Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease

Chronic cholestasis often results in premature death from liver failure with fibrosis; however, the molecular mechanisms contributing to biliary cirrhosis are not demonstrated. In this article, we show that the death signal mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) may be a key regulator of cholestatic liver injury. Agonistic anti-DR5 monoclonal antibody treatment triggered cholangiocyte apoptosis, and subsequently induced cholangitis and cholestatic liver injury in a mouse strain-specific manner. TRAIL- or DR5-deficient mice were relatively resistant to common bile duct ligation-induced cholestasis, and common bile duct ligation augmented DR5 expression on cholangiocytes, sensitizing mice to DR5-mediated cholangitis. Notably, anti-DR5 monoclonal antibody-induced cholangitis exhibited the typical histological appearance, reminiscent of human primary sclerosing cholangitis. Human cholangiocytes constitutively expressed DR5, and TRAIL expression and apoptosis were significantly elevated in cholangiocytes of human primary sclerosing cholangitis and primary biliary cirrhosis patients. Thus, TRAIL/DR5-mediated apoptosis may substantially contribute to chronic cholestatic disease, particularly primary sclerosing cholangitis.

Reduced serum chemerin in patients with more severe liver cirrhosis

Chemerin is a well-established modulator of immune cell function and its serum levels are induced in inflammatory diseases. Liver cirrhosis is associated with inflammation which is aggravated by portal hypertension. The objective of this study was to evaluate whether chemerin is induced in patients with more severe liver cirrhosis and portal hypertension. Chemerin has been measured by ELISA in the portal venous serum (PVS), systemic venous serum (SVS) and hepatic venous serum (HVS) of 45 patients with liver cirrhosis. Chemerin is higher in HVS compared to PVS in accordance with our recently published finding. SVS, HVS and PVS chemerin decline in patients with more advanced liver injury defined by the CHILD-PUGH score. Hepatic chemerin has been determined in a small cohort and is similarly expressed in normal and cirrhotic liver. MELD score and serum markers of liver and kidney function do not correlate with chemerin. There is a positive correlation of chemerin in all compartments with Quick prothrombin time and of SVS chemerin with systolic blood pressure. PVS chemerin is induced in patients with modest/massive ascites but this does not translate into higher HVS and SVS levels. Chemerin is not associated with variceal size. Reduction of portal pressure by transjugular intrahepatic portosystemic shunt does not affect chemerin levels. These data show that low chemerin in patients with more severe liver cirrhosis is associated with reduced Quick prothrombin time.

P2X1 regulated IL-22 secretion by innate lymphoid cells is required for efficient liver regeneration.

Paracrine signalling mediated via cytokine secretion is essential for liver regeneration after hepatic resection, yet the mechanisms of cellular crosstalk between immune and parenchymal cells are still elusive. Interleukin-22 (IL-22) is released by immune cells and mediates strong hepatoprotective functions. However, it remains unclear if IL-22 is critical for the crosstalk between liver lymphocytes and parenchymal cells during liver regeneration after partial hepatectomy. Here we found that plasma levels of IL-22 and its upstream cytokine IL-23 are highly elevated in patients after major liver resection. In a mouse model of partial hepatectomy, deletion of IL-22 was associated with significantly delayed hepatocellular proliferation and an increase of hepatocellular injury and endoplasmic reticulum stress. Using Rag1-/- and Rag2-/- γc-/- mice we show that the main producers of IL-22 post partial hepatectomy are conventional natural killer cells and innate lymphoid cells type 1. Extracellular ATP, a potent danger molecule, is elevated in patients immediately after major liver resection. Antagonism of the P2 type nucleotide receptors P2X1 and P2Y6 significantly decreased IL-22 secretion ex vivo. In vivo, specific inhibition of P2X1 was associated with decreased IL-22 secretion, elevated liver injury and impaired liver regeneration.

Atorvastatin attenuates hepatic fibrosis in rats after bile duct ligation via decreased turnover of hepatic stellate cells

Activation of hepatic stellate cells (HSC) and transdifferentiation to myofibroblasts following liver injury is the main culprit for hepatic fibrosis. Myofibroblasts show increased proliferation, migration, contraction, and production of extracellular matrix (ECM). In vitro, HMG-CoA reductase inhibitors (statins) inhibit proliferation and induce apoptosis of myofibroblastic HSC. To investigate the antifibrotic effects of atorvastatin in vivo we used bile duct ligated rats (BDL).

