Presentation and publication skills: How to review a paper.

We provide comprehensive insights into the peer review process and guide potential reviewers through the steps of reviewing scientific manuscripts. We discuss essential aspects such as the reviewer's responsibility in responding to invitations and maintaining confidentiality throughout the process, the criteria for accepting or rejecting papers, and efficient review of resubmissions. We emphasize the importance of prioritizing the review responsibility within other commitments, communication using professional and courteous language, and adherence to deadlines. We also offer practical tips on evaluating the abstract, introduction, materials and methods, results, and discussion section and summarizing the critiques in the review report.

Computational Modeling of Fructose Metabolism and Development in NAFLD.

Non-alcohol fatty liver disease (NAFLD) is a common disorder that has increased in prevalence 20-fold over the last three decades. It covers a spectrum of conditions resulting from excess lipid accumulation in the liver without alcohol abuse. Among all the risk factors, over-consumption of fructose has been repeatedly reported in both clinical and experimental studies to be highly associated with the development of NAFLD. However, studying in vivo systems is complicated, time consuming and expensive. A detailed kinetic model of fructose metabolism was constructed to investigate the metabolic mechanisms whereby fructose consumption can induce dyslipidaemia associated with NAFLD and to explore whether the pathological conditions can be reversed during the early stages of disease. The model contains biochemical components and reactions identified from the literature, including ~120 parameters, 25 variables, and 25 first order differential equations. Three scenarios were presented to demonstrate the behavior of the model. Scenario one predicts the acute effects of a change in carbohydrate input in lipid profiles. The results present progressive triglyceride accumulations in the liver and plasma for three diets. The rate of accumulation was greater in the fructose diet than that of the mixed or glucose only models. Scenario two explores the variability of metabolic reaction rate within the general population. Sensitivity analysis reveals that hepatic triglyceride concentration is most sensitive to the rate constant of pyruvate kinase and fructokinase. Scenario three tests the effect of one specific inhibitor that might be potentially administered. The simulations of fructokinase suppression provide a good model for potentially reversing simple steatosis induced by high fructose consumption, which can be corroborated by experimental studies. The predictions in these three scenarios suggest that the model is robust and it has sufficient detail to present the kinetic relationship between fructose and lipid in the liver.

Porcine model characterizing various parameters assessing the outcome after acetaminophen intoxication induced acute liver failure.

AIM: To investigate the changes of hemodynamic and laboratory parameters during the course of acute liver failure following acetaminophen overdose. METHODS: Eight pigs underwent a midline laparotomy following jejunal catheter placement for further acetaminophen intoxication and positioning of a portal vein Doppler flow-probe. Acute liver failure was realized by intrajejunal acetaminophen administration in six animals, two animals were sham operated. All animals were invasively monitored and received standardized intensive care support throughout the study. Portal blood flow, hemodynamic and ventilation parameters were continuously recorded. Laboratory parameters were analysed every eight hours. Liver biopsies were sampled every 24 h following intoxication and upon autopsy. RESULTS: Acute liver failure (ALF) occurred after 28 ± 5 h resulted in multiple organ failure and death despite maximal support after further 21 ± 1 h (study end). Portal blood flow (baseline 1100 ± 156 mL/min) increased to a maximum flow of 1873 ± 175 mL/min at manifestation of ALF, which was significantly elevated (P < 0.01). Immediately after peaking, portal flow declined rapidly to 283 ± 135 mL/min at study end. Thrombocyte values (baseline 307 × 103/µL ± 34 × 103/µL) of intoxicated animals declined slowly to values of 145 × 103/µL ± 46 × 103/µL when liver failure occurred. Subsequent appearance of severe thrombocytopenia in liver failure resulted in values of 11 × 103/µL ± 3 × 103/µL preceding fatality within few hours which was significant (P > 0.01). CONCLUSION: Declining portal blood flow and subsequent severe thrombocytopenia after acetaminophen intoxication precede fatality in a porcine acute liver failure model.

A Computational Model of Hepatic Energy Metabolism: Understanding Zonated Damage and Steatosis in NAFLD.

