Gavin Wright

Endotoxemia produces coma and brain swelling in bile duct ligated rats

This study explores the hypothesis that the inflammatory response induced by administration of lipopolysaccharide (LPS) exacerbates brain edema in cirrhotic rats; and if so whether this is associated with altered brain metabolism of ammonia or anatomical disturbance of the blood‐brain barrier. Adult Sprague‐Dawley rats 4 weeks after bile duct ligation (BDL)/Sham‐operation, or naïve rats fed a hyperammonemic diet (HD), were injected with LPS (0.5 mg/kg, intraperitoneally) or saline, and killed 3 hours later. LPS administration increased brain water in HD, BDL, and sham‐operated groups significantly (P < 0.05), but this was associated with progression to pre‐coma stages only in BDL rats. LPS induced cytotoxic brain swelling and maintained anatomical integrity of the blood‐brain barrier. Plasma/brain ammonia levels were higher in HD and BDL rats than in sham‐operated controls and did not change with LPS administration. Brain glutamine/myoinositol ratio was increased in the HD group but reduced in the BDL animals. There was a background pro‐inflammatory cytokine response in the brains of cirrhotic rats, and plasma/brain tumor necrosis factor alpha (TNF‐α) and IL‐6 significantly increased in LPS‐treated animals. Plasma nitrite/nitrate levels increased significantly in LPS groups compared with non‐LPS controls; however, frontal cortex nitrotyrosine levels only increased in the BDL + LPS rats (P < 0.005 versus BDL controls). Conclusion: Injection of LPS into cirrhotic rats induces pre‐coma and exacerbates cytotoxic edema because of the synergistic effect of hyperammonemia and the induced inflammatory response. Although the exact mechanism of how hyperammonemia and LPS facilitate cytotoxic edema and pre‐coma in cirrhosis is not clear, our data support an important role for the nitrosation of brain proteins. (HEPATOLOGY 2007.)

Relationship between activation of the sympathetic nervous system and renal blood flow autoregulation in cirrhosis

BACKGROUND & AIMS It has been proposed that activation of the sympathetic nervous system causes a rightward shift in the renal autoregulatory curve such that renal blood flow is critically dependent on renal perfusion pressure and that this contributes to the development of the hepatorenal syndrome. The aims of the study were to determine the relationship of renal blood flow and renal perfusion pressure in patients with liver cirrhosis and the effect on renal hemodynamics following insertion of a transjugular intrahepatic portosystemic shunt (TIPS). METHODS Fifty-six patients were recruited into groups (1) with no ascites, (2) with diuretic-responsive ascites, (3) with intractable ascites, (4) with type II hepatorenal syndrome, and (5) requiring a TIPSs for refractory ascites. We measured cardiac hemodynamics, renal blood flow, renal perfusion pressure, and portal pressure and norepinephrine levels and mathematically modeled the renal autoregulatory curve. RESULTS Renal blood flow correlated with renal perfusion pressure (r(2) = 0.78; P < .001) and inversely with the hepatic venous pressure gradient (r(2) = 0.61; P < .0001) and plasma norepinephrine levels (r(2) = 0.78; P < .0001). Norepinephrine levels increased with increasing disease severity, and this was associated with a rightward and downward shift of the renal blood flow/renal perfusion pressure autoregulatory curve. TIPS insertion reduced portal pressure and plasma norepinephrine levels (P < .001), and the renal blood flow/renal perfusion pressure curve was shifted upward. CONCLUSIONS The relationship between renal blood flow and renal perfusion pressure involves a critical interplay between the sympathetic nervous system and the kidney. TIPS insertion decreases sympathetic activation and improves renal function through positive effects on renal blood flow autoregulation.

A self-expanding metal stent for complicated variceal hemorrhage: experience at a single center

BACKGROUND Refractory variceal bleeding is associated with a high mortality. Existing salvage techniques such as transjugular intrahepatic portosystemic shunt (TIPS) and balloon tamponade (BT) have important limitations and may not be appropriate for all patients. OBJECTIVE To evaluate the safety and efficacy of a novel removable self-expanding metal stent in the management of refractory variceal bleeding. DESIGN Case series. SETTING Tertiary referral liver center. PATIENTS Ten patients with variceal hemorrhage with contraindications to TIPS insertion or BT. INTERVENTIONS Insertion of a self-expanding metal stent (SX-Ella DANIS stent). MAIN OUTCOME MEASURES Survival, failure to control bleeding, and complications. RESULTS Stent insertion was successful in 9 of 10 patients. Failure to control bleeding was observed in 3 patients (2 with gastric varices), with control of bleeding in the remainder. Overall survival at 42 days was 50%. Six patients survived the acute bleeding episode and had stents removed endoscopically at a median of 9 days after insertion. One patient had a minor ulceration of the esophagus caused by stent insertion. CONCLUSIONS Insertion of the SX-Ella DANIS stent in patients with refractory variceal bleeding or complications of previous therapy is effective for the control of bleeding. Stent insertion can be achieved in the majority of patients without fluoroscopic control and without major complications. In selected patients, SX-Ella DANIS stent insertion offers an alternative to other methods of salvage such as BT and TIPS and could be considered a substitute for BT after a prospective trial.

