Yh Oo

Entecavir treatment for chronic hepatitis B: Adaptation is not needed for the majority of naïve patients with a partial virological response†‡

Entecavir (ETV) is a potent inhibitor of viral replication in nucleos(t)ide analogue (NA)‐naïve chronic hepatitis B (CHB) patients. The aim of this study was to investigate the long term efficacy and safety of ETV in NA‐naïve CHB patients, particularly in those with detectable hepatitis B virus (HBV) DNA after 48 weeks, in whom treatment adaptation is suggested by current guidelines. In a multicenter cohort study, we investigated 333 CHB patients treated with entecavir monotherapy. The NA‐naïve population consisted of 243 patients, whereas 90 were NA‐experienced. Virological response (VR) (HBV DNA <80 IU/mL) was achieved in 48%, 76%, and 90% of hepatitis B e antigen (HBeAg)‐positive and in 89%, 98%, and 99% of HBeAg‐negative NA‐naïve patients at weeks 48, 96, and 144, respectively. Thirty‐six of 175 (21%) NA‐naïve patients with at least 48 weeks of follow‐up had a detectable load at week 48 (partial virological response [PVR]). Twenty‐nine (81%) patients with PVR reached VR during prolonged ETV monotherapy, and none of them developed ETV‐resistance. Among 22 patients with HBV DNA <1,000 IU/mL at week 48, VR was achieved in 21 (95%) patients, compared with eight of 14 (57%) patients with HBV DNA ≥1,000 IU/mL. Continuous HBV DNA decline was observed in most patients without VR during follow‐up, and in three patients adherence was suboptimal according to the treating physician. ETV was safe and did not affect renal function or cause lactic acidosis. Conclusion: ETV monotherapy can be continued in NA‐naïve patients with detectable HBV DNA at week 48, particularly in those with a low viral load because long‐term ETV leads to a virological response in the vast majority of patients. (HEPATOLOGY 2011;)

Entecavir treatment does not eliminate the risk of hepatocellular carcinoma in chronic hepatitis B: limited role for risk scores in Caucasians

Background Hepatocellular carcinoma (HCC) risk-scores may predict HCC in Asian entecavir (ETV)-treated patients. We aimed to study risk factors and performance of risk scores during ETV treatment in an ethnically diverse Western population. Methods We studied all HBV monoinfected patients treated with ETV from 11 European referral centres within the VIRGIL Network. Results A total of 744 patients were included; 42% Caucasian, 29% Asian, 19% other, 10% unknown. At baseline, 164 patients (22%) had cirrhosis. During a median follow-up of 167 (IQR 82–212) weeks, 14 patients developed HCC of whom nine (64%) had cirrhosis at baseline. The 5-year cumulative incidence rate of HCC was 2.1% for non-cirrhotic and 10.9% for cirrhotic patients (p<0.001). HCC incidence was higher in older patients (p<0.001) and patients with lower baseline platelet counts (p=0.02). Twelve patients who developed HCC achieved virologic response (HBV DNA <80 IU/mL) before HCC. At baseline, higher CU-HCC and GAG-HCC, but not REACH-B scores were associated with development of HCC. Discriminatory performance of HCC risk scores was low, with sensitivity ranging from 18% to 73%, and c-statistics from 0.71 to 0.85. Performance was further reduced in Caucasians with c-statistics from 0.54 to 0.74. Predicted risk of HCC based on risk-scores declined during ETV therapy (all p<0.001), but predictive performances after 1 year were comparable to those at baseline. Conclusions Cumulative incidence of HCC is low in patients treated with ETV, but ETV does not eliminate the risk of HCC. Discriminatory performance of HCC risk scores was limited, particularly in Caucasians, at baseline and during therapy.

Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice

Background Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. Methods Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. Results OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-β signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. Conclusions OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.

