Jane Macnaughtan

Treatment with non-selective beta blockers is associated with reduced severity of systemic inflammation and improved survival of patients with acute-on-chronic liver failure

BACKGROUND & AIMS Non-selective beta blockers (NSBBs) have been shown to have deleterious outcomes in patients with refractory ascites, alcoholic hepatitis and spontaneous bacterial peritonitis leading many physicians to stop the drug in these cases. Acute-on-chronic liver failure (ACLF) is characterized by systemic inflammation and high mortality. As NSBBs may have beneficial effects on gut motility and permeability and, systemic inflammation, the aims of this prospective, observational study were to determine whether ongoing use of NSBBs reduced 28-day mortality in ACLF patients. METHODS The study was performed in 349 patients with ACLF included in the CANONIC study, which is a prospective observational investigation in hospitalized cirrhotic patients with acute deterioration. The data about the use of NSBBs, its type and dosage was specifically recorded. Patient characteristics at enrollment significantly associated with treatment and mortality were taken into account as potential confounders to adjust for treatment effect. A logistic regression model was fitted. RESULTS 164 (47%) ACLF patients received NSBBs whereas 185 patients did not. Although the CLIF-C ACLF scores were similar at presentation, more patients in the NSBB treated group had lower grades of ACLF (p=0.047) at presentation and significantly more patients improved. Forty patients (24.4%) died in NSBB treated group compared with 63 patients (34.1%) (p=0.048) [estimated risk-reduction 0.596 (95%CI: 0.361-0.985; p=0.0436)]. This improvement in survival was associated with a significantly lower white cell count (NSBB: 8.5 (5.8); no NSBB: 10.8 (6.6); p=0.002). No long-term improvement in survival was observed. CONCLUSIONS This study shows for the first time that ongoing treatment with NSBBs in cirrhosis is safe and reduces the mortality if they develop ACLF. Careful thought should be given before stopping NSBBs in cirrhotic patients.

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

BACKGROUND & AIMS Superimposed 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. METHODS Sprague-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. RESULTS The 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. CONCLUSIONS The 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.

Increased renal expression and urinary excretion of TLR4 in acute kidney injury associated with cirrhosis

Patients with cirrhosis frequently develop renal dysfunction, a proportion of who do not fulfill criteria for hepatorenal syndrome (HRS). We hypothesized that the kidneys in these patients would exhibit histological and biomarker evidence of kidney injury. We looked specifically for TLR expression as they may mediate kidney injury.

Immunomodulatory and antioxidant function of albumin stabilises the endothelium and improves survival in a rodent model of chronic liver failure

BACKGROUND & AIMS Liver failure is characterized by endothelial dysfunction, which results in hemodynamic disturbances leading to renal failure. Albumin infusion improves hemodynamics and prevents renal dysfunction in advance liver failure. These effects are only partly explained by the oncotic properties of albumin. This study was designed to test the hypothesis that albumin exerts its beneficial effects by stabilising endothelial function. METHODS In vivo: systemic hemodynamics, renal function, markers of endothelial dysfunction (ADMA) and inflammation were studied in analbuminaemic and Sprague-Dawley rats, 6-weeks after sham/bile duct ligation surgery. In vitro: human umbilical vein endothelial cells were stimulated with LPS with or without albumin. We studied protein expression and gene expression of adhesion molecules, intracellular reactive oxygen species, and cell stress markers. RESULTS Compared to controls, analbuminaemic rats had significantly greater hemodynamic deterioration after bile duct ligation, resulting in worse renal function and shorter survival. This was associated with significantly greater plasma renin activity, worse endothelial function, and disturbed inflammatory response. In vitro studies showed that albumin was actively taken up by endothelial cells. Incubation of albumin pre-treated endothelial cells with LPS was associated with significantly less activation compared with untreated cells, decreased intracellular reactive oxygen species, and markers of cell stress. CONCLUSIONS These results show, for the first time, that absence of albumin is characterised by worse systemic hemodynamics, renal function and higher mortality in a rodent model of chronic liver failure and illustrates the important non-oncotic properties of albumin in protecting against endothelial dysfunction.

