Explanted Liver Tissue Research Articles

Detection of H-Pylori in the Explanted Liver Tissue and the Enlarged Perihepatic Lymph Nodes of Cirrhotic Patients with Decompensated End-Stage Liver Disease Recruited for Liver Transplantation.

Helicobacter pylori bacteria colonize the gastric mucosa and contribute to the occurrence and development of gastrointestinal diseases. According to the WHO, H. pylori bacteria are considered class I carcinogen. To detect Helicobacter pylori organisms by IHC expression of anti-H. Pylori antibodies in the explanted liver tissue; and enlarged perihepatic lymph nodes of cirrhotic liver; to detect any relation between the presence of the organism and histopathological findings in the liver tissue. This retrospective cross-sectional study included forty cases of cirrhotic patients with decompensated end-stage liver, recruited for liver transplantation based on combined clinical, radiological, and histological data. Samples were immunohistochemically analyzed for anti-H-Pylori antibodies to detect Helicobacter pylori organisms in the explanted liver tissue and enlarged perihepatic lymph nodes. The presence of the organism was correlated with clinic-pathologic variables. Eighty-five percent (34 cases) and seventy percent (28 cases) of cases were positive for anti-H-Pylori antibodies in the liver and lymph nodal tissues, respectively. More than eighty percent (14 cases) and half of the studied cases (8 cases) showed dysplasia in liver tissue expressing anti-H-Pylori-antibody in the liver tissue and the lymph nodes, respectively. All HCC cases expressed anti-H-Pylori antibody in the liver tissue and the lymph nodes. The relation between anti-H-Pylori antibody expression in lymph nodes and the presence of dysplasia or HCC in liver tissue was statistically significant (p-value = 0.037 and p-value = 0.041 respectively). Our results conclude that there is a pathogenic role of extra-gastric H-Pylori colonization in lymph nodal tissue and in liver tissue, and it may be preventable by treating H. pylori, especially if treatment can be started very early.

NGS of brush cytology samples improves the detection of high-grade dysplasia and cholangiocarcinoma in patients with primary sclerosing cholangitis: A retrospective and prospective study.

Biliary dysplasia, a precursor of cholangiocarcinoma (CCA), is a common complication of primary sclerosing cholangitis. Patients with high-grade dysplasia (HGD) or early CCA who have received oncological treatment are candidates for liver transplantation. The preoperative diagnosis of CCA or HGD is challenging, and the sensitivity of biliary brush cytology (BC) is limited. By using next-generation sequencing (NGS), we retrospectively analyzed archived tissue samples (n=62) obtained from explanted liver tissue and CCA samples to identify oncogenic mutations that occur during primary sclerosing cholangitis carcinogenesis. BC samples were prospectively collected from patients with primary sclerosing cholangitis (n=97) referred for endoscopic retrograde cholangiography to measure the diagnostic utility of NGS combined with BC compared with traditional cytology alone. Mutations in KRAS, GNAS, FLT3, RNF43, TP53, ATRX, and SMAD4 were detected in archived CCA or HGD samples. KRAS, GNAS, TP53, CDKN2A, FBXW7, BRAF, and ATM mutations were detected in prospectively collected brush samples from patients with histologically verified CCA or HGD. One patient with low-grade dysplasia in the explanted liver had KRAS and GNAS mutations in brush sample. No mutations were observed in brush samples or archived tissues in liver transplantation cases without biliary neoplasia. While KRAS mutations are common in biliary neoplasms, they were also observed in patients without biliary neoplasia during surveillance. In summary, NGS of BC samples increased the sensitivity of detecting biliary neoplasia compared with traditional cytology. Performing NGS on BC samples may help diagnose HGD or early CCA, benefiting the timing of liver transplantation.