PWE-145 Characterisation Of Circulating And Liver Infiltrating Mait Cells In Human Inflammatory Liver Diseases

Introduction Mucosal-Associated Invariant T (MAIT) cells are characterised by expression of the semi-invariant TCR α-chain Vα7.2-Jα33 and high expression of CD161 and shown to play a role at mucosal barriers. They display a limited T cell receptor repertoire being restricted by the MHC class 1-related molecule, MR1 but secrete high levels of pro-inflammatory cytokines suggesting they may play an important role in liver inflammation. We have shown before that the majority of MAIT cells in circulation are CD8+ MAIT cells. Recently, presence of MIAT cells have been described within human liver perfusate. However, very little is known about the phenotype and functions of liver infiltrating MAIT cells. In this study we investigated the frequencies and phenotypes of human liver infiltrating MAIT cells in healthy donors and diseased livers. Methods Peripheral blood and explanted liver infiltrating lymphocytes were freshly isolated and phenotyped by multicolour flow cytometry. The MAIT population was defined as CD3+CD16Hi Va7.2+. Results There was no difference in frequencies of circulating CD3PosCD161HiVa7.2Pos MAIT cells between patients with inflammatory liver disease and healthy controls (1.4 ± 0.7% vs. 2.3 ± 1.0%) and the majority were CD8Pos (82.3 ± 3.1%) with a smaller population of CD4Pos (2.7 ± 0.6%) and double negative CD8NegCD4Neg cells (14.8 ± 2.9%). Total CD3PosCD161HiVa7.2Pos MAIT frequencies were not significantly altered in inflamed liver tissue compared to blood (4.4 ± 1.0% vs. 1.4 ± 0.7%). However, in the inflamed liver, the CD8+ subset was reduced (61.2 ± 6.2 vs. 82.3 ± 3.1, P = 0.006) while the CD4+ MAIT subset was increased (15.9 ± 5.6 vs. 2.7 ± 0.6, P = 0.02). CXCR3, liver homing chemokine receptor was highly enriched on circulating and liver infiltrating CD3PosCD161HiVa7.2Pos MAIT cells (>75%). Liver infiltrating MAIT cells expressed chemokine receptors CCR5 (78.4 ± 7.2), CX3CR1 (51.3 ± 10), CCR6 (46.3 ± 14.8) and CXCR6 (36 ± 6.2%). Interestingly they expressed high levels of the integrin β7 (39.1 ± 3.6) and CD103 (19.6 ± 5.5%), which are associated with mucosal immune responses. They also expressed the cytokine receptors IL23R (27.1 ± 8.5%) and IL18Rα (76.7 ± 5%). Conclusion We have described for the first time that CD3PosCD161HiVa7.2Pos MAIT cells are present in inflamed human liver and express high levels of CXCR3 receptor implicated in lymphocyte recruitment to the liver and three other chemokine receptors CX3CR1 and CCR6 and CXCR6 that are associated with homing to portal tracts and bile ducts. Thus MAIT cells may play a role in biliary pathology. Disclosure of Interest None Declared.

Hepatocellular carcinoma surveillance in hepatitis B virus–infected individuals: Who and how?

