Roadmap to DILI research in Europe. A proposal from COST action ProEuroDILINet.

In the current article the aims for a constructive way forward in Drug-Induced Liver Injury (DILI) are to highlight the most important priorities in research and clinical science, therefore supporting a more informed, focused, and better funded future for European DILI research. This Roadmap aims to identify key challenges, define a shared vision across all stakeholders for the opportunities to overcome these challenges and propose a high-quality research program to achieve progress on the prediction, prevention, diagnosis and management of this condition and impact on healthcare practice in the field of DILI. This will involve 1. Creation of a database encompassing optimised case report form for prospectively identified DILI cases with well-characterised controls with competing diagnoses, biological samples, and imaging data; 2. Establishing of preclinical models to improve the assessment and prediction of hepatotoxicity in humans to guide future drug safety testing; 3. Emphasis on implementation science and 4. Enhanced collaboration between drug-developers, clinicians and regulatory scientists. This proposed operational framework will advance DILI research and may bring together basic, applied, translational and clinical research in DILI.

Biomarkers of idiosyncratic drug-induced liver injury (DILI) - a systematic review.

INTRODUCTION: Idiosyncratic drug-induced liver injury (DILI) is an unpredictable event, and there are no specific biomarkers that can distinguish DILI from alternative explanations or predict its clinical outcomes. AREAS COVERED: This systematic review summarizes the available evidence for all biomarkers proposed to have a role in the diagnosis or prognosis of DILI. Following a comprehensive search, we included all types of studies in humans. We included DILI cases based on any threshold criteria but excluded intrinsic DILI, commonly caused by paracetamol overdose. We classified studies into diagnostic and prognostic categories and assessed their methodological quality. After reviewing the literature, 14 studies were eligible. EXPERT OPINION: Diagnostic studies were heterogeneous with regard to the study population and outcomes measured. Prognostic models were developed by integrating novel biomarkers, risk scores, and traditional biomarkers, which increased their prognostic ability to predict death or transplantation by 6 months. This systematic review highlights the case of need for non-genetic biomarkers that distinguish DILI from acute liver injury related to alternative etiology. Biomarkers with the potential to identify serious adverse outcomes from acute DILI should be validated in independent prospective cohorts with a substantial number of cases.

Gender and gut microbiota composition determine hepatic bile acid, metabolic and inflammatory response to a single fast-food meal in healthy adults.

BACKGROUND & AIMS: Regular consumption of fast-food (FF) as a form of typical Western style diet is associated with obesity and the metabolic syndrome, including its hepatic manifestation nonalcoholic fatty liver disease. Currently, it remains unclear how intermittent excess FF consumption may influence liver metabolism. The study aimed to characterize the effects of a single FF binge on hepatic steatosis, inflammation, bile acid (BA), glucose and lipid metabolism. METHODS: Twenty-five healthy individuals received a FF meal and were asked to continue eating either for a two-hour period or until fully saturated. Serum levels of transaminases, fasting BA, lipid profile, glucose and cytokine levels as well as transient elastography and controlled attenuation parameter (CAP; to assess hepatic steatosis) were analyzed before (day 0) and the day after FF binge (day 1). Feces was collected prior and after the FF challenge for microbiota analysis. RESULTS: The FF meal induced a modest increase in CAP, which was accompanied by a robust increase of fasting serum BA levels. Surprisingly, levels of cholesterol and bilirubin were significantly lower after the FF meal. Differentiating individuals with a relevant delta BA (>1 µmol/l) increase vs. individuals without (delta BA ≤1 µmol/l), identified several gut microbiota, as well as gender to be associated with the BA increase and the observed alterations in liver function, metabolism and inflammation. CONCLUSION: A single binge FF meal leads to a robust increase in serum BA levels and alterations in parameters of liver injury and metabolism, indicating a novel metabolic aspect of the gut-liver axis.

c-Jun N-terminal kinase 2 promotes enterocyte survival and goblet cell differentiation in the inflamed intestine.