Abstract 1858: Enhanced delivery of anti PD-1 antibody to liver tumors in oncopig using pressure enabled drug delivery (PEDD) versus a systemic delivery

Abstract Background: Anti-PD-1 checkpoint inhibitors have been developed to mitigate immune suppression by reversing T cell exhaustion. Typically, checkpoint inhibitors are delivered systemically, which may be inadequate due to poor drug distribution and limited penetration into solid tumors due to high intra-tumoral pressure. In such tumors, interstitial fluid pressure and solid stress restricts blood flow and therapeutic agent distribution throughout the tumor stroma. Pressure enabled drug deliveryTM (PEDDTM) has been demonstrated to improve therapeutic delivery in pre-clinical models and in patients. PEDD has also been shown to reduce off-target tissue exposure to embolics while increasing intra-tumoral therapeutic concentration. We hypothesized that PEDD hepatic arterial infusion (HAI) using a TriNav device would enhance anti-PD-1 antibody delivery to porcine liver tumors when compared to systemic delivery. Methods: The study was conducted in transgenic pigs (oncopigs) 35-50kg in weight. Explanted liver tissue was treated to induce oncogene expression and re-implanted in the liver at 4 locations for tumor formation. A fixed dosage (1 mg/animal) of fluorescently labeled InVivoSIM anti-human PD-1 (AB_2894731) checkpoint inhibitor (CPI) was infused in 10ml of PBS at 2 ml/min. The TriNav was positioned within a HA branch supplying a hepatic lobe with a fully formed tumor for local-regional infusion. Systemic infusion was conducted through the femoral vein. Liver tissue was collected 1 hour following infusion and drug levels were quantified by nearIR fluorescence imaging on sequential 1 cm thick sections. NearIR images were overlaid on color images, tumor border delineated, and the mean fluorescent CPI signal was calculated in concentric rings relative to the tumor border. Results: A significant 2.6-to-5.4-fold increase in CPI signal intensity was observed from 15mm within the tumor to 30mm from the outer edge of tumor with PEDD using the TriNav device when compared to systemic delivery (p ≤ 0.05). PEDD delivery also resulted in significant 2.5-to-3.9-fold enrichment from 5mm within the tumor to 10mm surrounding the tumor (relative to normal tissue within the treated lobe, p ≤ 0.05). No significant difference in signal intensity was observed in peri-tumoral regions and a reduction in signal was observed in tissues 5mm to 15mm within the tumor relative to normal tissue (p ≤ 0.05) following systemic CPI infusion. Conclusions: This study demonstrates that HAI delivery of CPI into liver tumors in the oncopig via PEDD using a TriNav device resulted in superior delivery compared to systemic infusion. Importantly, PEDD significantly concentrated delivery of CPI into the tumors and within the rapidly growing margin of the tumors where target immune cells such as lymphocytes are present. Citation Format: David Benjamin Jaroch, Chandra C. Ghosh, Prajna Guha, Steven Katz, Bryan F. Cox, Thomas G. Hullinger. Enhanced delivery of anti PD-1 antibody to liver tumors in oncopig using pressure enabled drug delivery (PEDD) versus a systemic delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1858.

Hepatic Protein and Phosphoprotein Signatures of Alcohol-Associated Cirrhosis and Hepatitis

Alcohol-associated liver disease is a global health care burden, with alcohol-associated cirrhosis (AC) and alcohol-associated hepatitis (AH) being two clinical manifestations with poor prognosis. The limited efficacy of standard of care for AC and AH highlights a need for therapeutic targets and strategies. The current study aimed to address this need through the identification of hepatic proteome and phosphoproteome signatures of AC and AH. Proteomic and phosphoproteomic analyses were conducted on explant liver tissue (test cohort) and liver biopsies (validation cohort) from patients with AH. Changes in protein expression across AH severity and similarities and differences in AH and AC hepatic proteome were analyzed. Significant alterations in multiple proteins involved in various biological processes were observed in both AC and AH, including elevated expression of transcription factors involved in fibrogenesis (eg, Yes1-associated transcriptional regulator). Another finding was elevated levels of hepatic albumin (ALBU) concomitant with diminished ALBU phosphorylation, which may prevent ALBU release, leading to hypoalbuminemia. Furthermore, altered expression of proteins related to neutrophil function and chemotaxis, including elevated myeloperoxidase, cathelicidin antimicrobial peptide, complement C3, and complement C5 were observed in early AH, which declined at later stages. Finally, a loss in expression of mitochondria proteins, including enzymes responsible for the synthesis of cardiolipin was observed. The current study identified hepatic protein signatures of AC and AH as well as AH severity, which may facilitate the development of therapeutic strategies.

Aberrant hepatic trafficking of gut-derived T cells is not specific to primary sclerosing cholangitis.