In non-alcoholic fatty liver disease (NAFLD), lipid build-up and the resulting damage is known to occur more severely in pericentral cells. Due to the complexity of studying individual regions of the sinusoid, the causes of this zone specificity and its implications on treatment are largely ignored. In this study, a computational model of liver glucose and lipid metabolism is presented which treats the sinusoid as the repeating unit of the liver rather than the single hepatocyte. This allows for inclusion of zonated enzyme expression by splitting the sinusoid into periportal to pericentral compartments. By simulating insulin resistance (IR) and high intake diets leading to the development of steatosis in the model, we identify key differences between periportal and pericentral cells accounting for higher susceptibility to pericentral steatosis. Secondly, variation between individuals is seen in both susceptibility to steatosis and in its development across the sinusoid. Around 25% of obese individuals do not show excess liver fat, whilst 16% of lean individuals develop NAFLD. Furthermore, whilst pericentral cells tend to show higher lipid levels, variation is seen in the predominant location of steatosis from pericentral to pan-sinusoidal or azonal. Sensitivity analysis was used to identify the processes which have the largest effect on both total hepatic triglyceride levels and on the sinusoidal location of steatosis. As is seen in vivo, steatosis occurs when simulating IR in the model, predominantly due to increased uptake, along with an increase in de novo lipogenesis. Additionally, concentrations of glucose intermediates including glycerol-3-phosphate increased when simulating IR due to inhibited glycogen synthesis. Several differences between zones contributed to a higher susceptibility to steatosis in pericentral cells in the model simulations. Firstly, the periportal zonation of both glycogen synthase and the oxidative phosphorylation enzymes meant that the build-up of glucose intermediates was less severe in the periportal hepatocyte compartments. Secondly, the periportal zonation of the enzymes mediating ß-oxidation and oxidative phosphorylation resulted in excess fats being metabolised more rapidly in the periportal hepatocyte compartments. Finally, the pericentral expression of de novo lipogenesis contributed to pericentral steatosis when additionally simulating the increase in sterol-regulatory element binding protein 1c (SREBP-1c) seen in NAFLD patients in vivo. The hepatic triglyceride concentration was predicted to be most sensitive to inter-individual variation in the activity of enzymes which, either directly or indirectly, determine the rate of free fatty acid (FFA) oxidation. The concentration was most strongly dependent on the rate constants for ß-oxidation and oxidative phosphorylation. It also showed moderate sensitivity to the rate constants for processes which alter the allosteric inhibition of ß-oxidation by acetyl-CoA. The predominant sinusoidal location of steatosis meanwhile was most sensitive variations in the zonation of proteins mediating FFA uptake or triglyceride release as very low density lipoproteins (VLDL). Neither the total hepatic concentration nor the location of steatosis showed strong sensitivity to variations in the lipogenic rate constants.

Acute Citrulline-Malate Supplementation and High-Intensity Cycling Performance.

Cunniffe, B, Papageorgiou, M, O'Brien, B, Davies, NA, Grimble, GK, and Cardinale, M. Acute citrulline-malate supplementation and high-intensity cycling performance. J Strength Cond Res 30(9): 2638-2647, 2016-Dietary L-citrulline-malate (CM) consumption has been suggested to improve skeletal muscle metabolism and contractile efficiency, which would be expected to predispose exercising individuals to greater fatigue resistance. The purpose of this study was to examine the effects of CM supplementation on acid-base balance and high-intensity exercise performance. In a double-blind, placebo-controlled, crossover study, 10 well-trained males consumed either 12 g of CM (in 400 ml) or lemon sugar-free cordial (placebo [PL]) 60 minutes before completion of 2 exercise trials. Each trial consisted of subjects performing 10 (×15 seconds) maximal cycle sprints (with 30-second rest intervals) followed by 5 minutes recovery before completing a cycle time-to-exhaustion test (TTE) at 100% of individual peak power (PP). Significant increases in plasma concentrations of citrulline (8.8-fold), ornithine (3.9-fold), and glutamine (1.3-fold) were observed 60 minutes after supplementation in the CM trial only (p ≤ 0.05) and none of the subjects experienced gastrointestinal side-effects during testing. Significantly higher exercise heart rates were observed in CM condition (vs. PL) although no between trial differences in performance related variables (TTE: [120 ± 61 seconds CM vs. 113 ± 50 seconds PL]), PP or mean power, ([power fatigue index: 36 ± 16% CM vs. 28 ± 18% PL]), subjective rating of perceived exertion or measures of acid-base balance (pH, lactate, bicarbonate, base-excess) were observed (p > 0.05). This study demonstrated that acute supplementation of 12 g CM does not provide acute ergogenic benefits using the protocol implemented in this study in well-trained males.

Circulating microRNAs Reveal Time Course of Organ Injury in a Porcine Model of Acetaminophen-Induced Acute Liver Failure.