Clinical and pathophysiological consequences of alterations in the microbiome in cirrhosis.

Cirrhosis is a major cause of mortality worldwide. Exponential rises in prevalence have been observed secondary to increases in obesity and alcohol consumption. Multiple lines of evidence implicate gut-derived bacteria and bacterial ligands as a central driver of pathogenesis. Recent developments in culture-independent techniques have facilitated a more accurate description of microbiome composition in cirrhosis and led to the description of measures of dysbiosis shown to be associated with disease. More importantly, metagenomic studies are adding to an understanding of the functional contribution of the microbiota and may prove to be a more clinically relevant biomarker than phylogenetic studies. Much like other dysbiotic states such as inflammatory bowel disease, the microbiota in cirrhosis is characterized by a low microbial and genetic diversity. Therapeutic strategies to diminish this process are currently limited to selective intestinal decontamination with antibiotics. This review summarizes the available data and develops a framework for the use of current and future treatment strategies to diminish the consequences of dysbiosis in cirrhosis. Interventional strategies to bind bacterial products in the gut lumen and blood, and modulate the magnitude of host sensing mechanisms remain an unmet clinical need. A greater understanding of the host–microbiota interaction in cirrhosis is of key importance to inform future interventional strategies to diminish the currently escalating burden of the disease.

Corrigendum: Lipopolysaccharide-Induced Neutrophil Dysfunction Following Transjugular Intrahepatic Portosystemic Stent Shunt (TIPSS) Insertion is Associated with Organ Failure and Mortality

“Rajiv Jalan and Schalk van der Merwe were responsible for project design. Rajiv Jalan was responsible for data collection. Data analysis, interpretation and writing of the article was performed by Jane Macnaughtan and Rajiv Jalan. Jane Macnaughtan, Raj Mookerjee, Schalk van der Merwe and Rajiv Jalan contributed to critical revision of the article. Jane Macnaughtan and Rajiv Jalan were responsible for producing the final version of the manuscript for publication”.

PTH-095 Oral carbon therapy is associated with a selective modulation of the microbiome in cirrhotic rats which is associated with a significant reduction in inflammatory activation

Introduction The host-microbiome interaction is pathological in cirrhosis promoting a dysregulated inflammatory response resulting in organ injury and diminished survival. Yaq-001 is an oral non-absorpable carbon shown to result in improvement in hepatic haemodynamics and markers of liver injury but the mechanism of how this is achieved in unknown. The aims of this study were to determine whether the beneficial effects of Yaq-001 was related to the composition and associated functional state of the microbiome in bile-duct ligated cirrhotic animals. Method BDL (n = 12) or sham (n = 12) rats were randomised to treatment with Yaq-001 (Yaqrit Ltd. UK) from weeks 2–4. Analysis of urine samples was performed using 1NMRs. Bacterial DNA from stool was extracted, amplified and sequenced. Arterial plasma was co-incubated with HEK-Blue hTLR4 and IL-1β/IL-18 reporter cell lines. Constitutive ROS and LPS-induced ROS production from circulating monocyte populations was determined using flow cytometry. Results BDL was associated with a significant reduction in Peptidostreptococcaceae and Clostridium XI (p < 0.05) in stool compared to sham controls. Carbon therapy was associated with significant increases in firmicutes, in particular clostridia populations in stool with significant reductions in bacteroides populations (p < 0.05). Functionally, BDL rats were found to have significantly higher urinary bile acids, Trimethylamine N-oxide, benzoate, glycine, acetate and lactate than sham controls (p < 0.05). Significantly lower levels of citrate, dimethylamine (DMA) and creatinine were observed in BDL compared to sham animals (p < 0.05). Carbon treatment results in a significant increase in urinary creatinineand bile acids and reduction in urinary glycine (p < 0.05). A significant increase in IL18/IL1B expression was observed in untreated BDL rats compared to sham which was significantly attenuated with carbon treatment (p < 0.05). Monocyte LPS-induced ROS production was significantly higher in BDL rats but attenuated with carbon treatment (p < 0.05). Conclusion The results of this study show that Yaq-001, which is a non-specific adsorbent has substantial effects on the composition and function of the microbiome in cirrhotic rats and positively modulates the function of monocytes regarding ROS production and inflammasome activation. These data provide compelling evidence that the gut bacterial products are important in mediating immune dysfunction in cirrhosis and is a target of therapy. Disclosure of interest None Declared.