c-Jun N-terminal kinases (JNKs) contribute to immune signaling but their functional role during intestinal mucosal inflammation has remained ill defined. Using genetic mouse models, we characterized the role of JNK1 and JNK2 during homeostasis and acute colitis. Epithelial apoptosis, regeneration, differentiation, and barrier function were analyzed in intestinal epithelium-specific (ΔIEC) or complete JNK1 and bone marrow chimeric or complete JNK2 deficient mice as well as double-knockout animals (JNK1ΔIECJNK2-/-) during homeostasis and acute dextran sulfate sodium (DSS)-induced colitis. Results were confirmed using human HT-29 cells and wild-type or JNK2-deficient mouse intestinal organoid cultures. We show that nonhematopoietic JNK2 but not JNK1 expression confers protection from DSS-induced intestinal inflammation reducing epithelial barrier dysfunction and enterocyte apoptosis. JNK2 additionally enhanced Atonal homolog 1 expression, goblet cell and enteroendocrine cell differentiation, and mucus production under inflammatory conditions. Our results identify a protective role of epithelial JNK2 signaling to maintain mucosal barrier function, epithelial cell integrity, and mucus layer production in the event of inflammatory tissue damage.

Lack of gp130 expression in hepatocytes attenuates tumor progression in the DEN model.

Chronic liver inflammation is a crucial event in the development and growth of hepatocellular carcinoma (HCC). Compelling evidence has shown that interleukin-6 (IL-6)/gp130-dependent signaling has a fundamental role in liver carcinogenesis. Thus, in the present study we aimed to investigate the role of gp130 in hepatocytes for the initiation and progression of HCC. Hepatocyte-specific gp130 knockout mice (gp130(Δhepa)) and control animals (gp130(f/f)) were treated with diethylnitrosamine (DEN). The role of gp130 for acute injury (0-144 h post treatment), tumor initiation (24 weeks) and progression (40 weeks) was analyzed. After acute DEN-induced liver injury we observed a reduction in the inflammatory response in gp130(Δhepa) animals as reflected by decreased levels of IL-6 and oncostatin M. The loss of gp130 slightly attenuated the initiation of HCC 24 weeks after DEN treatment. In contrast, 40 weeks after DEN treatment, male and female gp130(Δhepa) mice showed smaller tumors and reduced tumor burden, indicating a role for hepatocyte-specific gp130 expression during HCC progression. Oxidative stress and DNA damage were substantially and similarly increased by DEN in both gp130(f/f) and gp130(Δhepa) animals. However, gp130(Δhepa) livers revealed aberrant STAT5 activation and decreased levels of transforming growth factor-ß (TGFß), pSMAD2/3 and SMAD2, whereas phosphorylation of STAT3 at Tyr705 and Ser727 was absent. Our results indicate that gp130 deletion in hepatocytes reduces progression, but not HCC initiation in the DEN model. Gp130 deletion resulted in STAT3 inhibition but increased STAT5 activation and diminished TGF-dependent signaling. Hence, blocking gp130 in hepatocytes might be an interesting therapeutic target to inhibit the growth of HCC.

TNFR1 determines progression of chronic liver injury in the IKKγ/Nemo genetic model.

Death receptor-mediated hepatocyte apoptosis is implicated in a wide range of liver diseases including viral and alcoholic hepatitis, ischemia/reperfusion injury, fulminant hepatic failure, cholestatic liver injury, as well as cancer. Deletion of NF-κB essential modulator in hepatocytes (IKKγ/Nemo) causes spontaneous progression of TNF-mediated chronic hepatitis to hepatocellular carcinoma (HCC). Thus, we analyzed the role of death receptors including TNFR1 and TRAIL in the regulation of cell death and the progression of liver injury in IKKγ/Nemo-deleted livers. We crossed hepatocyte-specific IKKγ/Nemo knockout mice (Nemo(Δhepa)) with constitutive TNFR1(-/-) and TRAIL(-/-) mice. Deletion of TNFR1, but not TRAIL, decreased apoptotic cell death, compensatory proliferation, liver fibrogenesis, infiltration of immune cells as well as pro-inflammatory cytokines, and indicators of tumor growth during the progression of chronic liver injury. These events were associated with diminished JNK activation. In contrast, deletion of TNFR1 in bone-marrow-derived cells promoted chronic liver injury. Our data demonstrate that TNF- and not TRAIL signaling determines the progression of IKKγ/Nemo-dependent chronic hepatitis. Additionally, we show that TNFR1 in hepatocytes and immune cells have different roles in chronic liver injury-a finding that has direct implications for treating chronic liver disease.

