D. Dhar

Prevention of acute kidney injury in a rodent model of cirrhosis following selective gut decontamination is associated with reduced renal TLR4 expression

BACKGROUND & AIMSnSuperimposed infection and/or inflammation precipitate renal failure in cirrhosis. This study aimed at testing the hypothesis that increased gut bacterial translocation in cirrhosis primes the kidney to the effect of superimposed inflammation by upregulating expression of Toll-like receptor 4 (TLR4), NFκB, and cytokines. A well-characterized bile-duct ligated (BDL) model of cirrhosis, which develops renal failure following superimposed inflammatory insult with lipopolysaccharide (LPS), was used and selective gut decontamination was performed using norfloxacin.nnnMETHODSnSprague-Dawley rats were studied: Sham, Sham+LPS; BDL, BDL+LPS; an additional BDL and BDL+LPS groups were selectively decontaminated with norfloxacin. Plasma biochemistry, plasma renin activity (PRA) and cytokines and, protein expression of TLR4, NFκB, and cytokines were measured in the kidney homogenate. The kidneys were stained for TLR4, TLR2, and caspase-3. Endotoxemia was measured using neutrophil burst and Limulus amoebocyte lysate (LAL) assays.nnnRESULTSnThe groups treated with norfloxacin showed significant attenuation of the increase in plasma creatinine, plasma and renal TNF-α and renal tubular injury on histology. The increased renal protein expression of TLR4, NFκB, and caspase-3 in the untreated animals was significantly attenuated in the norfloxacin treated animals. PRA was reduced in the treated animals and severity of endotoxemia was also reduced.nnnCONCLUSIONSnThe results show for the first time that kidneys in cirrhosis show an increased expression of TLR4, NFκB, and the pro-inflammatory cytokine TNF-α, which makes them susceptible to a further inflammatory insult. This increased susceptibility to LPS can be prevented with selective decontamination, providing novel insights into the pathophysiology of renal failure in cirrhosis.

Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure

BACKGROUND & AIMSnIn cirrhosis, superimposed inflammation often culminates in acute-on-chronic liver failure (ACLF) but the mechanism underlying this increased sensitivity is not clear. Cx43 is a ubiquitous gap junction protein that allows transmission of signals between cells at a much higher rate than the constitutively expressed gap junctions. The aims of the study were to test the hypothesis that inflammation drives the increased expression of hepatic Cx43 and to determine its role by Cx43 inhibition.nnnMETHODSnFour weeks after bile-duct ligation (BDL) or sham operation, rats were treated with an anti-TNF antibody, or saline; with or without LPS (1mg/kg); given 3h prior to termination. Biochemistry and cytokines were measured in the plasma and hepatic protein expression (NFkB, TNFα, iNOS, 4HNE, Cx26, 32, and 43) and confocal microscopy (Cx26, 32, and 43) were performed. The effect of a Cx43-specific inhibitory peptide was studied in a mouse BDL model.nnnRESULTSnBDL animals administered LPS developed typical features of ACLF but animals administered infliximab were relatively protected. Cx26/32 expression was significantly decreased in BDL animals while Cx43 was significantly increased and increased further following LPS. Infliximab treatment prevented this increase. However, inhibiting Cx43 in BDL mice produced detrimental effects with markedly greater hepatocellular necrosis.nnnCONCLUSIONSnThe results of this study show for the first time an increased expression of hepatic Cx43 in cirrhosis and ACLF, which was related to the severity of inflammation. This increased Cx43 expression is likely to be an adaptive protective response of the liver to allow better cell-to-cell communication.

Effect of toll‐like receptor 7 and 9 targeted therapy to prevent the development of hepatocellular carcinoma

Chronic liver disease is a predisposing factor for development of hepatocellular carcinoma (HCC). Toll‐like receptors play a crucial role in immunity against microbial pathogens and recent evidence suggests that they may also be important in pathogenesis of chronic liver disease. The purpose of this study was to determine whether TLR7 and TLR9 are potential targets for prevention and progression of HCC.

