Recombinant Alkaline Phosphatase Prevents Acute on Chronic Liver Failure

The lipopolysaccharide (LPS)– toll-like receptor-4 (TLR4) pathway plays an important role in liver failure. Recombinant alkaline phosphatase (recAP) deactivates LPS. The aim of this study was to determine whether recAP prevents the progression of acute and acute-on-chronic liver failure (ACLF). Eight groups of rats were studied 4-weeks after sham surgery or bile duct ligation and were injected with saline or LPS to mimic ACLF. Acute liver failure was induced with Galactosamine-LPS and in both models animals were treated with recAP prior to LPS administration. In the ACLF model, the severity of liver dysfunction and brain edema was attenuated by recAP, associated with reduction in cytokines, chemokines, liver cell death, and brain water. The activity of LPS was reduced by recAP. The treatment was not effective in acute liver failure. Hepatic TLR4 expression was reduced by recAP in ACLF but not acute liver failure. Increased sensitivity to endotoxins in cirrhosis is associated with upregulation of hepatic TLR4, which explains susceptibility to development of ACLF whereas acute liver failure is likely due to direct hepatoxicity. RecAP prevents multiple organ injury by reducing receptor expression and is a potential novel treatment option for prevention of ACLF but not acute liver failure

Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure

Background and aims: Toll-like receptor 4 (TLR4) plays an essential role in mediating organ injury in acute liver failure (ALF) and acute-on-chronic liver failure (ACLF). Here we assess whether inhibiting TLR4 signaling can ameliorate liver failure and serve as a potential treatment. / Material and Methods: Circulating TLR4 ligands and hepatic TLR4 expression was measured in plasma samples and liver biopsies from patients with cirrhosis. TAK-242 (TLR4 inhibitor) was tested in vivo with 10mg/Kg, i.p. in rodent models of ACLF (bile duct ligation + lipopolysaccharide (LPS); carbontetrachloride + LPS) and ALF (Galactosamine + LPS) and in vitro on immortalized human monocytes (THP-1) and hepatocytes (HHL5).. The in vivo therapeutic effect was assessed by coma free survival, organ injury and cytokine release and in vitro by measuring IL6, IL1b or cell injury (TUNEL), respectively. / Results: In patients with cirrhosis, hepatic TLR4 expression was upregulated and circulating TLR4 ligands were increased (p<0.001). ACLF in rodents was associated with a switch from apoptotic cell death in ALF to non-apoptotic forms of cell death. TAK-242 reduced LPS induced cytokine secretion and cell death (p=0.002) in hepatocytes and monocytes in vitro. In rodent models of ACLF, TAK-242 administration improved coma free survival, reduced the degree of hepatocyte cell death in liver p<0.001) and kidneys (p=0.048) and reduced circulating cytokine levels (IL1b p<0.001). In a rodent model of ALF TAK-242 prevented organ injury (p<0.001) and systemic inflammation (IL1b p<0.001). / Conclusion: This study shows that TLR4 signaling is a key factor in the development of both ACLF and ALF and its inhibition improves severity of organ injury and outcome. TAK-242 may be of therapeutic relevance in patients with liver failure

The role of RIPK1 mediated cell death in acute on chronic liver failure

Acute-on-chronic liver failure (ACLF) is characterized predominantly by non-apoptotic forms of hepatocyte cell death. Necroptosis is a form of programmed lytic cell death in which receptor interacting protein kinase (RIPK) 1, RIPK3 and phosphorylated mixed lineage kinase domain-like (pMLKL) are key components. This study was performed to determine the role of RIPK1 mediated cell death in ACLF. RIPK3 plasma levels and hepatic expression of RIPK1, RIPK3, and pMLKL were measured in healthy volunteers, stable patients with cirrhosis, and in hospitalized cirrhotic patients with acutely decompensated cirrhosis, with and without ACLF (AD). The role of necroptosis in ACLF was studied in two animal models of ACLF using inhibitors of RIPK1, necrostatin-1 (NEC-1) and SML2100 (RIPA56). Plasma RIPK3 levels predicted the risk of 28- and 90-day mortality (AUROC, 0.653 (95%CI 0.530–0.776), 0.696 (95%CI 0.593–0.799)] and also the progression of patients from no ACLF to ACLF [0.744 (95%CI 0.593–0.895)] and the results were validated in a 2nd patient cohort. This pattern was replicated in a rodent model of ACLF that was induced by administration of lipopolysaccharide (LPS) to bile-duct ligated rats and carbon tetrachloride-induced fibrosis mice administered galactosamine (CCL4/GalN). Suppression of caspase-8 activity in ACLF rodent model was observed suggesting a switch from caspase-dependent cell death to necroptosis. NEC-1 treatment prior to administration of LPS significantly reduced the severity of ACLF manifested by reduced liver, kidney, and brain injury mirrored by reduced hepatic and renal cell death. Similar hepato-protective effects were observed with RIPA56 in a murine model of ACLF induced by CCL4/GalN. These data demonstrate for the first time the importance of RIPK1 mediated cell death in human and rodent ACLF. Inhibition of RIPK1 is a potential novel therapeutic approach to prevent progression of susceptible patients from no ACLF to ACLF