A new model of interactive effects of alcohol and high-fat diet on hepatic fibrosis

Alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) are the most frequent conditions leading to elevated liver enzymes and liver cirrhosis, respectively, in the Western world. However, despite strong epidemiological evidence for combined effects on the progression of liver injury, the mutual interaction of the pathophysiological mechanisms is incompletely understood. The aim of this study was to establish and analyze an experimental murine model, where we combined chronic alcohol administration with a NASH-inducing high-fat (HF) diet.

[Pharmacotherapy of hyperthyreosis--adverse drug reactions]

The antithyroid drugs mainly include thioimidazole (carbimazole, methimazole=thiamazole) and propylthiouracil. After absorption, carbimazole is rapidly metabolized to methimazole and thus switching between these two drugs should not be considered in case of side effects. Furthermore, in case of side effects, sometimes even cross reactions between thioimidazoles and propylthiouracil occur. Common and typical adverse reactions of antithyroid drugs include dose dependent hypothyroidism and thus thyroid function should be repeatedly checked while the patient is on antithyroid drugs. Furthermore, pruritus and rash may develop. In this case, one might try to switch from thioimidazoles to propylthiouracil or vice versa. Antithyroid drugs may cause mild dose dependent neutropenia or severe allergy-mediated agranulocytosis, which typically occurs during the first three months of treatment, has an incidence of 3 per 10,000 patients and cross reactivity between thioimidazoles to propylthiouracil may occur. Rarely, antithyroid drugs can cause aplastic anemia. Mainly propylthiouracil, but sometimes also methimazole may lead to an asymptomatic transient increase in liver enzymes or to severe, even lethal liver injury of cholestatic or hepatocellular pattern. Since propylthiouracil associated liver injury was observed increasingly among children and adolescent, it has been suggested to prefer thioimidazoles for these patients. Because of these potential serious adverse effects, physicians should advise patients to immediately seek medical help if they get a fever or sore throat or malaise, abdominal complaints or jaundice, respectively. Furthermore, arthralgias may develop in 1-5% of patients under both antithyroid drugs. Since arthralgias may be the first symptom of more serious immunologic side effects, it is recommended to stop the antithyroid drug in this case. Drug induced polyarthritis mainly develops during the first month of therapy, whereas ANCA-positive vasculitis is generally observed only after long term exposure to propylthiouracil or very rarely with the thioimidazoles. The teratogenic risk of the thioimidazoles is somewhat higher (Aplasia cutis congenita), that is why one generally recommends preferring propylthiouracil during pregnancy. During breast feeding both, thioimidazoles or propylthiouracil, may be administered. Nowadays, perchlorate is only used short term in case of latent hyperthyroidism before administering iodine-containing contrast agents. Therefore, the known side effects, which usually are only observed after long term treatment, are not an issue any more.

Mechanisms of hepatocellular toxicity associated with dronedarone--a comparison to amiodarone

Dronedarone is a new antiarrhythmic drug with an amiodarone-like benzofuran structure. Shortly after its introduction, dronedarone became implicated in causing severe liver injury. Amiodarone is a well-known mitochondrial toxicant. The aim of our study was to investigate mechanisms of hepatotoxicity of dronedarone in vitro and to compare them with amiodarone. We used isolated rat liver mitochondria, primary human hepatocytes, and the human hepatoma cell line HepG2, which were exposed acutely or up to 24h. After exposure of primary hepatocytes or HepG2 cells for 24h, dronedarone and amiodarone caused cytotoxicity and apoptosis starting at 20 and 50 µM, respectively. The cellular ATP content started to decrease at 20 µM for both drugs, suggesting mitochondrial toxicity. Inhibition of the respiratory chain required concentrations of ~10 µM and was caused by an impairment of complexes I and II for both drugs. In parallel, mitochondrial accumulation of reactive oxygen species (ROS) was observed. In isolated rat liver mitochondria, acute treatment with dronedarone decreased the mitochondrial membrane potential, inhibited complex I, and uncoupled the respiratory chain. Furthermore, in acutely treated rat liver mitochondria and in HepG2 cells exposed for 24h, dronedarone started to inhibit mitochondrial β-oxidation at 10 µM and amiodarone at 20 µM. Similar to amiodarone, dronedarone is an uncoupler and an inhibitor of the mitochondrial respiratory chain and of β-oxidation both acutely and after exposure for 24h. Inhibition of mitochondrial function leads to accumulation of ROS and fatty acids, eventually leading to apoptosis and/or necrosis of hepatocytes. Mitochondrial toxicity may be an explanation for hepatotoxicity of dronedarone in vivo.