P35 Treatment with an Alpha 2a Adrenoreceptor Antagonist Modulates Hepatic Inflammation, Markedly Reduces Portal Pressure, and Improves Arterial Pressure and Hepatic Blood Flow in Cirrhotic Rats

Introduction Inflammation plays a pivotal role in modulating the severity of intrahepatic resistance in cirrhosis. Our studies have shown a close relationship between the activation of the sympathetic nervous system, inflammatory response and severity of portal hypertension. Stimulation of alpha 2a adrenergic (ADRA2a) receptors results in inflammation and vasodilation in resistance vasculature, and its antagonism has shown benefit in models of sepsis. Aim The aim of the study was to test the hypothesis that treating bile duct ligated rats (BDL) with an ADRA2a antagonist reduces hepatic inflammation and improves the haemodynamic abnormalities associated with cirrhosis. Method Male Sprague-Dawley rats (N=46) were studied 4-weeks after BDL surgery (N=29) or sham operation (N=17) and randomised to two doses of placebo or ADRA2a antagonist (BRL 44408, Sigma, UK, 10mg/kg s.c 24 hours prior to study). Portal vein and hepatic arterial blood flow, mean arterial (MAP) and portal pressure were measured directly. Plasma biochemistry was measured by colorimetry. ADRA2a and NFkB protein expression were determined by western blotting and immunohistochemistry (ADRA2a). Results BDL rats had significantly increased hepatic protein expression of ADRA2a compared with sham operated rats and this was mostly shown to be located on hepatocytes by immunohistochemistry. Following treatment with ADRA2a antagonist there was a significant increase in the MAP (p<0.05) and a significant reduction in portal pressure as compared to the placebo treated group (11.4±3.4 vs. 18.0±3.7 mmHg, p<0.001). The hepatic arterial blood flow was markedly increased in the treated group without significant change in the portal venous blood flow resulting in a significant reduction in intrahepatic resistance post treatment (1.1±0.2 vs. 0.5±0.1 mmHg/ml/min, p<0.05). Biochemical analysis showed a significant reduction in plasma lactate (p<0.05), AST (p<0.05) and a trend towards reduction in creatinine in treated animals. Hepatic phosphorylated NFκB expression was increased in BDL animals and this reduced significantly with ADRA2a antagonist treatment (p<0.05). Conclusion The results of this study show for the first time that modulating ADRA2a-mediated sympathetic tone and hepatic inflammation with an ADRA2a antagonist significantly improves systemic haemodynamics and reduces portal pressure, whilst also increasing hepatic blood flow. Our data provide the rationale for evaluating an ADRA2a antagonist in the treatment of portal hypertension.

P59 TLR4 antagonist in acute liver failure: a novel therapeutic strategy

Introduction Acetaminophen (APAP) toxicity is the commonest cause of acute liver failure (ALF) which is characterised by multiorgan failure manifested by rapid acute liver injury, severe brain oedema and renal failure. Without liver transplantation, about 40% patients die and its treatment is an unmet medical need. Unregulated inflammatory response plays an important role in its pathogenesis. A multitude of cytokines are released during ALF with consequent activation of NfKB and progressive liver injury. We hypothesised that Toll-like receptor-4 may be critical in the progression to multiorgan failure. Aim The aim of this study was to determine whether administration a novel TLR4 antagonist (STM28) to an APAP model of ALF in mice would prevent liver injury and its deleterious effect on the brain and the kidneys. (STM28 was kindly gifted by Professor Ken-ichi Tanamoto, Division of Microbilogy, National Institute of Health Sciences, Tokyo 158-8501, Japan). Method Three groups of Cd1 male mice were studied. Sham, APAP (acetaminophen, 500u2005mg/kg single dose IP after over night fasting), APAP+TLR4 antagonist (STM28; 20u2005ug IP, 1u2005h prior to the administration of APAP and 6u2005h later). All the mice were hydrated, administered 10% dextrose and kept in temperature controlled environment. Blood was collected for biochemistry and cytokine assay. Liver, kidney and brain were collected for western blotting and cytokine analyses and the brain frontal cortex for brain water. Animals were sacrificed at 12u2005h after APAP administration. Results Administration of APAP led to an increase in the level of liver enzymes, ALT (p=0.004) and AST (p=0.007) in comparison to the Shams, which was significantly reduced (ALT (p=0.01) and AST (p=0.008)) in the TLR4 antagonist group. ALF associated increase in ammonia (p=0.001) and brain water (p=0.04) were significantly reduced (ammonia (p=0.03); brain water (p=0.05)). ALF associated renal failure (p=0.004) was also markedly attenuated (p<0.09). Protein expression of NFkBp65 on western blot showed an increased expression in brain and kidney (p=0.03) and (p=0.02) respectively which was reduced to values that were not significantly different to Sham levels (brain (p=0.04) and kidney (p=0.02)). The expression of NFkBp65 protein expression on western blot in liver was down-regulated in ALF compared with sham (p=0.01) animals and restored in TLR4 antagonist group Sham values (p=0.02). Conclusion The results of this study show for the first time a role for TLR4 in pathogenesis of multiorgan failure in ALF. The data strongly support a potential therapeutic role for TLR4 antagonist in the prevention of progression of ALF.