Ammonia impairs neutrophil phagocytic function in liver disease

Hyperammonemia is a feature of liver failure, which is associated with increased risk of infection. The aims of the present study were to determine in vitro, in rats fed an ammoniagenic diet and in patients with cirrhosis, whether induction of hyperammonemia results in neutrophil dysfunction. As hyperammonemia produces cell swelling, we explored the role of the osmoregulating, p38 mitogen‐activated protein kinase (p38MAPK) pathway in mediating this neutrophil dysfunction. Neutrophils were isolated from blood of healthy volunteers and incubated with either 75 μM ammonia or phosphate‐buffered saline. Both groups were studied under hyponatremic conditions and/or with the addition of p38MAPK modulators. Neutrophil phagocytosis was measured in naive rats and rats fed an ammoniagenic diet and in patients with stable cirrhosis given placebo (n = 8) or an amino acid solution inducing hyperammonemia (n = 8). Cell volume and phagocytosis was analyzed by fluorescent‐activated cell sorting using fluorescein isothiocyanate–labeled E. coli. p38MAPK phosphorylation was measured by western blotting. In healthy neutrophils incubated with ammonia and in rats fed an ammoniagenic diet, neutrophils showed evidence of swelling, impaired phagocytosis, and increased spontaneous oxidative burst compared to controls. Phagocytosis was significantly impaired in patients with induced hyperammonemia compared to placebo. The effects of hyperammonemia and hyponatremia were synergistic. The p38MAPK intracellular signaling pathways were activated in healthy neutrophils exposed to ammonia in association with increased burst activity. Neutrophil phagocytic dysfunction was abrogated by the addition of a p38MAPK agonist. Conclusion: Ammonia produces neutrophil swelling and impairs neutrophil phagocytosis. The p38MAPK intracellular signaling pathway has been shown to be important in mediating the ammonia‐induced neutrophil dysfunction. (HEPATOLOGY 2008.)

Evaluation of coagulation abnormalities in acute liver failure

BACKGROUND & AIMS In acute liver failure (ALF), prothrombin time (PT) and its derivative prothrombin time ratio (PTR) are elevated, and are considered predictors of increased bleeding risk. We aimed at determining whether increased PT/PTR reflects the haemostatic potential and bleeding risk in ALF patients. METHODS Twenty consecutive ALF patients were recruited. Samples were analysed on admission for standard laboratory clotting tests (e.g. PT), thromboelastography (TEG), individual pro and anticoagulant factors and thrombin generation (TG) kinetics with and without Protac, a snake venom protein C activator, and microparticle assay. TG was also measured in 20 age and sex matched healthy volunteers. RESULTS PT was significantly raised (50.7s ± 7.2, p=0.0001) but did not correlate with TEG parameters. TEG tracings were consistent with a hypocoagulable state in 20%, normal in 45%, and hypercoagulable in 35% of the patients. There was a concomitant and proportional reduction in plasma levels of both procoagulants and natural anticoagulant proteins, in conjunction with a significant elevation in plasma levels of factors-VIII (FVIII) and Von Willebrand factor, and microparticles, culminating in an overall efficient, albeit reduced, thrombin generation capacity in comparison with healthy individuals. A heparin-like effect (HLE) was also noted in most patients. No significant clinical bleeding complications occurred and no blood transfusions were required. CONCLUSIONS In ALF, despite grossly deranged PT in all patients, estimation of bleeding risk suggests that the coagulation disturbance in ALF patients is complex and heterogeneous for which an individualised approach is required.