HLA haplotype determines hapten or p-i T cell reactivity to flucloxacillin

Drug-induced liver injury (DILI) is a main cause of drug withdrawal. A particularly interesting example is flucloxacillin (FLUX)-DILI, which is associated with the HLA-B*57:01 allele. At present, the mechanism of FLUX-DILI is not understood, but the HLA association suggests a role for activated T cells in the pathomechanism of liver damage. To understand the interaction among FLUX, HLA molecules, and T cells, we generated FLUX-reacting T cells from FLUX-naive HLA-B*57:01(+) and HLA-B*57:01(-) healthy donors and investigated the mechanism of T cell stimulation. We found that FLUX stimulates CD8(+) T cells in two distinct manners. On one hand, FLUX was stably presented on various HLA molecules, resistant to extensive washing and dependent on proteasomal processing, suggesting a hapten mechanism. On the other hand, in HLA-B*57:01(+) individuals, we observed a pharmacological interaction with immune receptors (p-i)-based T cell reactivity. FLUX was presented in a labile manner that was further characterized by independence of proteasomal processing and immediate T cell clone activation upon stimulation with FLUX in solution. This p-i-based T cell stimulation was restricted to the HLA-B*57:01 allele. We conclude that the presence of HLA-B*57:01 drives CD8(+) T cell responses to the penicillin-derivative FLUX toward nonhapten mechanism.

Oral imatinib treatment reduces early fibrogenesis but does not prevent progression in the long term

BACKGROUND/AIMS: Transactivated hepatic stellate cells (HSCs) represent the key source of extra cellular matrix (ECM) in fibrotic liver. Imatinib, a potent inhibitor of the PDGF receptor tyrosine kinase, reduces HSC proliferation and fibrogenesis when treatment is initiated before fibrosis has developed. We tested the antifibrotic potential of imatinib in ongoing liver injury and in established fibrosis. METHODS: BDL-rats were gavage fed with 20 mg/kg/d imatinib either early (days 0-21) or late (days 22-35) after BDL. Untreated BDL-rats served as controls. ECM and activated HSCs were quantified by morphometry. Tissue activity of MMP-2 was determined by gelatin zymography. mRNA expression of TIMP-1 and procollagen alpha1(I) were measured by RT-PCR. Liver tissue concentration of imatinib was measured by tandem mass spectrometry. RESULTS: Early imatinib reduced ECM formation by 30% (P=0.0455) but left numbers of activated HSCs and procollagen I expression unchanged. MMP-2 activity and TIMP-1 expression were reduced by 50%. Late imatinib treatment did not alter histological or molecular markers of fibrogenesis despite high imatinib tissue levels. CONCLUSIONS: The antifibrotic effectiveness of imatinib is limited to the early phase of fibrogenesis. In ongoing liver injury other mediators most likely compensate for the inhibited PDGF effect.

Hepatitis C coinfection is independently associated with decreased adherence to antiretroviral therapy in a population-based HIV cohort

