Imaging features for the prediction of clinical endpoints in chronic liver disease: a scoping review protocol.

INTRODUCTION: Chronic liver disease is a growing cause of morbidity and mortality in the UK. Acute presentation with advanced disease is common and prioritisation of resources to those at highest risk at earlier disease stages is essential to improving patient outcomes. Existing prognostic tools are of limited accuracy and to date no imaging-based tools are used in clinical practice, despite multiple anatomical imaging features that worsen with disease severity.In this paper, we outline our scoping review protocol that aims to provide an overview of existing prognostic factors and models that link anatomical imaging features with clinical endpoints in chronic liver disease. This will provide a summary of the number, type and methods used by existing imaging feature-based prognostic studies and indicate if there are sufficient studies to justify future systematic reviews. METHODS AND ANALYSIS: The protocol was developed in accordance with existing scoping review guidelines. Searches of MEDLINE and Embase will be conducted using titles, abstracts and Medical Subject Headings restricted to publications after 1980 to ensure imaging method relevance on OvidSP. Initial screening will be undertaken by two independent reviewers. Full-text data extraction will be undertaken by three pretrained reviewers who have participated in a group data extraction session to ensure reviewer consensus and reduce inter-rater variability. Where needed, data extraction queries will be resolved by reviewer team discussion. Reporting of results will be based on grouping of related factors and their cumulative frequencies. Prognostic anatomical imaging features and clinical endpoints will be reported using descriptive statistics to summarise the number of studies, study characteristics and the statistical methods used. ETHICS AND DISSEMINATION: Ethical approval is not required as this study is based on previously published work. Findings will be disseminated by peer-reviewed publication and/or conference presentations.

Haemodynamic changes in cirrhosis following terlipressin and induction of sepsis-a preclinical study using caval subtraction phase-contrast and cardiac MRI.

OBJECTIVES: Effects of liver disease on portal venous (PV), hepatic arterial (HA), total liver blood flow (TLBF), and cardiac function are poorly understood. Terlipressin modulates PV flow but effects on HA, TLBF, and sepsis/acute-on-chronic liver failure (ACLF)-induced haemodynamic changes are poorly characterised. In this study, we investigated the effects of terlipressin and sepsis/ACLF on hepatic haemodynamics and cardiac function in a rodent cirrhosis model using caval subtraction phase-contrast (PC) MRI and cardiac cine MRI. METHODS: Sprague-Dawley rats (n = 18 bile duct-ligated (BDL), n = 16 sham surgery controls) underwent caval subtraction PCMRI to estimate TLBF and HA flow and short-axis cardiac cine MRI for systolic function at baseline, following terlipressin and lipopolysaccharide (LPS) infusion, to model ACLF. RESULTS: All baseline hepatic haemodynamic/cardiac systolic function parameters (except heart rate and LV mass) were significantly different in BDL rats. Following terlipressin, baseline PV flow (sham 181.4 ± 12.1 ml/min/100 g; BDL 68.5 ± 10.1 ml/min/100 g) reduced (sham - 90.3 ± 11.1 ml/min/100 g, p < 0.0001; BDL - 31.0 ± 8.0 ml/min/100 g, p = 0.02), sham baseline HA flow (33.0 ± 11.3 ml/min/100 g) increased (+ 92.8 ± 21.3 ml/min/100 g, p = 0.0003), but BDL baseline HA flow (83.8 ml/min/100 g) decreased (- 34.4 ± 7.5 ml/min/100 g, p = 0.11). Sham baseline TLBF (214.3 ± 16.7 ml/min/100 g) was maintained (+ 2.5 ± 14.0 ml/min/100 g, p > 0.99) but BDL baseline TLBF (152.3 ± 18.7 ml/min/100 g) declined (- 65.5 ± 8.5 ml/min/100 g, p = 0.0004). Following LPS, there were significant differences between cohort and change in HA fraction (p = 0.03) and TLBF (p = 0.01) with BDL baseline HA fraction (46.2 ± 4.6%) reducing (- 20.9 ± 7.5%, p = 0.03) but sham baseline HA fraction (38.2 ± 2.0%) remaining unchanged (+ 2.9 ± 6.1%, p > 0.99). Animal cohort and change in systolic function interactions were significant only for heart rate (p = 0.01) and end-diastolic volume (p = 0.03). CONCLUSIONS: Caval subtraction PCMRI and cardiac MRI in a rodent model of cirrhosis demonstrate significant baseline hepatic haemodynamic/cardiac differences, failure of the HA buffer response post-terlipressin and an altered HA fraction response in sepsis, informing potential translation to ACLF patients. KEY POINTS: Caval subtraction phase-contrast and cardiac MRI demonstrate: • Significant differences between cirrhotic/non-cirrhotic rodent hepatic blood flow and cardiac systolic function at baseline. • Failure of the hepatic arterial buffer response in cirrhotic rodents in response to terlipressin. • Reductions in hepatic arterial flow fraction in the setting of acute-on-chronic liver failure.