L‐ornithine and phenylacetate synergistically produce sustained reduction in ammonia and brain water in cirrhotic rats

Treatment of hyperammonemia and hepatic encephalopathy in cirrhosis is an unmet clinical need. The aims of this study were to determine whether L‐ornithine and phenylacetate/phenylbutyrate (administered as the pro‐drug phenylbutyrate) (OP) combined are synergistic and produce sustained reduction in ammonia by L‐ornithine acting as a substrate for glutamine synthesis, thereby detoxifying ammonia, and the phenylacetate excreting the ornithine‐derived glutamine as phenylacetylglutamine in the urine. Sprague‐Dawley rats were studied 4 weeks after bile duct ligation (BDL) or sham operation. Study 1: Three hours before termination, an internal carotid sampling catheter was inserted, and intraperitoneal saline (placebo), OP, phenylbutyrate, or L‐ornithine were administered after randomization. BDL was associated with significantly higher arterial ammonia and brain water and lower brain myoinositol (P < 0.01, respectively), compared with sham‐operated controls, which was significantly improved in the OP‐treated animals; arterial ammonia (P < 0.001), brain water (P < 0.05), brain myoinositol (P < 0.001), and urinary phenylacetylglutamine (P < 0.01). Individually, L‐ornithine or phenylbutyrate were similar to the BDL group. In study 2, BDL rats were randomized to saline or OP administered intraperitoneally for 6 hours or 3, 5, or 10 days and were sacrificed between 4.5 and 5 weeks. The results showed that the administration of OP was associated with sustained reduction in arterial ammonia (P < 0.01) and brain water (P < 0.01) and markedly increased arterial glutamine (P < 0.01) and urinary excretion of phenylacetylglutamine (P < 0.01) in each of the OP treated groups. Conclusion: The results of this study provide proof of the concept that L‐ornithine and phenylbutyrate/phenylacetate act synergistically to produce sustained improvement in arterial ammonia, its brain metabolism, and brain water in cirrhotic rats. (HEPATOLOGY 2009.)

Interorgan ammonia metabolism in liver failure: the basis of current and future therapies.

Hepatic encephalopathy complicates the course of both acute and chronic liver disease and its treatment remains an unmet clinical need. Ammonia is thought to be central in its pathogenesis and remains an important target of current and future therapeutic approaches. In liver failure, the main detoxification pathway of ammonia metabolism is compromised leading to hyperammonaemia. In this situation, the other ammonia‐regulating pathways in multiple organs assume important significance. The present review focuses upon interorgan ammonia metabolism in health and disease describing the role of the key enzymes, glutamine synthase and glutaminase. Better understanding of these alternative pathways are leading to the development of new therapeutic approaches.

Blockade of PD1 and TIM3 Restores Innate and Adaptive Immunity in Patients with Acute Alcoholic Hepatitis

BACKGROUND & AIMS Susceptibility to bacterial infection is a feature of alcohol-related liver disease. Programmed cell death 1 (PD1), the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3, also known as hepatitis A virus cellular receptor 2), and their respective ligands-CD274 (also known as PD ligand 1 [PDL1]) and galectin-9-are inhibitory receptors that regulate the balance between protective immunity and host immune-mediated damage. However, their sustained hyperexpression promotes immune exhaustion and paralysis. We investigated the role of these immune inhibitory receptors in driving immune impairments in patients with alcoholic liver disease. METHODS In a prospective study, we collected blood samples from 20 patients with acute alcoholic hepatitis (AAH), 16 patients with stable advanced alcohol-related cirrhosis, and 12 healthy individuals (controls). Whole blood or peripheral blood mononuclear cells were assessed for expression of PD1, PDL1, TIM3, galectin-9, and Toll-like receptors on subsets of innate and adaptive immune effector cells. We measured antibacterial immune responses to lipopolysaccharide (endotoxin) using ELISpot assays, and used flow cytometry to quantify cytokine production, phagocytosis, and oxidative burst in the presence or absence of blocking antibodies against PD1 or TIM3. RESULTS Antibacterial innate and adaptive immune responses were greatly reduced in patients with AAH, compared with controls, and patients with alcohol-related cirrhosis had less severe dysfunctions in innate immune effector cells and preserved functional T-cell responses. Fewer T cells from patients with AAH produced interferon gamma in response to lipopolysaccharide, compared with controls. In addition, patients with AAH had greater numbers of interleukin 10-producing T cells, and reduced levels of neutrophil phagocytosis and oxidative burst in response to Escherichia coli stimulation, compared with controls. T cells from patients with AAH, but not alcohol-related cirrhosis, expressed higher levels of PD1 and PDL1, or TIM3 and galectin-9, than T cells from controls. Antibodies against PD1 and TIM3 restored T-cell production of interferon gamma, reduced the numbers of interleukin 10-producing T cells, and increased neutrophil antimicrobial activities. Circulating levels of endotoxin in plasma from patients with AAH caused over expression of immune inhibitory receptors on T cells via Toll-like receptor 4 binding to CD14(+) monocytes. CONCLUSIONS Antibacterial immune responses are impaired in patients with AAH. Lymphocytes from these patients express high levels of immune inhibitory receptors, produce lower levels of interferon gamma, and have increased IL10 production due to chronic endotoxin exposure. These effects can be reversed by blocking PD1 and TIM3, which increase the antimicrobial activities of T cells and neutrophils.