Supervivenica de hepatocitos alogénicos trasplantados en timo, en ausencia de terapia inmunosupresora / Survival o fallogeneic hepatocytes transplanted into thymus, without immunossuprresive therapy

El trasplante hepático constituye actualmente, una realidad clínica en situaciones de fallo hepático. Por otro lado, en el caso de diversos errores congénitos del metabolismo, en los cuales un solo fallo en una reacción de una ruta metabólica, conducea un fallo multiorgánico, la sustitución de un hígado morfológicamente normal parece una medida demasiado agresiva. Por ello, el uso de terapias celulares para el tratamiento de estas entidades está cada vez mas presente. Sin embargo, ambas estrategias terapéuticas se asocian a los efectos derivados de la inmunosupresión. La búsqueda de métodos que eliminen la necesidad de inmunosupresión es uno de los principales objetivos dentro del campode los trasplantes. La inyección intratímica de aloantígenos ha demostrado, en ciertos modelos experimentales, inducir tolerancia inmunológica. Sin embargo, los resultados con el trasplante intratímico de hepatocitos son contradictorios. Por este motivo, el objetivo de nuestro estudio ha sido estudiar el trasplante de hepatocitos en un órgano inmunológicamente privilegiado, como es el timo, donde se puede estudiar la funcionalidad y supervivencia de las células trasplantadas, así como estudiar la hipótesis de que el trasplante intratímico podría inducir un cierto grado de inmunomodulación en las células del sistemainmune. Se realizó trasplante de hepatocitos procedentes de ratas Fisher 344, en timo de ratas Gunn que habían recibido una dosis previa única de ciclosporina A. Después del trasplante, las ratas no recibieron nuevas dosis de ciclosporina, y fueron sacrificadas a diferentes tiempos. Se realizó microscopia óptica y se determinaron los niveles séricos y biliares de bilirrubina. También se estudió la presencia de mRNAs específicos y las poblaciones linfocitarias CD4 y CD8 en timo y sangre. Los datos obtenidos demuestran que los hepatocitos trasplantados sobreviven al menos 6 semanas, en los animales que habían recibido una dosis de ciclosporina A, antes del trasplante (AU)

Liver transplantation is currently a clinical reality, and it is well established as a treatment for acute organ failure. On the other hand, in the case of inborn errors of metabolism, in which a single step in a metabolic pathway leads to multiple organ failure, the replacement of a morphologically normal liver would be an aggressive measure. Thus, the use of cell therapy in these diseases is being considered. However, both strategies are associated with the effects derived from immunosuppression. The search for methods resulting in the avoidance of immunosupression is nowadays one of the main objectives within the field of transplantation. Intrathymic alloantigen injection has proved to induce immunological tolerance in certain experimental models. However, the results of intrathymic hepatocyte transplantation are contradictory. For this reason our objective was to study the transplantation of hepatocytes into an immunologically privileged organ such as the thymus, where we could assess the functionality and survival of the transplanted cells, as well as test the hypothesis that the intrathymic transplant could induce a certain immunomodulation in the cells of the immune system. Gunn rats, half of which received a single pre-transplant dose of cyclosporine A, were transplanted into the thymus with hepatocytes from Fisher 344 rats. After the transplant, rats did not receive any further dose of cyclosporine A, and they were sacrificed at different times. Light microscopy, bilirubin levels in both serum and bile, and presence of specific mRNAs were determined. Also, CD4 and CD8 lymphocyte subsets were studied in both thymus and blood. The results demonstrated thattransplanted hepatocytes survive for six weeks in animals which had been given cyclosporine A prior to the transplant (AU)

Kupffer cells and alcoholic liver disease.