PMO-127 Biological effects of oral nanoporous carbon in bile duct ligated rats

Introduction Gut-derived bacterial products and associated dysregulated inflammatory response play a central role in the pathogenesis of cirrhosis. Therapeutic options to target these factors are currently limited to long-term antibiotics. Nanoporous carbons are non-absorbable, synthetic materials which are safe with porosity manipulated for adsorption of middle and high molecular weight molecules and surface chemistry modified to alter adsorption capacity for biological molecules such as cytokines and endotoxin. We sought to determine their biological effects in bile-duct ligated rats as a model of cirrhosis. Methods 131 male Sprague-Dawley rats underwent bile duct-ligation or sham biliary surgery. Animals were pair fed with or without oral carbon therapy 2 weeks from surgery until completion of the experiment at 4–5 weeks. Intra-peritoneal lipopolysaccharide (LPS) was administered to four subgroups 3.5 h prior to completion of the study. The following groups were studied: Sham (n=16), Sham + carbon (n=15), Sham + LPS (n=11), Sham+LPS+carbon (n=10), BDL (n=27), BDL + carbon (n=26), BDL+LPS (n=10), BDL+LPS+carbon (n=16). Portal haemodynamics were performed on 93 rats and Kupffer cell (KC) numbers and Reactive oxygen species (ROS) production assessed by flow cytometry in a sub-group of animals. Liver biochemistry and portal venous cytokines were performed. Results A significant reduction in portal pressure was observed in BDL+LPS (mean 18.05±0.88 mm Hg untreated, 10.17±1.07 mm Hg with carbon, p<0.05) and BDL (mean 12.57±0.43 mm Hg untreated, 11.02±0.28 mm Hg with carbon, p<0.05) groups following carbon treatment. A significant reduction in ALT was observed in the carbon treated BDL+LPS (p<0.05) and BDL groups (p<0.05). Carbon treatment in BDL rats was associated with a significant reduction in LPS-induced ROS production. A trend towards reduction in portal venous IL-4 and IL-10 was observed in carbon-treated BDL rats. No significant difference in portal venous TNF-α was observed. Finally, a significant increase in body mass was observed in the BDL carbon-treated group (p<0.05). Conclusion Oral nanoporous carbon therapy results in a significant reduction in portal pressure and liver biochemistry associated with a reduction in endotoxin-induced KC ROS production. We postulate therefore, that the effect of nanoporous carbon is possibly via a reduction in endotoxin induced KC stimulation. Competing interests None declared.