Ammonia and inflammation in the pathogenesis of hepatic encephalopathy: Pandora's box?†‡

Hepatic encephalopathy (HE), the neuropsychiatric manifestation of liver disease, incorporates a spectrum of manifestations ranging from minimal derangements in neuropsychological function to confusion and coma. Over the past 10 years, studies have confirmed the strong association between hyperammonemia due to liver dysfunction and infection/inflammation in the pathogenesis of HE, in acute liver failure,1 cirrhosis,2 and more recently in acute-on-chronic liver failure.3 An improvement in HE following interventions which modulate inflammatory responses, such as hypothermia,4 and the use of indomethacin (cyclo-oxygenase pathway),5,6 albumin and albumin dialysis (free radicals, free metals, nonspecific protein-bound substances)7 and, probiotics (endotoxin)8 indicates that reducing inflammation is also a valid modality for treating HE. In this issue, Cauli et al.9 report in a rat model improved learning ability in animals with HE following the administration of the nonsteroidal anti-inflammatory drug (NSAID), ibuprofen. They showed that the chronic administration of ibuprofen (from day 10 up to 4 weeks after portacaval shunting [PCS]) at 5-6 times the therapeutic doses resulted in a “normalization” of cyclo-oxygenase (COX) and inducible nitric oxide synthase (iNOS) activity. Moreover, administration of ibuprofen was also associated with improvement in the glutamate–nitric oxide–cyclic guanosine monophosphate (Glu-NOcGMP) pathway. In PCS rats, brain interleukin-6 was elevated but tumor necrosis factor alpha (TNF ) remained unchanged, and controversially, TNF increased significantly in the ibuprofen-treated animals. It is important to note that this model is more akin to that observed in “minimal HE” as opposed to more severe forms of liver disease and therefore must be interpreted in this light. This study touches on the complex processes and multiple cell types involved in the pathogenesis of HE, highlighting the importance of inflammatory pathways and their modulation in the treatment of “minimal HE”. The questions that need to be addressed in the interpretation of the data presented and implications for pathogenic mechanisms and therapy are:

Role of aquaporin-4 in the development of brain oedema in liver failure

BACKGROUND & AIMS Liver failure is associated with progressive cytotoxic brain oedema (astrocyte swelling), which underlies hepatic encephalopathy (HE). Ammonia and superimposed inflammation are key synergistic factors in HE, but the mechanism(s) involved remain unknown. We aimed to determine whether aquaporin-4 (AQP4), an astrocyte endfeet bi-directional water channel, is associated with the brain oedema of HE. METHOD Rats (n=60) received sham-operation (sham), 5 days hyperammonaemia-inducing diet (HD), galactosamine (GALN) induced acute liver failure (ALF), 4 weeks bile duct-ligation (BDL) induced cirrhosis, or caecal ligation and puncture (CLP), a 24h model of bacterial peritonitis. Rats from every group (except CLP) were randomised to receive intraperitoneal injections of lipopolysaccharide (LPS; 1mg/kg) or saline, prior to termination 3h later. Brain water, AQP4 protein expression (western blot) and AQP4 localisation by immunogold electron microscopy were investigated. RESULTS Significant hyperammonaemia was observed in saline-injected BDL (p<0.05), GALN (p<0.01), and HD (p<0.01), compared to sham rats. LPS injection did not affect arterial ammonia or plasma biochemistry in any of the treatment groups. Increased brain water was observed in saline-injected GALN (p<0.05), HD (p<0.01), and CLP (p<0.001) compared to sham rats. Brain water was numerically increased in BDL rats, but this failed to reach significance (p=0.09). LPS treatment further increased oedema significantly in all treatment groups (p<0.05, respectively). AQP4 expression was significantly increased in saline-injected BDL (p<0.05), but not other treatment groups, compared to sham rats. Membrane polarisation was maintained in BDL rats. CONCLUSION The results suggest that AQP4 is not directly associated with the development of brain oedema in liver failure, hyperammonaemia, or sepsis. In cirrhosis, there is increased AQP4 protein expression, but membrane polarisation, is maintained, possibly in a compensatory attempt to limit severe brain oedema.