The "gut homing" hypothesis suggests the pathogenesis of primary sclerosing cholangitis (PSC) is driven by aberrant hepatic expression of gut adhesion molecules and subsequent recruitment of gut-derived T cells to the liver. However, inconsistencies lie within this theory including an absence of investigations and comparisons with other chronic liver diseases (CLD). Here, we examine "the gut homing theory" in patients with PSC with associated inflammatory bowel disease (PSC-IBD) and across multiple inflammatory liver diseases. Expression of MAdCAM-1, CCL25, and E-Cadherin were assessed histologically and using RT-PCR on explanted liver tissue from patients with CLD undergoing OLT and in normal liver. Liver mononuclear cells were isolated from explanted tissue samples and the expression of gut homing integrins and cytokines on hepatic infiltrating gut-derived T cells was assessed using flow cytometry. Hepatic expression of MAdCAM-1, CCL25 and E-Cadherin was up-regulated in all CLDs compared with normal liver. There were no differences between disease groups. Frequencies of α4β7, αEβ7, CCR9, and GPR15 expressing hepatic T cells was increased in PSC-IBD, but also in CLD controls, compared with normal liver. β7 expressing hepatic T cells displayed an increased inflammatory phenotype compared with β7 negative cells, although this inflammatory cytokine profile was present in both the inflamed and normal liver. These findings refute the widely accepted "gut homing" hypothesis as the primary driver of PSC and indicate that aberrant hepatic recruitment of gut-derived T cells is not unique to PSC, but is a panetiological feature of CLD.

Examining the Hepatic Immune System in Children With Liver Disease With Fine Needle Aspiration.

Liver biopsy is the standard in diagnosing liver diseases. Yet, it provides little space to perform comprehensive immune profiling of the liver. Hence, we explored whether fine needle aspirates (FNAs) could be used to elucidate the hepatic immunity in children. We enrolled 74 children undergoing diagnostic (n = 17) or protocol biopsy (n = 57) following liver transplantation (LT). Matched blood and FNAs were obtained. Additionally, explant liver tissue was collected from children (n = 14) undergoing LT. Immune cells were isolated from peripheral blood, FNAs and explanted livers. Immune-phenotypical profiling was done by flow cytometry. Biopsied patients (58% female) were at a median age of 46 months (interquartile range [IQR]: 12-118) and LT patients (71% female) were 48 months (IQR: 21-134, P = 0.78) old. CD69+, a hallmark of tissue-resident immune cells was expressed in 1.3% of CD3+ T cells from blood being higher in FNA (20%) and tissue (49%, P < 0.001). CD4+ T-cell frequencies in tissue (13%) and FNAs (20%) were lower compared to blood (35%, P < 0.001) whereas CD8+ T cells in tissue (33.5%) and FNA (32%) were higher than in blood (25%, P < 0.01). Mucosal associated invariant T cells were enriched in liver tissue (8.8%) and in the FNA (4.4%) compared to blood (1.7%, P < 0.001). Whereas the percentage of total Tregs (CD4+CD25+FOXP3+CD127low/-) decreased, the proportion of activated Tregs (CD4+CD45RA-FOXP3high) increased in FNA and explant. Breg (CD19+CD20+CD24highCD38high) frequencies were similar in all groups. FNA is a practical method to sample the liver immune system collecting even small cell subsets such as regulatory T/B cells.

Quantification of Viral RNA in Multiple Pieces of Explant Liver Tissue Shows Distinct Focal Differences in Hepatitis B Infection.

Hepatitis B virus (HBV) DNA and RNA were quantified by digital PCR assays in 20–30 tissue pieces from each of 4 liver explants with cirrhosis caused by HBV. The within-patient variability of HBV RNA levels between pieces was up to a 1000-fold. Core RNA and S RNA levels were similar and correlated strongly when replication was high, supporting that transcription was from covalently closed circular DNA (cccDNA). By contrast, enhanced expression of S RNA relative to cccDNA and core RNA in patients with medium-high or low replication supports that HBV surface antigen (HBsAg) can be expressed mainly from integrated HBV DNA in such patients.