91 TOLL LIKE RECEPTOR 4 ANTAGONIST PREVENTS ACETAMINOPHEN INDUCED ACUTE LIVER FAILURE IN MICE: A NOVEL THERAPEUTIC STRATEGY

Introduction Without transplantation, about 40% of patients with acute liver failure (ALF) die. Its treatment is an unmet need. Unregulated inflammation plays an important role in the pathogenesis. Our hypothesis is that Toll like receptor 4 (TLR 4) is critical in the progression of inflammation in ALF. The aims of the study were to determine whether (1) administration of a novel TLR4 antagonist to an acetaminophen (APAP) model of ALF in mice would prevent liver injury. (2) TLR4 antagonist in ALF prolongs survival. (3) TLR4 KO are protected from the liver injury induced by APAP. Methods Study 1 : 3 groups of CD1 mice were studied (n=6 in each group) Naive, APAP, 500u2005mg/kg single dose IP after overnight fasting). APAP+TLR4 antagonist, STM28 (Osaka, Japan); 20u2005μg IP, 1u2005h prior to administration of APAP and 6u2005h late). Study 2 : 3 groups of C57BL/6 mice were studied (n=6) in each group. Naive, APAP (500u2005mg/kg single dose IP), APAP+TLR4 antagonist; IAXO (Innaxon) 3u2005mg/kg IP, 1u2005h prior to administration of APAP and 6u2005h later. Study 3: C57BL/6 TLR4 KO were administered APAP 500u2005mg/kg and the respective naive controls were administered 500u2005mg/kg of saline. Biochemistry was measured using COBAS integra, Cytokines in plasma and tissue homogenate of liver, kidney and brain were measured using ELISA bead array. Brain water was measured using the dry-wet weight method. Results Both the TLR4 antagonist9s (STM28 and IAXO compound) reduced the plasma liver enzymes, ammonia and creatinine to the control level. The increase in the plasma TNF-α induced by APAP (45±3.2) was attenuated following TLR4 antagonist (20±2.3) (p Conclusion These data provides evidence for an important of TLR4 in APAP induced ALF and provide the rationale for a clinical trial of this strategy in ALF. Competing interests None declared.