Acute liver failure is a rare but catastrophic condition which can progress rapidly to multi-organ failure. Studies investigating the onset of individual organ injury such as the liver, kidneys and brain during the evolution of acute liver failure, are lacking. MicroRNAs are short, non-coding strands of RNA that are released into the circulation following tissue injury. In this study, we have characterised the release of both global microRNA and specific microRNA species into the plasma using a porcine model of acetaminophen-induced acute liver failure. Pigs were induced to acute liver failure with oral acetaminophen over 19h±2h and death occurred 13h±3h thereafter. Global microRNA concentrations increased 4h prior to acute liver failure in plasma (P<0.0001) but not in isolated exosomes, and were associated with increasing plasma levels of the damage-associated molecular pattern molecule, genomic DNA (P<0.0001). MiR122 increased around the time of onset of acute liver failure (P<0.0001) and was associated with increasing international normalised ratio (P<0.0001). MiR192 increased 8h after acute liver failure (P<0.0001) and was associated with increasing creatinine (P<0.0001). The increase in miR124-1 occurred concurrent with the pre-terminal increase in intracranial pressure (P<0.0001) and was associated with decreasing cerebral perfusion pressure (P<0.002). Conclusions: MicroRNAs were released passively into the circulation in response to acetaminophen-induced cellular damage. A significant increase in global microRNA was detectable prior to significant increases in miR122, miR192 and miR124-1, which were associated with clinical evidence of liver, kidney and brain injury respectively.

Extracorporeal liver assist device to exchange albumin and remove endotoxin in acute liver failure: Results of a pivotal pre-clinical study.

BACKGROUND & AIMS: In acute liver failure, severity of liver injury and clinical progression of disease are in part consequent upon activation of the innate immune system. Endotoxaemia contributes to innate immune system activation and the detoxifying function of albumin, critical to recovery from liver injury, is irreversibly destroyed in acute liver failure. University College London-Liver Dialysis Device is a novel artificial extracorporeal liver assist device, which is used with albumin infusion, to achieve removal and replacement of dysfunctional albumin and reduction in endotoxaemia. We aimed to test the effect of this device on survival in a pig model of acetaminophen-induced acute liver failure. METHODS: Pigs were randomised to three groups: Acetaminophen plus University College London-Liver Dialysis Device (n=9); Acetaminophen plus Control Device (n=7); and Control plus Control Device (n=4). Device treatment was initiated two h after onset of irreversible acute liver failure. RESULTS: The Liver Dialysis Device resulted in 67% reduced risk of death in acetaminophen-induced acute liver failure compared to Control Device (hazard ratio=0.33, p=0.0439). This was associated with 27% decrease in circulating irreversibly oxidised human non-mercaptalbumin-2 throughout treatment (p=0.046); 54% reduction in overall severity of endotoxaemia (p=0.024); delay in development of vasoplegia and acute lung injury; and delay in systemic activation of the TLR4 signalling pathway. Liver Dialysis Device-associated adverse clinical effects were not seen. CONCLUSIONS: The survival benefit and lack of adverse effects would support clinical trials of University College London-Liver Dialysis Device in acute liver failure patients.

Metabolic regulation of hepatitis B immunopathology by myeloid-derived suppressor cells.

Infection with hepatitis B virus (HBV) results in disparate degrees of tissue injury: the virus can either replicate without pathological consequences or trigger immune-mediated necroinflammatory liver damage. We investigated the potential for myeloid-derived suppressor cells (MDSCs) to suppress T cell-mediated immunopathology in this setting. Granulocytic MDSCs (gMDSCs) expanded transiently in acute resolving HBV, decreasing in frequency prior to peak hepatic injury. In persistent infection, arginase-expressing gMDSCs (and circulating arginase) increased most in disease phases characterized by HBV replication without immunopathology, whilst L-arginine decreased. gMDSCs expressed liver-homing chemokine receptors and accumulated in the liver, their expansion supported by hepatic stellate cells. We provide in vitro and ex vivo evidence that gMDSCs potently inhibited T cells in a partially arginase-dependent manner. L-arginine-deprived T cells upregulated system L amino acid transporters to increase uptake of essential nutrients and attempt metabolic reprogramming. These data demonstrate the capacity of expanded arginase-expressing gMDSCs to regulate liver immunopathology in HBV infection.

Caecal ligation and puncture induced sepsis in the rat results in increased brain water content and perimicrovessel oedema.

