ObjectiveImmune paresis in patients with acute-on-chronic liver failure (ACLF) accounts for infection susceptibility and increased mortality. Immunosuppressive mononuclear CD14+HLA-DR− myeloid-derived suppressor cells (M-MDSCs) have recently been identified to quell antimicrobial responses in immune-mediated diseases. We sought to delineate the function and derivation of M-MDSC in patients with ACLF, and explore potential targets to augment antimicrobial responses.DesignPatients with ACLF (n=41) were compared with healthy subjects (n=25) and patients with cirrhosis (n=22) or acute liver failure (n=30). CD14+CD15−CD11b+HLA-DR− cells were identified as per definition of M-MDSC and detailed immunophenotypic analyses were performed. Suppression of T cell activation was assessed by mixed lymphocyte reaction. Assessment of innate immune function included cytokine expression in response to Toll-like receptor (TLR-2, TLR-4 and TLR-9) stimulation and phagocytosis assays using flow cytometry and live cell imaging-based techniques.ResultsCirculating CD14+CD15−CD11b+HLA-DR− M-MDSCs were markedly expanded in patients with ACLF (55% of CD14+ cells). M-MDSC displayed immunosuppressive properties, significantly decreasing T cell proliferation (p=0.01), producing less tumour necrosis factor-alpha/interleukin-6 in response to TLR stimulation (all p<0.01), and reduced bacterial uptake of Escherichia coli (p<0.001). Persistently low expression of HLA-DR during disease evolution was linked to secondary infection and 28-day mortality. Recurrent TLR-2 and TLR-4 stimulation expanded M-MDSC in vitro. By contrast, TLR-3 agonism reconstituted HLA-DR expression and innate immune function ex vivo.ConclusionImmunosuppressive CD14+HLA-DR− M-MDSCs are expanded in patients with ACLF. They were depicted by suppressing T cell function, attenuated antimicrobial innate immune responses, linked to secondary infection, disease severity and prognosis. TLR-3 agonism reversed M-MDSC expansion and innate immune function and merits further evaluation as potential immunotherapeutic agent.

Objective To evaluate the role of hepatocellular and extrahepatic apoptosis during the evolution of Acetaminophen-induced acute liver failure (AALF). Design & Setting A prospective observational study in two tertiary liver transplant units. Patients 88 patients with AALF were recruited. Control groups included patients with non-AALF (n=13), non-hepatic multi-organ failure (MOF, n=28), chronic liver disease (CLD, n=19) and healthy controls (HC, n=11). Measurements Total and caspase-cleaved cytokeratin 18 (M65 and M30 measured on admission and sequentially on day 3, 7 and 10 following admission. Levels were also determined from hepatic, portal vein and systemic arterial blood in seven patients undergoing transplantation. Protein arrays of liver homogenates from AALF patients were assessed for apoptosis-associated proteins and histological assessment of liver tissue was performed. Main Results Admission M30 levels were significantly elevated in AALF and NALF patients compared to MOF, CLD and healthy controls. Admission M30 levels correlated with outcome with AUROC of 0.755 (0.639-0.885, p<0.001). Peak levels in ALF patients were seen on admission then fell significantly but did not normalize over ten days. A negative gradient of M30 from the portal to hepatic vein was demonstrated in AALF patients (p=0.042) at the time of liver transplant. Analysis of protein array data demonstrated lower apoptosis-associated protein and higher catalase concentrations in AALF liver compared to controls (p<0.05). Explant histological analysis revealed evidence of cellular proliferation with an absence of histological evidence of apoptosis. Conclusions Hepatocellular apoptosis occurs in the early phases of human AALF, peaking on day 1 of hospital admission and correlates strongly with poor outcome. Hepatic regenerative/tissue repair responses prevail during the later stages of ALF where elevated levels of M30 are likely to reflect epithelial cell death in extra-hepatic organs.

Background and aims Molecular analyses of biliary brushings using microarray and qPCR have the potential to provide valuable information on the biology of biliary diseases. Microarray analysis of biliary strictures has rarely been applied to endoscopic biliary brushings. Methods Biliary brushings were obtained from patients with benign and malignant biliary disease at the time of ERCP. Microarray analysis of mRNA isolated using brushings from ten patients was validated for a selection of genes by qPCR using the same source mRNA and a second fresh set of nine biliary brushings as well as surgical resection tissue. Cultured cholangiocytes were used to assess the impact of bile or x-ray contrast solution on RNA quality. Results RNA was of variable quantity (100–1500 ng) and poor quality (Agilent RNA Integrity Number (RIN) <5, estimated to be fragments 100 to 600 base pairs long). Reliable qPCR results required primer pairs designed to produce amplicons <130bp. Differential gene expression by microarray analysis identified 1,140 up-regulated genes and 1,001 down-regulated genes between benign and malignant biliary strictures. The trends in a selection of 45 upregulated genes, including various HOX genes, collagens, PVT1, MUC4, MUC5AC and LEF1, were validated by qPCR using RNA from biliary strictures with a moderate to strong correlation coefficient between microarray and qPCR (r=0.41 to r=0.57). Immunohistochemistry of surgical resection tissues (n=23) showed elevated CD9, SERPINA3 and PNMA2 protein expression in cancer samples. Conclusions RNA isolated from biliary brushings, is suitable for molecular analysis of biliary diseases using qPCR and microarray.