The Macrophage Activation Marker Soluble CD163 is Associated With Early Allograft Dysfunction After Liver Transplantation.

BACKGROUND/OBJECTIVES: Soluble CD163 (sCD163), a macrophage activation marker, is upregulated in conditions of macrophage proliferation and activation. Elevated sCD163 levels have been associated with liver disease severity and progression. During liver transplantation, the implanted liver is exposed to ischaemia and reperfusion injury, resulting in an acute inflammatory response and macrophage activation. The relationship between sCD163 levels during liver transplantation and the development of early allograft dysfunction (EAD) has not been investigated. METHODS: We included 27 cirrhosis patients (age 55 [range 32-72] years, 23 men) on the waiting list for liver transplantation. Alcohol consumption and viral hepatitis were the most frequent causes for cirrhosis. Patients were characterised by standard biochemical analysis and based on clinical disease severity scores. Information about donor, graft and course of the liver transplantation was recorded. sCD163 levels were measured at the time of liver transplantation before surgery, 2 h after reperfusion, and then at 24 h after transplantation. RESULTS: We observed above-normal sCD163 levels at baseline (5.9 mg/L [4.7-8.8]). Two hours after reperfusion, sCD163 levels increased significantly from baseline (8.4 mg/L [7.4-10.9]; P < 0.01). Twenty-four hours after transplantation, sCD163 levels were significantly reduced compared with baseline (3.7 mg/L [2.9-5.5]; P < 0.01). However, in patients with EAD (n = 16), sCD163 levels were increased compared with patients without EAD (4.1 [3.2-7.4] vs. 3.1 [2.8-3.8] mg/L; P = 0.03). CONCLUSIONS: We observed elevated sCD163 levels in patients with EAD after liver transplantation, confirming macrophage activation to play a role in EAD. Thus, sCD163 may be used as an early marker for EAD after liver transplantation, but larger studies are warranted to validate these findings.

Prognostic Role of Ammonia in Patients With Cirrhosis.

Ammonia is thought to be central to the pathogenesis of hepatic encephalopathy (HE), but its prognostic role in patients with cirrhosis and acute decompensation is unknown. The aims of this study were to determine the relationship between ammonia levels and severity of HE and its association with organ dysfunction and short-term mortality. We identified 498 patients from two institutions as part of prospective observational studies in patients with cirrhosis. Plasma ammonia levels were measured on admission and Chronic Liver Failure-Sequential Organ Failure Assessment criteria were used to determine the presence of organ failures. The 28-day patient survival was determined. Receiver operating characteristic analysis was used to identify the cutoff points for ammonia values, and multivariable analysis was performed using the Cox proportional hazard regression model. The 28-day mortality was 43.4%. Plasma ammonia correlated with severity of HE (P < 0.001), was significantly higher in nonsurvivors (93 [73-121] versus 67 [55-89] µmol/L, P < 0.001), and was an independent predictor of 28-day mortality (hazard ratio, 1.009, P < 0.001). An ammonia level of 79.5 µmol/L had sensitivity of 68.1% and specificity of 67.4% for predicting 28-day mortality. An ammonia level of ≥79.5 µmol/L was associated with a higher frequency of organ failures (liver [P = 0.004], coagulation [P < 0.001], kidney [P = 0.004], and respiratory [P < 0.001]). Lack of improvement in baseline ammonia at day 5 was associated with high mortality (70.6%). Conclusion: Ammonia level correlates with not only the severity of HE but also the failure of other organs and is an independent risk factor for mortality; lack of improvement in ammonia level is associated with high risk of death, making it an important biomarker and a therapeutic target.

Statins: Old drugs as new therapy for liver diseases?