To investigate brain water content and ultrastructure in a rat caecal ligation and puncture (CLP) model of sepsis, adult male Wistar rats were assigned to one of the following experimental groups: CLP, Un-operated or Sham. CLP was performed under anaesthesia, Sham rats were exposed to anaesthesia, laparotomy and caecal mobilisation and Un-operated rats did not experience anaesthesia or surgery. CLP and Sham rats were sacrificed 18-20 h following recovery from surgery and Un-operated rats were sacrificed at the same time. Frontal cortex samples (CLP n = 9; Un-operated n = 10; Sham n = 8) were taken immediately post mortem and their water content determined using gravimetry. Similar samples were taken from other rats (CLP n = 8; Un-operated n = 8; Sham n = 8), processed for electron microscopy and subjected to morphometric analysis. There was significantly more brain water in CLP than Un-operated (P < 0.01) and Sham (P < 0.05) rats. Electron microscopy revealed significantly more peri-microvessel oedema in CLP than Un-operated (P < 0.001) and Sham rats (P < 0.05). Microvessel endothelial cell lumen cross-sectional area was significantly smaller in CLP than Un-operated (P < 0.001) and Sham (P < 0.05) rats and microvessel endothelial cell cross-sectional area was significantly smaller in CLP than Un-operated (P < 0.05) rats. Significantly more endothelial cell cytoplasmic area was occupied by mitochondria in CLP than Un-operated (P < 0.05) and Sham (P < 0.05) rats. However, experimental group did not affect the number of mitochondria present in endothelial cell profiles, or their cross-sectional area. Therefore, sepsis-induced cerebral oedema involves an increase in and a redistribution of brain water, together with ultrastructural changes to cerebral microvessels and adjacent tissue.

Role of toll-like receptor 4 in mediating multiorgan dysfunction in mice with acetaminophen induced acute liver failure.

Strategies for the prevention of multiorgan dysfunction (MOD) in acetaminophen (APAP)-induced acute liver failure (ALF) are an unmet need. Our study tested the hypothesis that sterile inflammation induced by APAP in a mouse model would activate toll-like receptor 4 (TLR4) in the liver and extrahepatic organs and lead to the progression of ALF and MOD and that the administration of the novel TLR4 antagonist STM28 (a peptide formed of 17 amino-acids) would prevent liver injury and associated MOD. ALF and, subsequently, MOD were induced in TLR4-knockout (KO) mice (B6.B10ScN-Tlr4 (lpsdel) /JthJ) and wild-type (WT) mice (C57BL/6) with APAP (500 mg/kg). A second set of experiments was conducted to evaluate the effects of a pretreatment with a novel TLR4 antagonist, STM28, on APAP-induced MOD in CD1 mice. Animals were sacrificed at the coma stage, and plasma, peripheral blood cells, liver, kidneys, and brain were collected. Biochemistry values and cytokines were measured. Liver and kidneys were studied histologically and were stained for TLR4 and activated Kupffer cells, and the expression of nuclear factor kappa B-p65 was quantified with western blotting. Brain water was measured in the frontal cortex. After APAP administration, TLR4-KO (NFkBp65) mice were relatively protected from liver necrosis and end-organ dysfunction and had significantly better survival than WT controls (P < 0.01). STM28 attenuated liver injury and necrosis, reduced creatinine levels, and delayed the time to a coma significantly. The increases in cytokines in the plasma and liver, including TLR4 expression and the activation of Kupffer cells, after APAP administration were reduced significantly in the STM28-treated animals. The increased number of circulating myeloid cells was reduced significantly after STM28 treatment. In conclusion, these data provide evidence for an important role of the TLR4 antagonist in the prevention of the progression of APAP-induced ALF and MOD.

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

BACKGROUND & AIMS: In 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. METHODS: Four 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. RESULTS: BDL 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. CONCLUSIONS: The 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.

A reproducible, clinically relevant, intensively managed, pig model of acute liver failure for testing of therapies aimed to prolong survival.