EFFECTS OF ORAL NANOPOROUS CARBON THERAPY IN LEPTIN NULL MICE AS A MODEL OF NON-ALCOHOLIC STEATOHEPATITIS

Introduction Endotoxaemia is implicated in the pathogenesis of non-alcoholic fatty liver disease. Modulation of intra-luminal factors driving bacterial translocation may have the capacity to impact on the natural history of the disease. Nanoporous carbons are non-absorbable, synthetic materials which are safe with porosity manipulated for adsorption of middle and high molecular weight molecules and surface chemistry modified to alter adsorption capacity for biological molecules. We sought to determine their biological effects in leptin null mice, which are hyperphagic and obese with evident steatohepatitis and to ascertain whether nanoporous carbons can reverse established non-alcoholic steatohepatitis (NASH) in these animals. Methods 10 lep−/lep− null and 10 heterozygote male mice were randomised to receive powdered chow ± carbon (0.4 g/100 g body weight/day) for 4 weeks. Extent of liver injury was assessed by serum levels of ALT. Additionally, non-parenchymal cells were isolated and the Kupffer cell (KC) population characterised by flow cytometry as those cells expressing F4/80, CD68 and CD11b. Reactive oxygen species (ROS) production by isolated KCs was also assayed. Hepatic TLR-4 expression as a surrogate of endotoxaemia was determined by immunohistochemistry. Results In lep−/lep− mice, oral carbon treatment was associated with a significant reduction in ALT 889±280 IU/ml to 408±42 IU/ml (p<0.05). Total KC population was found to be increased in lep−/lep− mice compared to heterozygote control with a significant reduction observed with carbon treatment (p<0.05). A significant reduction in KCs ROS production was also observed in carbon treated lep−/lep− mice (p<0.05) compared to untreated lep−/lep− controls. A significant reduction in the F4/80+, CD68−, CD11b+ cell sub-population in lep−/lep− in the presence of carbon treatment group was also observed (p<0.05). Moreover, hepatic TLR-4 expression was reduced in carbon-treated lep−/lep− mice compared to non-treated controls. Finally, we observed a trend towards reduction in final body weight in carbon-treated lep−/lep− mice compared to untreated controls group (p=0.095). Conclusion Oral nanoporous carbon through modulating endotoxaemia and KC function may be a promising therapy in NASH. Competing interests None declared.

680 URINARY TLR4: A NOVEL BIOMARKER TO IDENTIFY PATIENTS WITH ACUTE KIDNEY INJURY IN PATIENTS WITH ACUTE ON CHRONIC LIVER FAILURE

Introduction Patients with stable cirrhosis often present with acute deterioration of cirrhosis secondary to precipitating illness which may progress to organ failure, a condition referred to as acute on chronic Liver failure (ACLF). A proportion of these patients develop renal dysfunction which do not fulfil criteria for the diagnosis of hepatorenal syndrome (HRS). We hypothesised that the kidneys in patients who develop renal dysfunction in ACLF would exhibit histological and biomarker evidence of acute kidney injury (AKI). Since ACLF is associated with systemic inflammatory response (SIRS) we aimed to look for Toll like receptor (TLR) 4 and 2 which recognise pathogens and when activated lead to apoptosis and production of cytokines. Methods Study 1 : 74 subjects [healthy volunteers (6), compensated alcoholic cirrhosis (11), acute deterioration of alcoholic cirrhosis (57)] were included prospectively. Urinary biomarkers, kidney injury molecule-1 (KIM-1, a marker of renal injury), Glutathione S-Transferase (πGST, αGST; markers of proximal and distal tubular injury) (Commercial ELISA), and urinary TLR4 (Western Blotting) were measured. Study 2 : Renal biopsies were available from 8 alcoholic cirrhosis patients (3 AKI; 5 HRS) which were stained for TLR4, TLR2 and, Caspase-3. Results Study 1 : Nine patients developed AKI on the background of acute deterioration of cirrhosis and 3 had HRS. KIM-1, πGST and αGST were higher in patients with acute deterioration of cirrhosis compared with controls but did not differ in those with and without AKI. Urinary TLR4 values were significantly higher in patients with acute deterioration of cirrhosis with AKI (4.7±1.1) compared to controls (0.38±0.04) and stable cirrhosis (0.32±0.08) and patients with acute deterioration of cirrhosis without renal dysfunction (1.6±0.32) (p Conclusion These data provide evidence for severe tubular injury and apoptosis in patients with AKI and identifies urinary TLR4, as a novel biomarker to identify AKI in Acute deterioration of cirrhosis. Competing interests None declared.