Liver disease is a major cause of illness and death worldwide. A central component in the complex network leading to the development of alcoholic liver disease is the activation of Kupffer cells by endotoxin and other soluble mediators. Alcohol consumption induces a state of "leaky gut increasing plasma and liver endotoxin levels. When Kupffer cells become activated, they interact with a complex of proteins located on the extracellular membrane signaling to produce a wide array of soluble factors, including cytokines, chemokines, growth factors, cyclooxygenase and lipoxygenase metabolites, and reactive oxygen species such as superoxide anion, hydrogen peroxide, and nitric oxide, all of which provide physiologically diverse and pivotal paracrine effects on all other liver cell types and, ultimately, liver injury. Kupffer cells are also central to the liver homeostatic response to injury as upon cellular degenerative changes, they immediately respond to the insult and release mediators to orchestrate inflammatory and reparative responses. Thus, the homeostatic responses are initiated by Kupffer cell-derived mediators at the cellular level and underlie the liver s defense and reparative mechanisms against injury. In order to understand better the role of Kupffer cells in the onset of liver injury, animal models in which Kupffer cells are inactivated, and cell culture settings (e.g. co-cultures) are being used with promising results that advance our understanding of alcoholic liver disease.

Células de Kupffer y hepatopatía alcohólica / Kupffer cells and alcoholic liver disease

Las hepatopatías son una de las principales causas de morbilidady mortalidad en todo el mundo. Uno de los componentesesenciales de la compleja red que lleva al desarrollo de la hepatopatíaalcohólica es la activación de las células de Kupffer por endotoxinasy otros mediadores solubles. El consumo de alcohol induceun estado de "escapes digestivos" que aumenta los niveles deendotoxinas en plasma e hígado. Cuando las células de Kupffer seactivan, interactúan con un complejo de proteínas situado en lavía de señalización de las membranas extracelulares para producirtoda una serie de factores solubles, como citocinas, quimiocinas,factores de crecimiento, metabolitos de la ciclooxigenasa y la lipooxigenasay especies reactivas del oxígeno como el anión superóxido,el peróxido de hidrógeno y el óxido nítrico, todos los cualesejercen efectos paracrinos fisiológicamente diversos y fundamentalessobre todos los demás tipos de células del hígado, produciendoen última instancia la lesión hepática. Las células de Kupfferson también esenciales para la respuesta homeostática del hígadoa las lesiones, ya que, al presentarse cambios celulares degenerativos,responden de inmediato a la agresión y liberan mediadoresque orquestan las reacciones inflamatorias y reparadoras. Así, lasrespuestas homeostáticas comienzan por los mediadores que procedende las células de Kupffer, que, a nivel celular, se encuentranen la base de los mecanismos de defensa y reparadores del hígadofrente a las lesiones. Para poder comprender mejor el papel de lascélulas de Kupffer en el inicio de la lesión hepática, se están empleandocultivos celulares (p. ej., co-cultivos) y modelos animalesen los que dichas células de Kupffer se encuentran inactivadas. Estosestudios están proporcionando resultados prometedores, yaque están contribuyendo a progresar en nuestros conocimientossobre la hepatopatía alcohólica

Liver disease is a major cause of illness and death worldwide. Acentral component in the complex network leading to the developmentof alcoholic liver disease is the activation of Kupffer cellsby endotoxin and other soluble mediators. Alcohol consumptioninduces a state of ‘leaky gut’ increasing plasma and liver endotoxinlevels. When Kupffer cells become activated, they interact witha complex of proteins located on the extracellular membrane signalingto produce a wide array of soluble factors, including cytokines,chemokines, growth factors, cyclooxygenase and lipoxygenasemetabolites, and reactive oxygen species such assuperoxide anion, hydrogen peroxide, and nitric oxide, all ofwhich provide physiologically diverse and pivotal paracrine effectson all other liver cell types and, ultimately, liver injury. Kupffercells are also central to the liver homeostatic response to injury asupon cellular degenerative changes, they immediately respond tothe insult and release mediators to orchestrate inflammatory andreparative responses. Thus, the homeostatic responses are initiatedby Kupffer cell-derived mediators at the cellular level and underliethe liver’s defense and reparative mechanisms against injury.In order to understand better the role of Kupffer cells in theonset of liver injury, animal models in which Kupffer cells are inactivated,and cell culture settings (e.g. co-cultures) are being usedwith promising results that advance our understanding of alcoholicliver disease

Evolution of the activity of UGT1A1 throughout the development and adult life in a rat.