BACKGROUND: A clinically relevant, translational large animal model of acute liver failure (ALF) is required for testing of novel therapies to prolong survival in acute liver failure, to permit spontaneous liver recovery or to act as a bridge to transplantation. AIMS: The aim was to establish a pig model of acetaminophen-induced ALF that mimics the human clinical syndrome, is managed as in a human intensive care unit and has a predictable survival time. METHODS: Nine female pigs were anaesthetised and instrumented for continuous intensive care monitoring and management using: target-driven protocols for treatment of cardiovascular collapse, metabolic acidosis and electrolyte abnormalities; intermittent positive pressure ventilation; and continuous renal replacement therapy. Six animals were induced to ALF with acetaminophen (paracetamol). Three animals acted as controls. RESULTS: Irreversible acute liver failure, defined as rise in prothrombin time >3 times normal, occurred 19.3 ± 1.8 h after the onset of acetaminophen administration. Death occurred predictably 12.6 ± 2.7 h thereafter, with acute hepatocellular necrosis in all animals. Clinical progression of liver failure mimicked the human condition including development of coagulopathy, intracranial hypertension, hyperammonaemia, cardiovascular collapse, elevation in creatinine, metabolic acidosis and hyperlactataemia. In addition, cardiovascular monitoring clearly demonstrated progressive cardiac dysfunction in ALF. CONCLUSIONS: A reproducible, clinically relevant, intensively managed, large animal model of acute liver failure, with death as a result of multi-organ failure, has been successfully validated for translational studies of disease progression and therapies designed to prolong survival in man.

Serum autotaxin is increased in pruritus of cholestasis, but not of other origin, and responds to therapeutic interventions.

UNLABELLED: Pruritus is a seriously disabling symptom accompanying many cholestatic liver disorders. Recent experimental evidence implicated the lysophospholipase, autotaxin (ATX), and its product, lysophosphatidic acid (LPA), as potential mediators of cholestatic pruritus. In this study, we highlight that increased serum ATX levels are specific for pruritus of cholestasis, but not pruritus of uremia, Hodgkin's disease, or atopic dermatitis. Treatment of patients with cholestasis with the bile salt sequestrant, colesevelam, but not placebo, effectively reduced total serum bile salts and fibroblast growth factor 19 levels, but only marginally altered pruritus intensity and ATX activity. Rifampicin (RMP) significantly reduced itch intensity and ATX activity in patients with pruritus not responding to bile salt sequestrants. In vitro, RMP inhibited ATX expression in human HepG2 hepatoma cells and hepatoma cells overexpressing the pregnane X receptor (PXR), but not in hepatoma cells in which PXR was knocked down. Treatment of severe, refractory pruritus by the molecular adsorbents recirculation system or nasobiliary drainage improved itch intensity, which, again, correlated with the reduction of ATX levels. Upon reoccurrence of pruritus, ATX activity returned to pretreatment values. CONCLUSION: Serum ATX activity is specifically increased in patients with cholestatic, but not other forms of, systemic pruritus and closely correlates with the effectiveness of therapeutic interventions. The beneficial antipruritic action of RMP may be explained, at least partly, by the PXR-dependent transcriptional inhibition of ATX expression. Thus, ATX likely represents a novel therapeutic target for pruritus of cholestasis.

Liver replacement therapy.

The realization of a support device able to effectively replace liver function in patients with hepatic failure has thus far been an elusive goal. The complexity of liver metabolic, synthetic, detoxifying, and excretory functions make artificial hepatic support extremely challenging. Currently, no specific treatment is available to reverse acute or acute-on-chronic liver failure, and morbidity and mortality of these syndromes are still high. Present management strategies are supportive, while waiting for spontaneous liver regeneration or liver transplant. Because of the scarcity of donor organs, liver support strategies are needed for patients with inadequate liver function until an appropriate organ becomes available for transplantation or until their liver recovers from injury. Currently available liver support systems comprise nonbiological systems (e.g., hemodiafiltration, albumin dialysis, and plasma exchange) and bioartificial systems utilizing viable liver cells. The role for these novel systems and their impact on survival or other clinically important outcomes are controversial. Development and use of bioartificial systems are limited by the inherent cost.

Nitric oxide and L-arginine metabolism in a devascularized porcine model of acute liver failure.

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.

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.

Reduction in hyperammonaemia by ornithine phenylacetate prevents lipopolysaccharide-induced brain edema and coma in cirrhotic rats.