In addition to lowering cholesterol levels, statins have pleiotropic effects, particularly anti-inflammatory, antiangiogenic, and antifibrotic, that may be beneficial in some chronic inflammatory conditions. Statins have only recently been investigated as a potential treatment option in chronic liver diseases because of concerns related to their safety in patients with impaired liver function. A number of experimental studies in animal models of liver diseases have shown that statins decrease hepatic inflammation, fibrogenesis and portal pressure. In addition, retrospective cohort studies in large populations of patients with cirrhosis and pre-cirrhotic conditions have shown that treatment with statins, with the purpose of decreasing high cholesterol levels, was associated with a reduced risk of disease progression, hepatic decompensation, hepatocellular carcinoma development, and death. These beneficial effects persisted after adjustment for disease severity and other potential confounders. Finally, a few randomised controlled trials have shown that treatment with simvastatin decreases portal pressure (two studies) and mortality (one study). Statin treatment was generally well tolerated but a few patients developed severe side effects, particularly rhabdomyolysis. Despite these promising beneficial effects, further randomised controlled trials in large series of patients with hard clinical endpoints should be performed before statins can be recommended for use in clinical practice.

Ammonia: A novel target for the treatment of non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver diseases ranging from steatosis, through non-alcoholic steatohepatitis (NASH) to cirrhosis. The development of fibrosis is the most important factor contributing to NASH-associated morbidity and mortality. Hepatic stellate cells (HSCs) are responsible for extracellular matrix deposition in conditions of frank hepatocellular injury and are key cells involved in the development of fibrosis. In experimental models and patients with NASH, urea cycle enzyme gene and protein expression is reduced resulting in functional reduction in the in vivo capacity for ureagenesis and subsequent hyperammonemia at a pre-cirrhotic stage. Ammonia has been shown to activate HSCs in vivo and in vitro. Hyperammonemia in the context of NASH may therefore favour the progression of fibrosis and the disease. We therefore hypothesise that ammonia is a potential target for prevention of fibrosis progression of patients with NASH.

FXR agonism protects against liver injury in a rat model of intestinal failure-associated liver disease.

BACKGROUND: Intestinal failure-associated liver disease (IFALD) is a clinical challenge. The pathophysiol-ogy is multifactorial and remains poorly understood. Disturbed recirculation of bile salts, e.g. due to loss of bile via an enterocutaneous fistula, is considered a major contributing factor. We hypothesize that impaired signaling via the bile salt receptor FXR underlies the development of IFALD. The aim of this study was to investigate whether activation of FXR improves liver homeostasis during chronic loss of bile in rats. METHODS: To study consequences of chronic loss of bile, rats underwent external biliary drainage (EBD) or sham surgery for seven days, and the prophylactic potential of the FXR agonist INT-747 was assessed. RESULTS: EBD for 7 days resulted in liver test abnormalities and histological liver damage. Expression of the intestinal FXR target gene Fgf15 was undetectable after EBD, and this was accompanied by an anticipated increase in hepatic Cyp7a1 expression, indicating increased bile salt synthesis. Treatment with INT-747 improved serum biochemistry, reduced loss of bile fluid in drained rats and prevented development of drainage-associated histological liver injury. CONCLUSIONS: EBD results in extensive hepatobiliary injury and cholestasis. These data suggest that FXR activation might be a novel therapy in preventing liver dysfunction in patients with intestinal failure. RELEVANCE FOR PATIENTS: This study demonstrates that chronic loss of bile causes liver injury in rats. Abro-gated recycling of bile salts impairing of enterohepatic bile salt/FXR signaling underlies these pathological changes, as administration of FXR agonist INT747 prevents biliary drainage-induced liver damage. Phar-macological activation of FXR might be a therapeutic strategy to treat disorders accompanied by a per-turbed enterohepatic circulation such as intestinal failure-associated liver disease.

Improved hepatic arterial fraction estimation using cardiac output correction of arterial input functions for liver DCE MRI.