Biliary excretion is the main route of disposal of bilirubin and impaired excretion results in jaundice, a well recognisable symptom of liver disease. Conjugation of bilirubin in the liver is essential for its clearance. The glucuronidation of bilirubin is catalysed by the microsomal UDP-glucuronosyltransferase UGT1A1. Patients with Crigler-Najjar syndrome type 1 and Gunn rats, mutant strain of the Wistar rats, bear an autosomal recessive disorder resulting in hyperbilirubinemia. The aim of this work is to add new data about activity of UGT1A1 during the perinatal period and adult life. The results showed that activity of UGT1A1 is detectable from day 22 of the gestation. After birth, activity of UGT1A1 gradually increases and reaches the levels of adult life. Furthermore, bilirubin azopigments have been separated and characterized by thin layer chromatography. We have found that concentration of samples by evaporation and ulterior storing at -20 degrees C seemed to be suitable for the maintenance of samples.

Hepatic proliferation in Gunn rats transplanted with hepatocytes: effect of retrorsine and tri-iodothyronine.

Hepatocyte transplantation would offer an attractive alternative to liver transplantation in the treatment of inborn errors of liver metabolism. However, a major problem in most transplantation studies to date has been the limited growth of transplanted cells in the recipient organ. We performed a strategy for selective proliferation of transplanted cells by interfering with the proliferative capacity of resident hepatocytes, using the pyrrolizidine alkaloid retrorsine and then transplanting liver cells in conjunction with repeated administration of triiodothyronine, an inducer of hepatocyte proliferation in rats. In the present study, foetal and adult syngeneic hepatocyte transplantation into spleen was performed in retrorsine-treated hyperbilirubinemic Gunn rats. In parallel, repeated injections of triiodothyronine were given to recipients. Rats were sacrificed at 1, 7, 30 and 90 days after transplantation and blood and bile samples were taken to assess the functionality of transplanted cells. The proliferative activity of transplanted hepatocytes was evaluated using proliferating cell nuclear antigen labelling index. In summary, both adult and foetal hepatocyte transplantation were effective in correcting a metabolic abnormality in Gunn rats for as long as 3 months. The RS/T3 model, as a measure to increase graft function, could represent an important advance to future clinical application of hepatocyte transplantation.

A compared study on the purification of fetal and adult rat hepatocyte suspensions by biomagnetic isolation techniques and flow cytometry analysis.

An analysis of liver cell populations from both adult and 21 day pregnancy rat fetuses (E21) was carried out. The results show that E21 hepatocytes express OX-43, as do endothelial cells but not adult hepatocytes. OX-43 could be used in future as a cell marker for the hepatocyte maturation.

Expression of bilirubin UDP-glucuronosyltransferase (bUGT) throughout fetal development: intrasplenic transplantation into Gunn rats to correct enzymatic deficiency.

The aim of this work was to determine the pattern of expression of hepatic bilirubin UDP-glucuronosyltransferase throughout fetal development in rats, with the purpose of using fetal hepatocytes at the most appropiate stage of development for transplantation into Gunn rats lacking bilirubin UDP-glucuronosyltransferase activity and then assessing the therapeutic capacity of the implants. The results show that at day 13 of gestational life there is already bilirubin UDP-glucuronosyltransferase gene expression. Twenty-one-day fetal hepatocyte transplantation was also performed into the spleens of hyperbilirubinemic Gunn rats, when alpha-fetoprotein mRNA is still detectable. At 15, 30, and 90 days after transplantation, a mild decrease in total bilirubin serum levels was observed. An increase in bile conjugated bilirubin also was observed at 30 and 90 days. These data suggest the favorable evolution of transplanted cells and show its feasibility for therapy.