OBJECTIVE: In liver failure, inflammation synergistically exacerbates the deleterious cerebral effects of ammonia. The aims were to test whether treatment with the ammonia-lowering agent ornithine phenylacetate (OP) and/or anti-TNF-α (infliximab) prevent the deleterious brain consequences of lipopolysaccharide (LPS) in cirrhotic rats. DESIGN: Rats 4 weeks following bile duct-ligation (BDL), sham-operation (sham) and/or 7 days hyperammonemic feed (HD), were randomized to receive LPS (1 mg/kg) or saline, and treatment with either 3 days intraperitoneal injections of OP (0.6 g/kg) and/or infliximab, 10 mg/kg. Animals were sacrificed at coma stages or at 3 h. RESULTS: In sham rats, both HD and LPS increased brain water, with an increase in ammonia in the former and brain cytokines in the latter but with no effect on consciousness. BDL + HD rats caused significantly higher plasma ammonia, TNF-α and IL-6 levels compared to sham. LPS significantly worsened coma stage, increased brain water and plasma and brain TNF-α. OP significantly delayed LPS-induced progression to coma stages (P < 0.009), reduced arterial ammonia and brain water (P < 0.001 and P < 0.01 respectively), which was associated with a significant reduction in cytokines. Infliximab significantly reduced plasma and brain cytokines, but not brain water. OP + infliximab attenuated increase in brain water and delayed occurrence of coma, which was not different to OP alone. In BDL rats, OP reduced the expression of brain iNOS and NFκB. CONCLUSION: Reduction in ammonia with OP in cirrhotic rats prevents LPS-induced brain edema and delays coma, suggesting that ammonia may prime the brain to the deleterious effect of LPS, possibly through effects on iNOS and NFκB related mechanisms.

Ammonia reduction with ornithine phenylacetate restores brain eNOS activity via the DDAH-ADMA pathway in bile duct-ligated cirrhotic rats.

Ammonia is central in the pathogenesis of hepatic encephalopathy, which is associated with dysfunction of the nitric oxide (NO) signaling pathway. Ornithine phenylacetate (OP) reduces hyperammonemia and brain water in cirrhotic animals. This study aimed to determine whether endothelial NO synthase activity is altered in the brain of cirrhotic animals, whether this is associated with changes in the endogenous inhibitor, asymmetric-dimethylarginine (ADMA) and its regulating enzyme, dimethylarginine-dimethylaminohydrolase (DDAH-1), and whether these abnormalities are restored by ammonia reduction using OP. Sprague-Dawley rats were studied 4-wk after bile duct ligation (BDL) (n = 16) or sham operation (n = 8) and treated with placebo or OP (0.6 g/kg). Arterial ammonia, brain water, TNF-α, plasma, and brain ADMA were measured using standard techniques. NOS activity was measured radiometrically, and protein expression for NOS enzymes, ADMA, DDAH-1, 4-hydroxynonenol ((4)HNE), and NADPH oxidase (NOX)-1 were measured by Western blotting. BDL significantly increased arterial ammonia (P < 0.0001), brain water (P < 0.05), and brain TNF-α (P < 0.01). These were reduced significantly by OP treatment. The estimated eNOS component of constitutive NOS activity was significantly lower (P < 0.05) in BDL rat, and this was significantly attenuated in OP-treated animals. Brain ADMA levels were significantly higher and brain DDAH-1 significantly lower in BDL compared with sham (P < 0.01) and restored toward normal following treatment with OP. Brain (4)HNE and NOX-1 protein expression were significantly increased in BDL rat brain, which were significantly decreased following OP administration. We show a marked abnormality of NO regulation in cirrhotic rat brains, which can be restored by reduction in ammonia concentration using OP.

BK large conductance Ca²+-activated K+ channel-deficient mice are not resistant to hypotension and display reduced survival benefit following polymicrobial sepsis.

Nitric oxide-mediated activation of large conductance calcium-activated potassium (BK) channels is considered an important underlying mechanism of sepsis-induced hypotension. Indeed, the nonselective K-channel inhibitor, tetraethylammonium chloride (TEA), has been proposed as a potential treatment to raise blood pressure in septic shock by virtue of its ability to inhibit BK channels. As experimental evidence has so far relied on pharmacological inhibition, we examined the effects of channel deletion using BKα subunit knockout (α, Slo) mice in two mouse models of polymicrobial sepsis, namely, intraperitoneal fecal slurry and cecal ligation and puncture. Comparison was made against TEA treatment in wild-type (WT) mice. Following slurry, BKα and WT mice developed similar degrees of hypotension over 10 h with no difference in cardiac output as assessed by echocardiography between groups. Tetraethylammonium chloride raised blood pressure significantly in septic WT mice, but had no effect on survival. However, following cecal ligation and puncture, a significantly reduced survival was seen in both BKα mice and (high-dose) TEA-treated WT mice compared with untreated WT animals. In conclusion, the BK channel does not appear to be integral to sepsis-induced hypotension but does affect survival through other mechanisms. The pressor effect of TEA may be related to effects on other potassium channels.