. We have been able to characterize covalent protein adducts in man formed by reactive drug metabolites (nevirapine, diclofenac) using techniques developed for β -lactam antibiotics that associate with various types of drug hypersensitivity. We have also developed in vitro models to look at the interplay between, genetic, chemical, and immunologic factors using cells from both hypersensitive and drug-naive individuals. Such techniques and concepts should enable us to elucidate various mechanistic pathways and thus define both chemical and biological variables that underpin this rare but serious form of DILI. Disclosure of Interest: None declared.

Toxicological Implications of Modulation of Gene Expression by MicroRNAs

MicroRNAs (miRNAs) are a large family of non-coding RNAs that are evolutionarily conserved, endogenous, and 21-23 nucleotides in length. miRNAs regulate gene expression by targeting messenger RNAs (mRNAs) by binding to complementary regions of transcripts to repress their translation or mRNA degradation. miRNAs are encoded by the genome, and more than 1000 human miRNAs have been identified so far. miRNAs are predicted to target ∼60% of human mRNAs and are expressed in all animal cells and have fundamental roles in cellular responses to xenobiotic stresses, which affect a large range of physiological processes such as development, immune responses, metabolism, tumor formation as well as toxicological outcomes. Recently, many reports concerning miRNAs related to cancer have been published; however, the miRNA research in the metabolism of xenobiotics and endobiotics and in toxicology has only recently been established. This review describes the current knowledge on the miRNA-dependent regulation of drug-metabolizing enzymes and nuclear receptors and its potential toxicological implications. In this review, miRNAs with reference to target prediction, potential modulation of toxicology-related changes of miRNA expression, role of miRNA in immune-mediated drug-induced liver injury, miRNA in plasma as potential toxicological biomarkers, and relevance of miRNA-related genetic polymorphisms are discussed.

Identification of a critical CYP2E1 epitope in patients with drug-induced liver injury from anesthetics (93.16)

Abstract Susceptible individuals develop immune-mediated drug-induced liver injury (DILI) after receiving halogenated anesthetics, anti-seizure medications, antibiotics or non-steroidal anti-inflammatory drugs. In anesthetic DILI, serum cytochrome P4502E1 (CYP2E1) autoantibodies and diminished CYP2E1 activity assist in confirming the diagnosis, while responsible epitopes are unknown. We hypothesize that anesthetic DILI is initiated by pathogenic CYP2E1 epitopes centered on the Ser129 active site and presented by MHC II molecules in susceptible haplotypes. To test our hypothesis, we identified 15 mer human CYP2E1 candidate epitopes centered on Ser129 that bind to mouse MHC II using competitive inhibition of OVA323 - 329 in I-Ad DO11.10 T cell hybridomas + LS102.9 activation. Next we measured splenocyte T cell proliferation to candidate epitopes using our model of anesthetic DILI in BALB/c mice two weeks following TFA-S100 immunizations. Two CYP2E1 epitopes were detected by competitive inhibition; however, only one epitope Gly113 - Leu135 was recognized by proliferation and human sera ELISA assays. SYFPEITHI prediction of the probability of binding confirmed H2-Ad and H2-Ed haplotypes in BALB/c mice and identified 6 human MHC II haplotypes that could bind this epitope: HLA-DRB1*0101, HLA-DRB1*0301 HLA-DRB1*0401, HLA-DRB1*0701 HLADRB*1101 and HLADRB1*1501. This is the first demonstration of a CYP2E1 epitope and MHC II haplotypes that may increase the risk of developing anesthetic DILI.