Deep sequencing of liver explant transcriptomes reveals extensive expression from integrated hepatitis B virus DNA.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Integration of HBV DNA into the human genome may contribute to oncogenesis and to the production of the hepatitis B surface antigen (HBsAg). Whether integrations contribute to HBsAg levels in the blood is poorly known. Here, we characterize the HBV RNA profile of HBV integrations in liver tissue in patients with chronic HBV infection, with or without concurrent hepatitis D infection, by transcriptome deep sequencing. Transcriptomes were determined in liver tissue by deep sequencing providing 200 million reads per sample. Integration points were identified using a bioinformatic pipeline. Explanted liver tissue from five patients with end-stage liver disease caused by HBV or HBV/HDV was studied along with publicly available transcriptomes from 21 patients. Almost all HBV RNA profiles were devoid of reads in the core and the 3' redundancy (nt 1830-1927) regions, and contained a large number of chimeric viral/human reads. Hence, HBV transcripts from integrated HBV DNA rather than from covalently closed circular HBV DNA (cccDNA) predominated in late-stage HBV infection, in particular in cases with hepatitis D virus co-infection. The findings support the suggestion that integrated HBV DNA can be a significant source of HBsAg in humans.

Instant Outcome Evaluation of Microwave Ablation With Subtraction CT in an In Vivo Porcine Model.

The aim of this study was to investigate whether the accuracy of multislice contrast-enhanced computed tomography (MS-CECT) may be improved by performing additional subtraction CT. Thirty-five microwave ablations were performed under CT guidance in 12 healthy and anesthetized pigs. Preablation and postablation MS-CECT scans were obtained in arterial and venous contrast phases. These scans were reconstructed and subtracted from each other. Lesion size was measured in a region of interest drawn around the ablation area. Computed tomography measurements were compared with standardized macroscopic images of explanted liver tissue, obtained immediately after ablation. Paired correlation and Bland-Altman analyses were performed for assessing agreement between modalities and ratings. The correlation between lesion size measured in CT and histology was very strong for subtracted images (r = 0.91; 95% confidence interval [CI], 0.8-0.96) and strong for standard MS-CECT images (r = 0.85; 95% CI, 0.68-0.93). Interrater agreement for all measurements was excellent (intraclass correlation coefficient, 0.99; 95% CI, 0.98-0.99 for subtraction and intraclass correlation coefficient, 0.99; 95% CI, 0.98-1.00 for MS-CECT). All differences were statistically significant (P < 0.05). Subtraction CT was superior to nonsubtracted MS-CECT in measurement of liver lesion size after microwave ablation in a porcine model, achieving a very strong correlation with pathologic measurement and a significantly lower overestimation of lesion size compared with MS-CECT.

Purinoceptor expression in hepatocellular virus (HCV)-induced and non-HCV hepatocellular carcinoma: an insight into the proviral role of the P2X4 receptor.

The basic idea behind this study was to discover the association and prevalence of purinoceptors in hepatitis C virus (HCV) and non-HCV hepatocellular carcinoma (HCC). Immunohistochemistry was performed to study the expression of P2X4 and P2X7 receptors on ex-planted liver tissue samples that were collected from HCC patients. Antibodies specific for the P2X4 and P2X7 receptors were used to target the specific receptors and secondary antibody was used with 3,3'-diaminobenzidine (DAB) detection system to visualize the color change in case of any positive expression There was a substantial increase in P2X4 receptor expression in HCV induced HCC as compared to non-HCV HCC. Surprisingly, there was no increase in the P2X7 receptor expression in both HCV HCC and non-HCV HCC. We conclude that P2X4 receptor expression was significant in the presence of HCV HCC. This may confirms the potential role of P2X4 receptor in the presence of virus in liver pathology. However insignificant expression of P2X7 receptor may avert our attention towards understanding the role of this receptor in pro-inflammatory and immune responses.

Helicobacter Pylori Status in Explant Livers of Liver Transplant Recipients in a European Patient Cohort

Background and study aims Helicobacter pylori (H. pylori) are known to be associated with several intestinal and extra-intestinal diseases such as peptic ulcers, mucosa-associated lymphoid tissue lymphoma, gastric cancer and/or idiopathic thrombocytopenic purpura. Recently, H. pylori genomic DNA has been detected in liver tissue of patients with chronic liver diseases. Furthermore, some studies suggested that detection of H. pylori is associated with progressive liver disease. However, the influence of this organism on the progression of chronic liver diseases remains unclear. The aim of this study was to evaluate the role of H. pylori in patients undergoing Liver Transplantation (LT) owing to end-stage liver disease.Patients and methods: This retrospective study enrolled 50 explant liver tissue from consecutive patients undergoing LT at the University Hospital of Muenster between January 2011 and December 2012. H. pylori DNA was detected in specimens by PCR.Results :We successfully extracted DNA from every liver tissue sample; however, H.pylori were not detectable in any liver specimen tested.Conclusion: In our patient cohort, H. pylori were not detectable in explant liver. Based on these results, H. pylori do not appear to have a significant impact on the progression of liver disease in our patient cohort.

Clear Cell Hepatocellular Carcinoma with Associated Tuberculosis-A Case Report

Clear cell hepatocellular carcinoma (CCHCC) is a rare variant of classical hepatocellular carcinoma (CHCC). Most of these cases pose a histological diagnostic dilemma with broad range of differentials of tumor with clear cell morphology. Use of immunostains or presence of classical hepatocellular carcinoma morphology focally aids in accurate diagnosis of these lesions. We report a case of a 62-year-old male who presented with loss of appetite and altered bowel habits for last 3 months. On investigation he was found to be HCV positive with genotype 1a and viral load of 3853865 IU/ml. His CECT revealed a well-defined hyper dense lesion of size 38 x 34 mm in segment V with a thin hypo dense rim. Contrast studies were suggestive of hepatocellular carcinoma with chronic liver disease. His PET CT revealed an FDG avid lesion in lung, biopsy from which showed caseating epithelioid granulomas. He underwent living donor related liver transplant. Explant liver tissue examined showed CCHCC with cirrhosis and several epithelioid granulomas. Occurrence of CCHCC with tuberculosis is a rare with no similar case has been reported in indexed English literature till date. We herein report the first case of CCHCC with coincidental Tuberculosis in an elderly male.

Stress-activated miR-21/miR-21* in hepatocytes promotes lipid and glucose metabolic disorders associated with high-fat diet consumption

ObjectivemiR-21 is an oncomir highly upregulated in hepatocellular carcinoma and in early stages of liver diseases characterised by the presence of steatosis. Whether upregulation of miR-21 contributes to hepatic metabolic...

Mechanisms of fibrosis in acute liver failure.

Background & AimsAcute liver failure (ALF) is a condition with high mortality and morbidity. Fibrosis in chronic liver disease was extensively researched, whereas fibrosis and underlying mechanism in acute liver failure remains unclear.MethodsHepatitis B virus related ALF patients were recruited to investigate if there was ongoing fibrosis by liver histology and liver stiffness measurement(LSM) analysis as well as fibrosis markers assay. Sera HMGB1 were kinetically detected in progression and remission stage of ALF. Hepatic stellate cell(HSC) activation by HMGB1 was explored by testing mRNA and protein level of α‐SMA and collagen 1a1 by using qPCR and western blot. Autophagy induction by HMGB1 was explored by LC3‐II conversion, autophagy flux and fluorescence.ResultsFirstly, ongoing fibrosis in progression stage of ALF was confirmed by histological analysis, LS measurement as well as fibrosis markers detection. HSC activation and autophagy induction in explanted liver tissue also revealed. Next, kinetic monitoring sera HMGB1 revealed elevated HMGB1 in progression stage of ALF vs HBsAg carrier, and drop back to base level in remission stage. Thirdly, rHMGB1 dose dependently activated HSCs, as indicated by increased mRNA and proteins level in α‐SMA and collagen 1a1. Moreover, autophagy was induced in HSC treated with rHMGB1, as illustrated by increased LC3 lipidation, elevated autophagy flux and GFP‐LC3 puncta.ConclusionsAcute liver failure is accompanied by ongoing fibrosis, HSC activation and autophagy induction. Increased HMGB1 activates HSC via autophagy induction. Those findings integrate HMGB1, HSCs activation, autophagy into a common framework that underlies the fibrosis in ALF.

PTU-130 Regulatory T Cells In Acute Liver Failure Are Functionally Intact And May Contribute To Retention Of Neutrophils In Areas Of Hepatic Necrosis

IntroductionAcute liver failure (ALF) is a sterile inflammation with a high mortality. The immunological response to acute liver injury is initiated by the infiltration of innate neutrophils and is followed...

Section 14. Combination of entecavir plus low-dose on-demand hepatitis B immunoglobulin is effective with very low hepatitis B recurrence after liver transplantation.

Antiviral prophylaxis with hepatitis B immunoglobulin (HBIg) plus lamivudine reduces the risk of hepatitis B virus (HBV) recurrence after HBV-related liver transplant (LT). However, HBIg is expensive, and lamivudine therapy is limited by drug resistance. This study assessed a pilot study of entecavir plus low-dose, on-demand HBIg in preventing HBV recurrence after LT. Between 2006 and May 2011, approximately 145 patients undergoing HBV-related LT and receiving entecavir plus low-dose, on-demand HBIg were enrolled and followed for a median of 36 months. A historical control group of 171 patients undergoing HBV-related LT between 1998 and 2010 and receiving lamivudine plus HBIg were followed for a median of 77 months. The primary end point was the proportion of patients with recurrent HBsAg-positivity. In the entecavir cohort, 2 (1.37%) of 145 patients experienced HBV recurrence, none of which had evidence of viral resistance. In the lamivudine cohort, 11 (6.4%) of 171 cases of HBV recurrence were observed, 5 of which were associated with lamivudine resistance. The cumulative probabilities of HBV recurrence were significantly different between both cohorts (P=0.055). HBsAg recurrence was associated with lower overall survival (P<0.001), even in patients with undetectable HBV DNA. Using pooled data from both cohorts, predictors of HBV recurrence were nucleoside selection, pre-LT hepatocellular carcinoma, post-LT low anti-HBs, male sex, and HBsAg-positivity in the explant liver tissue. Entecavir plus low-dose, on-demand HBIg resulted in a low rate of HBV recurrence without evidence of resistance development and provided an effective and cost-saving strategy for patients having HBV-related LT.

Therapeutic potential of vascular adhesion protein in primary sclerosing cholangitis

BackgroundVascular adhesion protein 1 (VAP1) is an adhesion molecule that also posseses potent amine oxidase activity; it produces hydrogen peroxide via the deamination of dietary amines. Through this function, VAP1 leads to activation of NFκB in hepatic sinusoidal endothelial cells (HSEC) resulting in expression of mucosal-vascular cell-adhesion molecule 1 (MAdCAM1), a mechanism proposed to contribute to the homing of gut-tropic lymphocytes expressing α4β7 to the liver. In view of the putative role of this pathway in hepatic diseases complicating inflammatory bowel disease, we aimed to quantify circulating or soluble (sVAP1) as well as intrahepatic VAP1 enzyme activity in patients with primary sclerosing cholangitis, and assess the functional consequence of its inhibition on MAdCAM1-dependent lymphocyte recruitment to HSEC. MethodsTotal VAP1 concentration was measured by ELISA. VAP1 amine oxidase activity was quantified in human serum and explanted liver tissue with the amplex red assay. We used flow-based adhesion assays with human HSEC isolated from liver explants, activated through tumour necrosis factor α and methylamine (VAP1 substrate), before and after treatment with VAP1 antibody or semicarbazide (VAP1 enzyme inhibitor). Fluorescence-activated cell sorted peripheral blood lymphocytes expressing α4β7 were perfused over HSEC under flow rates simulating physiological shear (0·05 kPa) and differences in adhesion and transmigration measured. FindingsPatients with primary sclerosing cholangitis had higher circulating median VAP1 enzyme activity (114·5 pmol hydrogen peroxide produced per min/mL serum, IQR 100·6–134·7) than patients with inflammatory bowel disease (60·3, 38·5–73·0; p=0·006), healthy controls (84·0, 77·7–105·7; p=0·02), and individuals with primary biliary cirrhosis (53·9, 33·0–90·9; p=0·006) and autoimmune hepatitis (77·6, 51·0–124·5; p=0·2, Mann-Whitney). Total sVAP1 concentration correlated well with sVAP1 enzyme activity (r2=0·75). Intrahepatic median VAP1 activity was also significantly higher in primary sclerosing cholangitis than in primary biliary cirrhosis (97·6 pmol [IQR 69·5–114·5] vs 24·6 [18·7–27·8], p=0·029) and autoimmune hepatitis (32·3 [23·3–35·6], p=0·028). HSEC pretreatment with semicarbazide and antibody led to a reduction in total α4β7+ lymphocyte adhesion (by 50% and 25%, respectively); however, both antibody and enzyme inhibition independently reduced transmigration by about 50% compared with untreated HSEC. InterpretationWe found that sVAP1 enzyme activity was greater in primary sclerosing cholangitis than in inflammatory bowel disease alone, healthy controls, and other immune-mediated liver diseases. Intrahepatic VAP1 enzyme activity was significantly higher in primary sclerosing cholangitis than in autoimmune hepatitis and primary biliary cirrhosis. Inhibition of VAP1 led to abrogation of α4β7-mediated adhesion to HSEC and is therefore a putative target for therapeutic intervention in primary sclerosing cholangitis. FundingWellcome Trust.