We recently reported that, in a large cohort of biopsy-proven patients with nonalcoholic fatty liver disease, severe steatosis, diagnosed by histology or by ultrasound, is independently linked to increased liver stiffness measurement (LSM) values. As a consequence, we reported higher rates of false-positive LSM results for the noninvasive assessment of F2-F4 and F3-F4 fibrosis by transient elastography in patients with severe steatosis compared with their counterparts. Accordingly, we suggested that in nonalcoholic fatty liver disease patients the presence of severe steatosis, per se or evaluated by ultrasound, should always be taken into account in order to avoid overestimations of liver fibrosis. Notably, we reported similar results in the setting of chronic hepatitis C patients as well. We thank Lupsor and colleagues for their interest in our study. According to our hypothesis, they tested whether steatosis noninvasively assessed by the controlled attenuated parameter (CAP), a measure recorded using a different software in the FibroScan, overestimates LSM in a small cohort of 30 patients with F2 fibrosis and with chronic liver disease due to different etiologies. Notably, other than to confirm the expected association between CAP and body mass index, they reported significantly higher LSM values in patients with CAP >280 m/second compared with their counterparts. However, in multivariate analysis they did not confirm CAP as an independent predictor of overestimation of F3-F4 fibrosis, using LSM>9.5 as a threshold. We think that data from Lupsor and colleagues are interesting and can represent a starting point to test, in large cohorts of patients well characterized for liver damage and discriminated for etiology of liver disease—nonalcoholic fatty liver disease and chronic hepatitis C considered separately are the most relevant clinical settings—whether the combination of LSM with CAP could improve the diagnostic accuracy of LSM for staging fibrosis. Concerning the lack of evidence in multivariate analysis of CAP as an independent predictor of fibrosis overestimation reported by the authors, we think that the value of a multivariate analysis in a very small number of patients is too limited to draw conclusions.

OC-029 Phenotype of Human Intrahepatic Innate Lymphoid Cell Subsets in Heatlh and Disease

Introduction Innate lymphoid cells (ILCs) involve in the initiation, regulation and resolution of inflammation. Although the role of ILCs in murine liver fibrosis and biliary proliferation has been reported, the phenotypic characteristics and functional role of these cells in human liver disease remains undefined. We explored the detailed phenotype of human intrahepatic ILCs to gain insight on their function. Methods Liver infiltrating lymphocytes from normal liver tissue, autoimmune liver diseases (AILD), alcoholic liver disease (ALD) and non-alcoholic steatohepatitis (NASH) explants were phenotyped with flow cytometry. Results Total intrahepatic ILC (CD3− lineage− CD45− CD127+ ) comprised 1% (IQR 0.4–1.7%) of the CD3− CD45+ population in normal liver and 0.4% for ALD/NASH and autoimmune livers. The ILC1 subset constituted the majority of intrahepatic ILC and its frequency is higher in normal liver compared to diseased livers (85% vs. 67%) (p < 0.05). In contrast, CD161+CRTH2+ ILC2 subset and ILC3 subset frequency are higher in diseased livers compared to normal livers. Interestingly, frequency of ILC3 is significantly higher in ALD compared to normal liver (p < 0.01). Intrahepatic ILC subsets are in activated and tissue resident state (CD69 75–90%). All subsets expressed the liver tissue homing chemokine receptor, CXCR3. Expression frequency is highest on diseased ILC1 (AILD 50%, ALD/NASH 56%,) compared to ILC2 (AILD 24%; ALD/NASH 15%) and ILC3 (AILD 38%, ALD/NASH 28%). Biliary tropic receptor CCR6 was expressed more highly by ILC subsets in diseased compared to normal livers. The fibronectin receptor, VLA5 and laminin receptor VLA6 were both highly expressed on all intrahepatic ILC subsets (60–90% and 55–80% respectively). In terms of the cytokine receptors expressed, on all ILC subsets, IL-6R was near undetectable (2%) and low expressions of ST2 (3–6%), IL-25R (2–5%) and IL-23R (2–7%) were detected. In contrast there was almost ubiquitous expression of IL18Ralpha. CD25 was also very highly expressed. Of note, CD25 was significantly higher in ILC1 and ILC3 subsets from normal compared to AILD (p < 0.01) livers (40% vs. 80%; 66% vs. 93%) while intrahepatic ILC2 and ILC3 subsets in diseased states highly expressed CD25 (>90%). Of likely functional importance, intrahepatic ILC1 expressed IFN-γ (40%) while ILC2 expressed IL-13 (20–50%) in diseased states. Conclusion We report for the first time the presence of all three ILC subsets within the human liver immune cell infiltrates. CXCR3+ IFN-γ expressing ILC1 subset is enriched in both normal and inflamed diseased livers. Higher frequencies of CCR6+ VLA5+ VLA6+ IL-13 expressing ILC2 are observed in diseased livers suggesting that this subset may play a role in biliary pathology and peri-biliary fibrosis. Disclosure of Interest None Declared