OBJECTIVE: To characterize the impact of hepatitis C (HCV) serostatus on adherence to antiretroviral treatment (ART) among HIV-infected adults initiating ART. METHODS: The British Columbia HIV/AIDS Drug Treatment Program distributes, at no cost, all ART in this Canadian province. Eligible individuals used triple combination ART as their first HIV therapy and had documented HCV serology. Statistical analyses used parametric and non-parametric methods, including multivariate logistic regression. The primary outcome was > or = 95% adherence, defined as receiving > or = 95% of prescription refills during the first year of antiretroviral therapy. RESULTS: There were 1186 patients eligible for analysis, including 606 (51%) positive for HCV antibody and 580 (49%) who were negative. In adjusted analyses, adherence was independently associated with HCV seropositivity [adjusted odds ratio (AOR), 0.48; 95% confidence interval (CI), 0.23-0.97; P = 0.003], higher plasma albumin levels (AOR, 1.07; 95% CI, 1.01-1.12; P = 0.002) and male gender (AOR, 2.53; 95% CI, 1.04-6.15; P = 0.017), but not with injection drug use (IDU), age or other markers of liver injury. There was no evidence of an interaction between HCV and liver injury in adjusted analyses; comparing different strata of HCV and IDU confirmed that HCV was associated with poor adherence independent of IDU. CONCLUSIONS: HCV-coinfected individuals and those with lower albumin are less likely to be adherent to their ART.

Natural killer T cell dysfunction in CD39-null mice protects against concanavalin A-induced hepatitis

Concanavalin A (Con A)-induced injury is an established natural killer T (NKT) cell-mediated model of inflammation that has been used in studies of immune liver disease. Extracellular nucleotides, such as adenosine triphosphate, are released by Con A-stimulated cells and bind to specific purinergic type 2 receptors to modulate immune activation responses. Levels of extracellular nucleotides are in turn closely regulated by ectonucleotidases, such as CD39/NTPDase1. Effects of extracellular nucleotides and CD39 on NKT cell activation and upon hepatic inflammation have been largely unexplored to date. Here, we show that NKT cells express both CD39 and CD73/ecto-5'-nucleotidase and can therefore generate adenosine from extracellular nucleotides, whereas natural killer cells do not express CD73. In vivo, mice null for CD39 are protected from Con A-induced liver injury and show substantively lower serum levels of interleukin-4 and interferon-gamma when compared with matched wild-type mice. Numbers of hepatic NKT cells are significantly decreased in CD39 null mice after Con A administration. Hepatic NKT cells express most P2X and P2Y receptors; exceptions include P2X3 and P2Y11. Heightened levels of apoptosis of CD39 null NKT cells in vivo and in vitro appear to be driven by unimpeded activation of the P2X7 receptor. CONCLUSION: CD39 and CD73 are novel phenotypic markers of NKT cells. Deletion of CD39 modulates nucleotide-mediated cytokine production by, and limits apoptosis of, hepatic NKT cells providing protection against Con A-induced hepatitis. This study illustrates a further role for purinergic signaling in NKT-mediated mechanisms that result in liver immune injury.

Severe hepatotoxicity following ingestion of Herbalife nutritional supplements contaminated with Bacillus subtilis

BACKGROUND/AIMS: Nutritional supplements are widely used. Recently, liver injury after consumption of Herbalife preparations was reported but the underlying pathogenesis remained cryptic. METHODS: Two patients presented with cholestatic hepatitis and pruritus, and cirrhosis, respectively. Viral, alcoholic, metabolic, autoimmune, neoplastic, vascular liver diseases and synthetic drugs as the precipitating causes of liver injury were excluded. However, both patients reported long-term consumption of Herbalife products. All Herbalife products were tested for contamination with drugs, pesticides, heavy metals, and softeners, and examined for microbial contamination according to standard laboratory procedures. Bacteria isolated from the samples were identified as Bacillus subtilis by sequencing the 16S rRNA and gyrB genes. RESULTS: Causality between consumption of Herbalife products and disease according to CIOMS was scored "probable" in both cases. Histology showed cholestatic and lobular/portal hepatitis with cirrhosis in one patient, and biliary fibrosis with ductopenia in the other. No contamination with chemicals or heavy metals was detected, and immunological testing showed no drug hypersensitivity. However, samples of Herbalife products ingested by both patients showed growth of Bacillus subtilis of which culture supernatants showed dose- and time-dependent hepatotoxicity. CONCLUSIONS: Two novel incidents of severe hepatic injury following intake of Herbalife products contaminated with Bacillus subtilis emphasize its potential hepatotoxicity.