Liver dynamic contrast enhanced (DCE) MRI pharmacokinetic modelling could be useful in the assessment of diffuse liver disease and focal liver lesions, but is compromised by errors in arterial input function (AIF) sampling. In this study, we apply cardiac output correction to arterial input functions (AIFs) for liver DCE MRI and investigate the effect on dual-input single compartment hepatic perfusion parameter estimation and reproducibility. Thirteen healthy volunteers (28.7 ± 1.94 years, seven males) underwent liver DCE MRI and cardiac output measurement using aortic root phase contrast MRI (PCMRI), with reproducibility (n = 9) measured at 7 d. Cardiac output AIF correction was undertaken by constraining the first pass AIF enhancement curve using the indicator-dilution principle. Hepatic perfusion parameters with and without cardiac output AIF correction were compared and 7 d reproducibility assessed. Differences between cardiac output corrected and uncorrected liver DCE MRI portal venous (PV) perfusion (p = 0.066), total liver blood flow (TLBF) (p = 0.101), hepatic arterial (HA) fraction (p = 0.895), mean transit time (MTT) (p = 0.646), distribution volume (DV) (p = 0.890) were not significantly different. Seven day corrected HA fraction reproducibility was improved (mean difference 0.3%, Bland-Altman 95% limits-of-agreement (BA95%LoA) ±27.9%, coefficient of variation (CoV) 61.4% versus 9.3%, ±35.5%, 81.7% respectively without correction). Seven day uncorrected PV perfusion was also improved (mean difference 9.3 ml min-1/100 g, BA95%LoA ±506.1 ml min-1/100 g, CoV 64.1% versus 0.9 ml min-1/100 g, ±562.8 ml min-1/100 g, 65.1% respectively with correction) as was uncorrected TLBF (mean difference 43.8 ml min-1/100 g, BA95%LoA ±586.7 ml min-1/ 100 g, CoV 58.3% versus 13.3 ml min-1/100 g, ±661.5 ml min-1/100 g, 60.9% respectively with correction). Reproducibility of uncorrected MTT was similar (uncorrected mean difference 2.4 s, BA95%LoA ±26.7 s, CoV 60.8% uncorrected versus 3.7 s, ±27.8 s, 62.0% respectively with correction), as was and DV (uncorrected mean difference 14.1%, BA95%LoA ±48.2%, CoV 24.7% versus 10.3%, ±46.0%, 23.9% respectively with correction). Cardiac output AIF correction does not significantly affect the estimation of hepatic perfusion parameters but demonstrates improvements in normal volunteer 7 d HA fraction reproducibility, but deterioration in PV perfusion and TLBF reproducibility. Improved HA fraction reproducibility maybe important as arterialisation of liver perfusion is increased in chronic liver disease and within malignant liver lesions.

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.

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

BACKGROUND & AIMS: 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. METHODS: Tissue microarrays containing liver samples from patients with cirrhosis, viral hepatitis and HCC were examined for expression of TLR7 and TLR9 and the data obtained was validated in liver specimens from the hospital archives. Proliferation of human HCC cell lines was studied following stimulation of TLR7 and TLR9 using agonists (imiquimod and CpG-ODN respectively) and inhibition with a specific antagonist (IRS-954) or chloroquine. The effect of these interventions was confirmed in a xenograft model and diethylnitrosamine (DEN)/nitrosomorpholine (NMOR)-induced model of HCC. RESULTS: TLR7 and TLR9 expression was up-regulated in human HCC tissue. Proliferation of HuH7 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 and TLR9 inhibition using IRS-954 or chloroquine significantly reduced HuH7 cell proliferation in vitro and inhibited tumour growth in the mouse xenograft model. HCC development in the DEN/NMOR rat model was also significantly inhibited by chloroquine (P < 0.001). CONCLUSION: The data suggest that inhibiting TLR7 and TLR9 with IRS-954 or chloroquine could potentially be used as a novel therapeutic approach for preventing HCC development and/or progression in susceptible patients.

Hepatic dimethylarginine-dimethylaminohydrolase1 is reduced in cirrhosis and is a target for therapy in portal hypertension.

BACKGROUND & AIMS: Portal hypertension is characterized by reduced hepatic eNOS activity. Asymmetric-dimethylarginine (ADMA), an eNOS inhibitor, is elevated in cirrhosis and correlates with the severity of portal hypertension. Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is the key enzyme metabolizing hepatic ADMA. This study characterized DDAH-1 in cirrhosis, and explored hepatic DDAH-1 reconstitution through farnesoid X receptor (FXR) agonism and DDAH-1 gene therapy. METHODS: DDAH-1 immunohistochemistry was conducted on human cirrhosis and healthy liver tissue. Subsequently, sham-operated or bile-duct-ligated (BDL) cirrhosis rats were treated with the FXR agonist obeticholic acid (OA, 5 mg/kg) or vehicle for 5 days. Further, animals underwent hydrodynamic injection with DDAH-1-expressing plasmid or saline control, which resulted in the following groups: sham+saline, BDL+saline, BDL+DDAH-1-plasmid. Portal pressure (PP) measurements were performed. Plasma ALT was measured by COBAS INTEGRA, DDAH-1 expression by qPCR and Western blot, eNOS activity by radiometric assay. RESULTS: Immunohistochemistry and Western-blotting confirmed hepatic DDAH-1 was restricted to hepatocytes, and expression decreased significantly in cirrhosis. In BDL rats, reduced DDAH-1 expression was associated with elevated hepatic ADMA, reduced eNOS activity and high PP. OA treatment significantly increased DDAH-1 expression, reduced hepatic tissue ADMA, and increased liver NO generation. PP was significantly reduced in BDL+OA vs. BDL+vehicle (8±1 vs. 13.5±0.6 mmHg; p<0.01) with no change in the mean arterial pressure (MAP). Similarly, DDAH-1 hydrodynamic injection significantly increased hepatic DDAH-1 gene and protein expression, and significantly reduced PP in BDL+DDAH-1 vs. BDL+saline (p<0.01). CONCLUSIONS: This study demonstrates DDAH-1 is a specific molecular target for portal pressure reduction, through actions on ADMA-mediated regulation of eNOS activity. Our data support translational studies, targeting DDAH-1 in cirrhosis and portal hypertension.

A histologic scoring system for prognosis of patients with alcoholic hepatitis.

BACKGROUND & AIMS: There is no histologic classification system to determine prognoses of patients with alcoholic hepatitis (AH). We identified histologic features associated with disease severity and created a histologic scoring system to predict short-term (90-day) mortality. METHODS: We analyzed data from 121 patients admitted to the Liver Unit (Hospital Clinic, Barcelona, Spain) from January 2000 to January 2008 with features of AH and developed a histologic scoring system to determine the risk of death using logistic regression. The system was tested and updated in a test set of 96 patients from 5 academic centers in the United States and Europe, and a semiquantitative scoring system called the Alcoholic Hepatitis Histologic Score (AHHS) was developed. The system was validated in an independent set of 109 patients. Interobserver agreement was evaluated by weighted κ statistical analysis. RESULTS: The degree of fibrosis, degree of neutrophil infiltration, type of bilirubinostasis, and presence of megamitochondria were independently associated with 90-day mortality. We used these 4 parameters to develop the AHHS to identify patients with a low (0-3 points), moderate (4-5 points), or high (6-9 points) risk of death within 90 days (3%, 19%, and 51%, respectively; P < .0001). The AHHS estimated 90-day mortality in the training and test sets with an area under the receiver operating characteristic value of 0.77 (95% confidence interval, 0.71-0.83). Interrater agreement values were 0.65 for fibrosis, 0.86 for bilirubinostasis, 0.60 for neutrophil infiltration, and 0.46 for megamitochondria. Interestingly, the type of bilirubinostasis predicted the development of bacterial infections. CONCLUSIONS: We identified histologic features associated with the severity of AH and developed a patient classification system that might be used in clinical decision making.

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.

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.

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.

Association between hepatic histopathologic lesions and clinical findings in dogs undergoing surgical attenuation of a congenital portosystemic shunt: 38 cases (2000-2004).

OBJECTIVE: To review hepatic histopathologic lesions in dogs undergoing surgical attenuation of a congenital portosystemic shunt (CPSS) in relation to clinical findings and tolerance of complete surgical attenuation. DESIGN: Retrospective case series. ANIMALS: 38 dogs that underwent surgical attenuation of a CPSS. PROCEDURES: Hepatic histologic examination findings and medical records of dogs undergoing surgical attenuation of a single CPSS between August 2000 and July 2004 were reviewed. Liver biopsy specimens were obtained from 38 dogs during surgery prior to complete (n = 16) or partial (22) attenuation of a CPSS and from 13 of the same dogs a median of 3 months following surgical attenuation. RESULTS: Portal tracts were inadequate for interpretation in 2 liver biopsy specimens. Liver biopsy specimens obtained prior to surgical attenuation of a CPSS had a lack of identifiable portal veins (13/36 dogs), hepatic arteriolar proliferation (25/36), ductular reaction (5/36), steatosis (16/38), and iron accumulation (32/38). Lack of identifiable portal veins on histologic examination was associated with increased hepatic arteriolar proliferation, decreased tolerance to complete surgical CPSS attenuation, and decreased opacification of intrahepatic portal vessels on portovenography. Ductular reaction was always associated with failure to tolerate complete surgical attenuation of a CPSS. Surgical CPSS attenuation resulted in significant clinical, serum biochemical, and portovenographic changes indicative of improved liver function, but only subtle changes in hepatic histologic examination findings. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs without identifiable intrahepatic portal veins that had a ductular reaction on hepatic histologic examination were less likely to tolerate complete attenuation of a CPSS.

The role of liver biopsy in the diagnosis and prognosis of patients with acute deterioration of alcoholic cirrhosis.

BACKGROUND & AIMS: The aim of this study was to systematically assess the diagnostic and prognostic value of early liver biopsy in patients who require hospital admission with acute deterioration of alcoholic cirrhosis. METHODS: Sixty-eight patients with acute deterioration of alcoholic cirrhosis underwent a liver biopsy within 7 days and the biopsies were processed using routine stains and K8/18 immunohistochemistry to characterize balloon degeneration. The biopsies were scored by two independent histopathologists using pre-defined criteria. The patients were managed according to institutional protocols and followed until the time of hospital discharge or death. RESULTS: With use of K8/18 immunohistochemistry, very high concordance rate for the diagnosis of balloon degeneration was reached (r = 0.7; p = 0.0001). The presence of a systemic inflammatory response (SIRS) suggestive of acute alcoholic steatohepatitis (ASH), predicts severe ASH histologically in only 50% patients. Moreover, in 41% of SIRS negative patients who were thought not to have ASH, a diagnosis of ASH was subsequently confirmed on histological grading. Patients that have SIRS criteria but no evidence of histological ASH are more likely to develop infection which may be indicated by the severity of canalicular cholestasis. Nineteen patients died during follow up. Patients manifesting ASH on biopsy who were also SIRS positive, had a significantly greater risk of mortality compared to those that were SIRS positive but ASH negative (p < 0.01) and those that were SIRS negative (p < 0.0001). CONCLUSIONS: The use of K8/18 immunostaining allows grading of the severity of alcoholic steatohepatitis. Early liver biopsy in these patients presenting with acute deterioration of cirrhosis is safe and provides important diagnostic and prognostic information.

Effect of probiotic treatment on deranged neutrophil function and cytokine responses in patients with compensated alcoholic cirrhosis.

BACKGROUND/AIM: Endotoxaemia contributes to neutrophil dysfunction, infection risk and mortality in patients with alcoholic cirrhosis. As probiotics may decrease Gram-negative gut organisms, we hypothesised that probiotic treatment would restore neutrophil function. METHODS: In an open-label study, patients with alcoholic cirrhosis (n=12) received Lactobacillus casei Shirota (6.5 x 10(9)) 3 times daily for 4 weeks. Data were compared to healthy controls (n=13) and cirrhotic patients (n=8) who did not receive probiotics. Neutrophil oxidative burst, phagocytosis, toll-like-receptor (TLR) expression, plasma cytokines and ex vivo endotoxin-stimulated cytokine production were measured. RESULTS: Baseline neutrophil phagocytic capacity in patients was significantly lower compared to healthy controls (73% versus 98%, p<0.05), but normalised at the end of the study (n=10, 100%, p<0.05). No improvement was seen in disease controls. Soluble TNF-receptor (sTNFR)-1 and-2 and interleukin (IL)10 were significantly elevated in patients' plasma but did not change during the study. Ex vivo endotoxin-stimulated levels of sTNFR1, sTNFR2 and IL10 were significantly lower at the end of the study (p<0.05). TLR2, 4 and 9 were overexpressed in patients. TLR4 expression normalised by the end of the study. CONCLUSIONS: Our data provide a proof-of-concept that probiotics restore neutrophil phagocytic capacity in cirrhosis, possibly by changing IL10 secretion and TLR4 expression, warranting larger randomised controlled and mechanistic studies.