294 TLR 9 INHIBITION: A NOVEL TARGET OF THERAPY FOR PRIMARY LIVER CANCER

Introduction Toll like receptor 9 (TLR9) is a member of the nucleotide-sensing endosomal TLR family which is critical to the innate immune defense against invading pathogens. TLR9 is activated by unmethylated CpG which is highly specific for bacterial DNA. Upon activation, TLR9 traffics from the endoplasmic reticulum (ER) to endosomes TLR9 signalling is inhibited by the aminoquinolone drug chloroquine. Aims (1) assess changes in TLR9 subcellular distribution. (2) Detect any changes in the endolysosomal system. (3) Determine the effects on cell proliferation in hepatocellular carcinoma (HCC) and cholangio carcinoma cell (CC) lines upon stimulation and inhibition of TLR9 signalling in each case. Methods Huh7D and HUCCT cells were treated with unmethylated CpG (ODN 2006) to stimulate, or chloroquine and Dynavax; IRS compound to inhibit TLR9 signalling. Cells were also treated with the TLR9 antagonist iODN. Cell growth was assessed and confocal immunofluorescence microscopy was used to determine TLR9 subcellular localisation using EEA1 and LAMP1, markers of the endolysosomal system. Results Confocal microscopy indicated a marked nuclear translocation of TLR9 in HUCCT and Huh7D when stimulated with CpG, while unstimulated controls showed cytoplasmic TLR9 localisation. TLR9 inhibition by iODN and chloroquine resulted in decreased cytoplasmic TLR9 meanwhile Dynavax treatment caused translocation of TLR9 to the perinuclear membranes. Dramatic changes were also observed in the distribution of LAMP1 and EEA1, which were found to be localise to juxtanuclear punctae on TLR9 stimulation. While following inhibition they translocated to perinuclear membranes. Huh7D cell counts the CpG treated cells, iODN, chloroquine and Dynavax compound were 4.5×10 5 2.1×10 5 , 1.5×10 5 and 1.7×10 5 per ml respectively, compared with the untreated cells 3×10 5 per ml which indicate a significant increase in proliferation with increased TLR9 stimulation and a significant decrease with TLR9 inhibition (p CpG treated cells, iODN, chloroquine and Dynavax were respectively 3.3×10 5 , 1.8×10 5 , 1.4×10 5 and 1.5×10 5 per ml compared with the untreated cells at 1.7×10 5 per ml. Conclusion Our study indicates that TLR9 activation increases cell proliferation whereas inhibition reduces it. Our data suggest that TLR9 may be associated with tumour proliferation and may provide a potential target for therapy in liver tumours. Competing interests None declared.

293 SELECTIVE GUT DECOMTAMINATION REDUCES HEPATIC EXPRESSION OF TOLL-LIKE RECEPTOR (TLR) 4 AND DEVELOPMENT OF CIRRHOSIS BUT DOES NOT PREVENT DEVELOPMENT OF HEPATOCELLULAR CARCINOMA (HCC)

Introduction Recent studies suggest that TLR4 inhibition may prevent fibrosis in murine models. Chronic antigenic stimulation due to increased bacterial gut translocation leads to upregulation of hepatic TLR4 and this may lead to fibrosis, cirrhosis and HCC. The aims of this study were to determine whether gut decontamination with Norfloxacin prevents cirrhosis and HCC in rodent model of cirrhosis and HCC. Methods 18 Fisher rats divided into three groups; 1st treated with DEN and NMOR (carcinogens). 2nd was treated with the same carcinogens + Norfloxacin from the beginning to 14u2005weeks (end of experiment). 3rd group was control. H&E, reticulin and Immunohistochemistry were done also liver function and TNF-α were measured. Results All the rats in 1st and 2nd groups developed HCC, the severity of which was not different between groups. With reticulin stain there was significantly reduced fibrosis in the group treated with Norfloxacin (score: 1 (0–2)) compared with the untreated group (score: 4 (3–5)) (p ** ). No difference inTLR4 expression was observed in the HCC nodules in both groups. There was a reduction of ALT (p Conclusion The results of this study suggest that selective decontamination of the gut may be a novel strategy to prevent cirrhosis probably by inhibiting hepatic TLR4 expression. In this model of cirrhosis and HCC, reduction of hepatic TLR4 does not prevent development of HCC suggesting that the mechanisms of its development are unrelated to severity of fibrosis and TLR4 related mechanisms. Competing interests None declared.

MODULATION OF TOLL-LIKE RECEPTOR GENES WITH SELECTIVE GUT DECONTAMINATION IN CIRRHOTIC ANIMALS PREVENTS THE DEVELOPMENT OF ACUTE-ON CHRONIC LIVER FAILURE (ACLF)