Impaired brain glymphatic flow in experimental hepatic encephalopathy

BACKGROUND AND AIMS: Neuronal function is exquisitely sensitive to alterations in extracellular environment. In patients with hepatic encephalopathy (HE), accumulation of metabolic waste products and noxious substances in the interstitial fluid of the brain is thought to be a consequence of the liver disease and may contribute to neuronal dysfunction and cognitive impairment. This study was designed to test the hypothesis that the accumulation of these substances, such as bile acids, may result from reduced clearance from the brain. METHODS: In a rat model of chronic liver disease with minimal HE (the bile duct ligation (BDL) model), we used emerging dynamic contrast-enhanced MRI and mass-spectroscopy techniques to assess the efficacy of the glymphatic system, which facilitates clearance of solutes from the brain. Immunofluorescence of aquaporin-4 (AQP4) and behavioural experiments were also performed. RESULTS: We identified discrete brain regions (olfactory bulb, prefrontal cortex and hippocampus) of altered glymphatic clearance in BDL rats, which aligned with cognitive/behavioural deficits. A significantly lower level of AQP4 expression was observed compared to the vasculature marker in both the olfactory bulb and prefrontal cortex in HE, which could be a contributing factor to the pathophysiological mechanisms underlying the impairment in glymphatic function observed in BDL rats. CONCLUSIONS: This study provides the first experimental evidence of impaired glymphatic flow in HE, potentially mediated by decreased AQP4 expression in the affected regions. LAY SUMMARY: The 'glymphatic system' is a newly discovered brain-wide pathway that facilitates clearance of various substances that accumulate in the brain due to its activity. This study evaluated whether the function of this system is altered in a model of brain dysfunction that occurs in cirrhosis. For the first time, we identified that the clearance of substances from the brain in cirrhosis is reduced because this clearance system is defective. This study proposes a new mechanism of brain dysfunction in patients with cirrhosis and provides new targets for therapy

Inhibition of TLR7 and TLR9 Reduces Human Cholangiocarcinoma Cell Proliferation and Tumor Development

Background: Toll-like receptors (TLRs) are key players in innate immunity and modulation of TLR signaling has been demonstrated to profoundly affect proliferation and growth in different types of cancer. However, the role of TLRs in human intrahepatic cholangiocarcinoma (ICC) pathogenesis remains largely unexplored. / Aims: We set out to determine if TLRs play any role in ICCs which could potentially make them useful treatment targets. / Methods: Tissue microarrays containing samples from 9 human ICCs and normal livers were examined immunohistochemically for TLR4, TLR7, and TLR9 expression. Proliferation of human ICC cell line HuCCT1 was measured by MTS assay following treatment with CpG-ODN (TLR9 agonist), imiquimod (TLR7 agonist), chloroquine (TLR7 and TLR9 inhibitor) and IRS-954 (TLR7 and TLR9 antagonist). The in vivo effects of CQ and IRS-954 on tumor development were also examined in a NOD-SCID mouse xenograft model of human ICC. / Results: TLR4 was expressed in all normal human bile duct epithelium but absent in the majority (60%) of ICCs. TLR7 and TLR9 were expressed in 80% of human ICCs. However, TLR7 was absent in all cases of normal human bile duct epithelium and only one was TLR9 positive. HuCCT1 cell proliferation in vitro significantly increased following IMQ or CpG-ODN treatment (P < 0.03 and P < 0.002, respectively) but decreased with CQ (P < 0.02). In the mouse xenograft model there was significant reduction in size of tumors from CQ and IRS-954 treated mice compared to untreated controls. / Conclusion: TLR7 and TLR9 should be further explored for their potential as actionable targets in the treatment of ICC

Ornithine Phenylacetate targets alterations in the expression and activity of Glutamine Synthase and Glutaminase to reduce ammonia levels in bile duct ligated rats

Background & Aims: In liver failure, ammonia homeostasis is dependent upon the function of the ammonia metabolising enzymes, glutamine synthetase (GS) and glutaminase (GA) but data about their protein expression and activity are lacking. The aims of this study were to determine the protein expression and activity of GS and GA in individual organs in a rat model of chronic liver disease and to test whether the treatment with the ammonia-lowering agent ornithine phenylacetate (OP) modulates their activities. Methods: 49 SD rats were studied 35 days after sham-operation or bile duct ligation (BDL). The BDL group received: L-ornithine (0.6 mg/kg/day), Phenylacetate (0.6 mg/kg/day), OP (0.6 mg/kg/day) or placebo (saline) for 5 days prior to sacrifice. Arterial ammonia, amino acids and liver biochemistry were measured. Expressions of GS and GA were determined by Western-blotting and activities by end-point methods in liver, muscle, gut, kidney, lung, and frontal cortex. Results: In BDL rats, hepatic GS enzyme activity was reduced by more than 80% compared to sham rats. Further, in BDL rats GA activity was reduced in liver but increased in the gut, muscle and frontal cortex compared to sham rats. OP treatment resulted in a reduction in hyperammonemia in BDL rats, associated with increased GS activity in the muscle and reduced gut GA activity. Conclusions: In a rat model of chronic liver failure, hyperammonemia is associated with inadequate compensation by liver and muscle GS activity and increased gut GA activity. OP reduces plasma ammonia by increasing GS in the muscle and reducing GA activity in the gut providing additional insights into its mechanism of its action. GS and GA may serve as important future therapeutic targets for hyperammonemia in liver failure. (C) 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved