E. Triantafyllou

High-volume plasma exchange in patients with acute liver failure: An open randomised controlled trial.

BACKGROUND & AIMSnAcute liver failure (ALF) often results in cardiovascular instability, renal failure, brain oedema and death either due to irreversible shock, cerebral herniation or development of multiple organ failure. High-volume plasma exchange (HVP), defined as exchange of 8-12 or 15% of ideal body weight with fresh frozen plasma in case series improves systemic, cerebral and splanchnic parameters.nnnMETHODSnIn this prospective, randomised, controlled, multicentre trial we randomly assigned 182 patients with ALF to receive either standard medical therapy (SMT; 90 patients) or SMT plus HVP for three days (92 patients). The baseline characteristics of the groups were similar. The primary endpoint was liver transplantation-free survival during hospital stay. Secondary-endpoints included survival after liver transplantation with or without HVP with intention-to-treat analysis. A proof-of-principle study evaluating the effect of HVP on the immune cell function was also undertaken.nnnRESULTSnFor the entire patient population, overall hospital survival was 58.7% for patients treated with HVP vs. 47.8% for the control group (hazard ratio (HR), with stratification for liver transplantation: 0.56; 95% confidence interval (CI), 0.36-0.86; p=0.0083). HVP prior to transplantation did not improve survival compared with patients who received SMT alone (CI 0.37 to 3.98; p=0.75). The incidence of severe adverse events was similar in the two groups. Systemic inflammatory response syndrome (SIRS) and sequential organ failure assessment (SOFA) scores fell in the treated group compared to control group, over the study period (p<0.001).nnnCONCLUSIONSnTreatment with HVP improves outcome in patients with ALF by increasing liver transplant-free survival. This is attributable to attenuation of innate immune activation and amelioration of multi-organ dysfunction.

Secretory leukocyte protease inhibitor: a pivotal mediator of anti-inflammatory responses in acetaminophen-induced acute liver failure.

Acetaminophen‐induced acute liver failure (AALF) is characterized both by activation of innate immune responses and susceptibility to sepsis. Circulating monocytes and hepatic macrophages are central mediators of inflammatory responses and tissue repair processes during human AALF. Secretory leukocyte protease inhibitor (SLPI) modulates monocyte/macrophage function through inhibition of nuclear factor kappa B (NF‐κB) signaling. The aims of this study were to establish the role of SLPI in AALF. Circulating levels of SLPI, monocyte cluster of differentiation 163 (CD163), human leukocyte antigen‐DR (HLA‐DR), and lipopolysaccharide (LPS)‐stimulated levels of NF‐κBp65, tumor necrosis factor alpha (TNF‐α) and interleukin (IL)‐6 were determined in patients with AALF, chronic liver disease, and healthy controls. Immunohistochemistry and multispectral imaging of AALF explant tissue determined the cellular sources of SLPI and hepatic macrophage phenotype. The phenotype and function of monocytes and macrophages was determined following culture with recombinant human (rh)‐SLPI, liver homogenates, and plasma derived from AALF patients in the presence and absence of antihuman (α)SLPI. Hepatic and circulatory concentrations of SLPI were elevated in AALF and immunohistochemistry revealed SLPI expression in biliary epithelial cells and within hepatic macrophages (h‐mψ) in areas of necrosis. H‐mψ and circulating monocytes in AALF exhibited an anti‐inflammatory phenotype and functional characteristics; typified by reductions in NF‐κBp65, TNF‐α, and IL‐6 and preserved IL‐10 secretion following LPS challenge. Culture of healthy monocytes with AALF liver homogenates, plasma, or rhSLPI induced monocytes with strikingly similar anti‐inflammatory characteristics which were reversed by inhibiting the activity of SLPI. Conclusion: SLPI is a pivotal mediator of anti‐inflammatory responses in AALF through modulation of monocyte/macrophage function, which may account for the susceptibility to sepsis in AALF. (Hepatology 2014;59:1564‐1576)

Multivariate metabotyping of plasma predicts survival in patients with decompensated cirrhosis.

Graphical abstract

Increased Expression of Cytotoxic T-Lymphocyte−Associated Protein 4 by T Cells, Induced by B7 in Sera, Reduces Adaptive Immunity in Patients With Acute Liver Failure

Background & Aims Patients with acute liver failure (ALF) have defects in innate immune responses to microbes (immune paresis) and are susceptible to sepsis. Cytotoxic T-lymphocyte−associated protein 4 (CTLA4), which interacts with the membrane receptor B7 (also called CD80 and CD86), is a negative regulator of T-cell activation. We collected T cells from patients with ALF and investigated whether inhibitory signals down-regulate adaptive immune responses in patients with ALF. Methods We collected peripheral blood mononuclear cells from patients with ALF and controls from September 2013 through September 2015 (45 patients with ALF, 20 patients with acute-on-chronic liver failure, 15 patients with cirrhosis with no evidence of acute decompensation, 20 patients with septic shock but no cirrhosis or liver disease, and 20 healthy individuals). Circulating CD4+ T cells were isolated and analyzed by flow cytometry. CD4+ T cells were incubated with antigen, or agonist to CD3 and dendritic cells, with or without antibody against CTLA4; T-cell proliferation and protein expression were quantified. We measured levels of soluble B7 molecules in supernatants of isolated primary hepatocytes, hepatic sinusoidal endothelial cells, and biliary epithelial cells from healthy or diseased liver tissues. We also measured levels of soluble B7 serum samples from patients and controls, and mice with acetaminophen-induced liver injury using enzyme-linked immunosorbent assays. Results Peripheral blood samples from patients with ALF had a higher proportion of CD4+ CTLA4+ T cells than controls; patients with infections had the highest proportions. CD4+ T cells from patients with ALF had a reduced proliferative response to antigen or CD3 stimulation compared to cells from controls; incubation of CD4+ T cells from patients with ALF with an antibody against CTLA4 increased their proliferative response to antigen and to CD3 stimulation, to the same levels as cells from controls. CD4+ T cells from controls up-regulated expression of CTLA4 after 24−48 hours culture with sera from patients with ALF; these sera were found to have increased concentrations of soluble B7 compared to sera from controls. Necrotic human primary hepatocytes exposed to acetaminophen, but not hepatic sinusoidal endothelial cells and biliary epithelial cells from patients with ALF, secreted high levels of soluble B7. Sera from mice with acetaminophen-induced liver injury contained high levels of soluble B7 compared to sera from mice without liver injury. Plasma exchange reduced circulating levels of soluble B7 in patients with ALF and expression of CTLA4 on T cells. Conclusions Peripheral CD4+ T cells from patients with ALF have increased expression of CTLA4 compared to individuals without ALF; these cells have a reduced response to antigen and CD3 stimulation. We found sera of patients with ALF and from mice with liver injury to have high concentrations of soluble B7, which up-regulates CTLA4 expression by T cells and reduces their response to antigen. Plasma exchange reduces levels of B7 in sera from patients with ALF and might be used to restore antimicrobial responses to patients.

PTH-106 Plasma S100A8/A9: a novel mechanistic biomarker in innate immune activation in acute-on-chronic liver failure

Introduction Acute-on-chronic liver failure (ACLF) is driven by systemic inflammation but lacks reliable diagnostic or prognostic biomarkers. Circulating S100A8/A9 heterodimer (calprotectin) is secreted by activated myeloid cells to activate and propagate innate immune responses and organ dysfunction. This study aims to evaluate circulating levels of S100A8/A9 in ACLF and determine its effect on myeloid cell function. Methods Plasma S100A8/A9 concentration of 92 patients at admission was analysed using enzyme-linked immunosorbent assay (ELISA) in ACLF (n=62), cirrhosis without organ failure (n=28) and healthy control (n=30) groups. Baseline plasma cytokines were measured by multiplex immunoassay. Indices of disease severity and survival was evaluated with Kaplan Meier analysis. Phenotype (CD11b, HLA-DR, Mer tyrosine kinase [MerTK], CD163 and CD206) of healthy CD14 +monocytes cultured with S100A8/A9 in vitro for 24u2009hours at 0, 1000 and 2500u2009ng/ml was assessed using flow cytometry (n=6). Results Admission plasma S100A8/A9 was higher in ACLF (median 2000u2009ng/ml) compared with cirrhotics without organ failure (934.8u2009ng/ml p=0.007) and healthy control (963u2009ng/ml p=0.003) (figure 1). S100A8/A9 was higher in patients with systemic inflammatory response syndrome (SIRS) (p=0.045) and non-survivors (p=0.01). Baseline interleukin-1β (IL-1β) was elevated in ACLF compared to healthy (0.07 vs. 0.36u2009pg/ml p=0.009), correlating with S100A8/A9 concentration (r=0.508 p=0.01). Area under the receiver operating characteristic curve (AUROC) for S100A8/A9 to detect the presence of ACLF was 0.681 (p=0.009). For 90u2009day mortality in ACLF, AUROC was 0.694 (p=0.014) but highest for the CLIF-ACLF score (0.767, p=0.001). S100A8/A9>1406u2009ng/ml (sensitivity 0.73 specificity 0.61) was associated with decreased transplant-free survival (log rank p=0.02) (figure 2). S100A8/A9 predicted 90u2009day mortality (p=0.018) on univariate analysis, remaining significant in a multivariate logistic regression model (OR 1.0 p=0.04). In flow cytometric analysis, activated CD11b+HLA-DRhighMerTKlow myeloid cells (%) significantly increased (p=0.01, Friedman’s ANOVA) as S100A8/A9 concentration increased from 1000 to 2500u2009ng/ml with a trend to reduction in CD206 (p=0.13) (figure 3).Abstract PTH-106 Figure 1Abstract PTH-106 Figure 2Abstract PTH-106 Figure 3 Conclusions Plasma S100A8/A9 is significantly elevated in ACLF, correlating strongly with activation of pro-inflammatory mediators and indices of disease severity, extra-hepatic organ failure and outcome. Our in vitro data indicate that this mediator promotes inflammation and represents a novel therapeutic target in ACLF.

P138 SERONEGATIVE ACUTE LIVER FAILURE REPRESENTS A MACROPHAGE–T CELL ACTIVATION SYNDROME

P136 DEFICIENCY OF IL-33 SENSITIZES TO SEVERE LIVER INJURY INDUCED BY ConA BUT NOT BY CCl4 IN MICE M.I. Arshad, A. Filliol, V. Genet, C. Lucas-Clerc, J.-P. Girard, C. Piquet-Pellorce, M. Samson. Inserm U 1085, Institut de Recherche Sante Environnement & Travail (IRSET), Rennes, France; Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan; Service de Biochimie CHU Rennes, Universite de Rennes 1, Rennes, Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique (IPBS-CNRS), Universite de Toulouse, Toulouse, Structure Federative BioSit UMS 3480 CNRS-US 18 Inserm, Rennes, France E-mail: michel.samson@univ-rennes1.fr

519 CELLULAR TH1 AND TH2 IMMUNE RESPONSES TO ALCOHOL DEHYDROGENASE WITHIN THE LIVER OF PATIENTS WITH ALCOHOL-RELATED CIRRHOSIS DESPITE ABSTINENCE

Methods: Reference hepatologists were identified in Argentina, Uruguay, Chile, Brazil, Mexico, Peru, Venezuela and Bolivia, who in turn were commissioned to establish national specialist networks contributing to the project. Data would be obtained using the methodology in place at the Spanish DILI Registry. Identified cases would be remitted to the coordinating centre in Malaga for causality assessment and information storage. Results: Seventy-three DILI cases have been analyzed up to November 2012, having a mean age of 52 years (range 15–86) and female predominance (60%). The therapeutic groups most frequently implicated were NSAIDs (22%) including nimesulide (5 cases) and diclofenac (4 cases); antiinfectives (19%) including nitrofurantoin (3 cases), herbal remedies (12%) including Morinda citrifolia, Peumus boldus and Monascus purpureus; hormonal therapy (12%) including cyproterone acetate (4 cases); and central nervous system drugs (11%). Hepatocellular injury (50%) was the most common type of liver damage. Jaundice was seen in 71% of cases, 53% required hospitalization and 38% fulfilled Hy’s Law criteria (66% of hormonal therapy cases, 44% of herbal cases). Positive autoantibody titers were present in 29% of cases, mainly antinuclear. Six cases were autoimmune hepatitis DILI (8%) and five cases had experienced a second DILI episode (7%). Conclusions: This initial analysis demonstrates similar phenotypic characteristics as observed in registers outside Latin America with respect to type of injury and severity. However, female cases seem to predominate in Latin America. With regards to causative agents, elevated representation of NSAIDs, hormonal treatments and herbal remedies were evidenced. Funding: Agencia Espanola del Medicamento. Proyecto Excelencia SAS P10CTS-6470. iSAEC. CIBERehd is funded by ISCIII.