PWE-124 Vap-1 Activity is Elevated in PSC and Modulates A4B7-Dependent Lymphocyte Adhesion to HSEC Under Flow

IntroductionVascular adhesion protein (VAP)-1 is an adhesion molecule which possesses potent amine oxidase activity, and deaminates dietary amines resulting in the production of H2O2. Through this function, VAP-1 leads to...

Study of ethanol induced toxicity in liver explants using microfluidic devices

Current in vitro methodologies for the culture and analysis of liver specific responses lack the sophistication of in vivo dynamics. In this work, a microfluidic based experimental methodology has been utilized to reproduce a biomimetic microenvironment in which pseudo in vivo liver tissue studies can be carried out under in vitro conditions. This innovative technique, which exploits the inherent advantages of microfluidic technology, has been utilised to study the viability and functionality of explant liver tissue over four days in the presence of varying concentrations of ethanol. Concentrations of ethanol as low as 20 mM have produced a decrease in WST-1 metabolism, a marker of mitochondrial activity, and an increase lactose dehydrogenase release, reflecting cell death, in the explant samples; these effects increase with higher ethanol concentrations. A concomitant decrease in albumin and urea synthesis was also observed. We believe the proposed methodology is widely applicable and is clearly of relevance to biological and clinical research including drug development and toxicity, as well as enabling better fundamental understanding of tissue/cell processes.

OC-016 Sequential activation of CXCR3 and CCR6 mediates recruitment and positioning of interleukin 17 producing T cells in the liver

<h3>Introduction</h3> IL-17 secreting CD4 T cells (Th17) are a distinct lineage of pro-inflammatory lymphocytes that develop under control of the transcription factor RORγt. Th17 cells are pathogenic in models of autoimmunity and have been implicated in the pathogenesis of several forms of liver inflammation including primary biliary cirrhosis, alcoholic and viral hepatitis. Despite their importance in perpetuating inflammation, there is little known about the molecular basis for their recruitment and positioning to specific sites such as the liver. <h3>Methods</h3> Human intrahepatic Th17 cells were isolated from explanted liver tissue without cytokine expansion and analysed by multi colour flow cytometry. Murine liver injury models of Con A hepatitis, MCD Diet and repeated CCL4 injections were used for intravital microscopic assessment of Th17 cell recruitment to the liver. <h3>Results</h3> Less that 0.25% of T cells in normal human liver expressed IL-17 but we detected IL-17 secreting cells in inflamed human liver samples with the highest frequencies detected in patients with seronegative fulminant hepatitis (median 3.7%; IQR 3.4–3.9) non-alcoholic steatohepatitis (median 3.3%; IQR 2.7–3.5%) and auto-immune hepatitis (median 2.8%; IQR 2.6–3.3%). Intrahepatic IL-17+ cells expressed RORγt and the IL-23 receptor consistent with a Th17 lineage. They secreted IL-22 and IFNγ in addition to IL-17 and expressed high levels of the chemokine receptors CCR5, CCR6, CXCR3 and CXCR6. Intravital microscopy in murine models of acute liver injury (con-A hepatitis), steatohepatitis (MCD) and chronic liver injury (CCl4) demonstrated enhanced recruitment of adoptively transferred Th17 cells to the hepatic parenchyma via the sinusoids. Recruitment was significantly reduced by a blocking the CXCR3 ligand CXCL10 which also ameliorated liver injury. The CCR6 ligand, CCL20, was restricted to biliary epithelium and secreted by cholangiocytes in vitro suggesting a further recruitment signal positions Th17 cells near bile ducts in portal tracts. <h3>Conclusion</h3> Th17 cells are recruited to sites of inflammation in the liver by sequential interactions in which the CXCR3 ligand, CXCL10 promotes recruitment into the parenchyma from blood via the sinusoids and the CCR6 ligand, CCL20 positions cells at the biliary epithelium.