Introduction Acute deterioration in the liver function following precipitating illness in patients with cirrhosis often lead to multiple organ failure. Inflammation is thought to be a major contributing factor. We hypothesised that toll like receptor9s (TLRs) play a pivotal role in lipopolysaccharide (LPS) mediated cytokine surge in patients with cirrhosis culminating in acute-on-chronic liver failure (ACLF). Selective gut decontamination with Norfloxacin would dampen this inflammatory cascade. Methods Global hepatic gene expression was studied in a rodent model of cirrhosis induced by bile duct ligation (BDL) (Agilent microarray) and compared the results to sham animals. TLR related genes were among the differentially expressed genes in the liver. We validated gene expression in the Liver and Kidney in a second set of experiment in Sham, BDL, BDL+LPS, BDL+Norfloxacin, BDL+Norfloxacin+LPS groups using the quantitative real-time PCR. Cytokines (Becton Dickenson) and biochemistry were measured (COBAS Integra 400). Results Among the several genes with high expression in the BDL group as detected by the microarray, both TLR 4 and 2 had significantly higher expression in liver and Kidney compared with the sham group. We also found a marked upregulation of the C-C motif ligand 2 (CCl2) and C-X-C motif ligand 2(CxCl2) Furthermore, LPS administration in BDL animals accentuated not only the TLRs (4 and 2) expression in both kidney and liver but also the Cxcl2 and CCl2 in both these organs associated with deterioration organ function as suggested with a rise in the liver enzyme, creatinine and a rise in plasma and tissue cytokines. Norfloxacin pre-treatment in BDL group (BDL+Norfloxacin) attenuated the TLR4 and TLR2 expression in both liver and kidney. Selective decontamination with Norfloxacin in BDL +LPS animals limited the upregulation of TLR4 and TLR2 in the Liver and Kidney. It also prevented the upregulation of the CxcL2 and CCl2 in both these organs. This was associated with significant improvement in liver enzymes, creatinine and cytokines. Conclusion This study shows that the liver and kidneys in cirrhotic animals are primed by upregulation of TLR9s and its downstream inflammatory mediators in the Liver and Kidney which make them exquisitely sensitive to the effects of superimposed infection/inflammation leading to organ failure. Selective decontamination with Norfloxacin prevents the progression of ACLF by reducing gene expression of TLR 2 and 4 and its associated inflammatory adaptors. Competing interests None declared.

PMO-092 TLR7 expression is increased in hepatocellular cancer (HCC) and its modulation is associated with alterations in tumour growth: a novel therapeutic target

Introduction We have previously described upregulation of TLR9, which is mainly located in the endosomes, in human HCC and cell lines. Their inhibition or stimulation was associated with alteration in tumour growth. As TLR7 is also expressed on the endosomes we hypothesised that its expression may also be altered in HCC. The aim of the study was to determine whether TLR7 is expressed in human HCC and whether its modulation alters tumour growth. Methods Study 1. Human tissue array platforms which included 102 cores of liver tissue (including 9 normal livers, 26 Hepatitis B and C, 25 HBV and HCV cirrhosis and 42 HCC) and liver tissue obtained from a DEN/NMORE model of HCC were stained for TLR7. The scoring was performed in a blinded fashion by two individual pathologists TLR7 was scored 2 when found in ≥1/3 of hepatocyte nucleus and 1 in <1/3. Study 2. Human HCC cell lines (HepG2 and Huh7) were tested for the localisation of TLR7 receptors using immunoflurocense antibody, confocal microscopy and response to stimulation was tested in the presence of a specific TLR7 agonist (Imiqumoid, Invivogen) and promega proliferation assay technique. Results Study 1. TLR7 was expressed in the nucleus of hepatocytes in 34/42 HCCs with intense staining in 24; four out 25 positive in cirrhosis only one showed intense staining. In HBV and HCV, 5/26 were positive and 0/9 in normal [p<0.001]. In rat tissues, TLR7 was found in all HCC tumour cells only while the background either normal, dysplastic or cirrhotic was negative. Study 2. Using confocal microscopy, TLR7 was found in the cytoplasm and the nucleus of both HepG2 and Huh7 and with stimulation of TLR7 agonist the cellular proliferation significantly increased compared to control p<0.05. Conclusion The data show that TLR7 is highly expressed in human HCCs, animal model of HCC and in cell lines. Importantly, the background cirrhotic liver does not express TLR7. Their stimulation is associated with marked increase in proliferation. These data suggest that TLR7 may be a future target of therapy in HCC. Competing interests None declared.