Concomitant injuries are an important determinant of outcome of high-grade blunt hepatic trauma

BACKGROUND: Little is known about the clinical importance of concomitant injuries in polytraumatized patients with high-grade blunt liver injury. A retrospective single-centre study was performed to investigate the safety of non-operative management of liver injury and the impact of concomitant intra- and extra-abdominal injuries on clinical outcome. METHODS: Some 183 patients with blunt liver injury were admitted to Berne University Hospital, Switzerland, between January 2000 and December 2006. Grade 3-5 injuries were considered to be high grade. RESULTS: Immediate laparotomy was required by 35 patients (19.1 per cent), owing to extrahepatic intra-abdominal injury (splenic and vascular injuries, perforations) in 21 cases. The mortality rate was 16.9 per cent; 22 of the 31 deaths were due to concomitant lesions. Of 81 patients with high-grade liver injury, 63 (78 per cent) were managed without surgery; liver-related and extra-abdominal complication rates in these patients were 11 and 17 per cent respectively. Grades 4 and 5 liver injury were associated with hepatic-related and extra-abdominal complications. CONCLUSION: Concomitant injuries are a major determinant of outcome in patients with blunt hepatic injury and should be given high priority by trauma surgeons. An algorithm for the management of blunt liver injury is proposed.

Eculizumab hepatotoxicity in pediatric aHUS.

BACKGROUND Eculizumab is a humanized anti-C5 antibody approved for the treatment of atypical hemolytic uremic syndrome (aHUS). Its use is increasing in children following reports of its safety and efficacy. METHODS We reviewed biochemical and clinical data related to possible drug-induced liver injury in 11 children treated with eculizumab for aHUS in a single center. RESULTS Elevated aminotransferases were observed in 7 children aged 6 to 11 years following eculizumab treatment for aHUS. Internationally accepted liver enzyme thresholds for drug-induced liver injury were exceeded in 5 cases. In all cases, liver injury was classified as mixed hepatocellular and cholestatic. Infectious and other causes were excluded in each case. One patient with no pre-existing liver disease developed tender hepatomegaly and liver enzyme derangement exceeding 20 times the upper limit of normal following initiation of eculizumab. Recurrent liver injury following re-challenge with eculizumab necessitated its discontinuation and transition to plasma therapy. CONCLUSIONS Hepatotoxicity in association with eculizumab is a potentially important yet previously unreported adverse event. We recommend monitoring liver enzymes in all patients receiving eculizumab. Further research is required to clarify the impact of this adverse event, to characterize the mechanism of potential hepatotoxicity, and to identify which patients are most at risk.

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

Extracellular nucleotides (e.g. ATP, UTP, ADP) are released by activated endothelium, leukocytes and platelets within the injured vasculature and bind specific cell-surface type-2 purinergic (P2) receptors. This process drives vascular inflammation and thrombosis within grafted organs. Importantly, there are also vascular ectonucleotidases i.e. ectoenzymes that hydrolyze extracellular nucleotides in the blood to generate nucleosides (viz. adenosine). Endothelial cell NTPDase1/CD39 has been shown to critically modulate levels of circulating nucleotides. This process tends to limit the activation of platelet and leukocyte expressed P2 receptors and also generates adenosine to reverse inflammatory events. This vascular protective CD39 activity is rapidly inhibited by oxidative reactions, such as is observed with liver ischemia reperfusion injury. In this review, we chiefly address the impact of these signaling cascades following liver transplantation. Interestingly, the hepatic vasculature, hepatocytes and all non-parenchymal cell types express several components co-ordinating the purinergic signaling response. With hepatic and vascular dysfunction, we note heightened P2- expression and alterations in ectonucleotidase expression and function that may predispose to progression of disease. In addition to documented impacts upon the vasculature during engraftment, extracellular nucleotides also have direct influences upon liver function and bile flow (both under physiological and pathological states). We have recently shown that alterations in purinergic signaling mediated by altered CD39 expression have major impacts upon hepatic metabolism, repair mechanisms, regeneration and associated immune responses. Future clinical applications in transplantation might involve new therapeutic modalities using soluble recombinant forms of CD39, altering expression of this ectonucleotidase by drugs and/or using small molecules to inhibit deleterious P2-mediated signaling while augmenting beneficial adenosine-mediated effects within the transplanted liver.