PTU-140 Intrahepatic Tregs Are Plastic But Functional And Biliary Epithelial Cells Support Their Fate

Introduction Regulatory T cells (Tregs) are crucial in maintaining peripheral tolerance. Tregs control T effector CD8, CD4, Th1 cells along with other immune cells to maintain hepatic tolerance. They are implicated in both human and murine model of hepatic inflammation including autoimmune hepatitis, viral hepatitis, liver cancer and post-transplantation tolerance. However little is known about the lineage stability, function and fate of human intrahepatic Tregs in the inflamed microenvironment. Methods Human liver infiltrating (LI) lymphocytes were freshly isolated from explanted liver tissues. LITregs cells surface phenotype, chemokine and cytokine receptor expression, intracellular-cytokine secretion was assessed ex-vivo by flow cytometry. Function and plasticity of post-endothelial transmigrated (PEM) Tregs in the inflamed microenvironment was assessed by suppression assays and flow cytometry. Distribution and localisation of LITregs in tissue was determined using dual immunohistochemistry and confocal microscopy. Cytokine expressions by the liver microenvironment were studied in vitro using Luminx. Real time PCR was used to study the mRNA expression. Survival and proliferation of PEM Tregs in microenvironment was studied in-vitro using co-culture assays using primary human biliary epithelial cells. Results LITregs highly express CD39 (57 ± 11%), CD95 (83 ± 4%), CD27 (73 ± 3%), CD44 (90 ± 3%) and low expression of CD40 (6.813 ± 3.25%). Cytokine receptors expression was (31 ± 15%) for IL15R, (17 ± 15%) for IL6R-α. Hepatic microenvironment is highly enriched with IL-1β (363 ± 88 pg/ml), IL-6 (8,960±pg/ml), IL-12 (44 ± 35 pg/ml), IFN-γ (21 ± 8.33 pg/ml). Minimal level of IL-2 was detected in inflamed liver supernatant. Post-endothelial migrated (PEM) Tregs and Tregs in the inflamed microenvironment are functional but suppression capacity was reduced in Tregs residing in the inflamed liver. Plasticity to other T cells lineage is minimal for Tregs in the inflamed microenvironment. LITregs reside close to bile ducts at the portal tract. Co-culture experiment of PEM Tregs and with biliary epithelial cells suggested that Tregs survival depends on FAS-FASL pathway and IL-2. Conclusion LITregs are plastic but functional in the inflamed intrahepatic microenvironment and their fate around biliary epithelial cells is supported via IL-2 cytokine and CD95-CD95 ligand pathway. Disclosure of Interest None Declared.

PTU-119 Phenotypic Characteristics And Localisation Of Novel Human Liver Infiltrating Nkp46 Subsets

Introduction CD56+Natural killer cells are the principal effector cells of the innate immune system and have a well-established role in tumour surveillance and anti-viral immunity. Expression of NKp46 has been shown to correlate closely with the severity of liver inflammation, viral resistance to IFN treatment and the attenuation of liver fibrosis. CD56+NKp46 cells expressing IL-17 and IL-22 have also been described as a family of innate lymphoid cells in humans. Although the role of intrahepatic NK cells has been well described, little is known about the function and phenotype of intrahepatic NKp46 subsets. Thus, We aim to investigate the phenotypic characteristics of CD56+ NKp46 cells in the inflamed human liver, with a view to exploring their functional role. Methods Liver infiltrating lymphocytes were freshly isolated from explanted human liver tissue from our transplant program and phenotyped with multicolor flow cytometry. Cellular localization was investigated by immunohistochemistry and confocal microscopy Results Human liver infiltrating NK cells reside predominantly around biliary epithelial cells at the portal tract close to regulatory T cells. We observed two populations of liver-infiltrating CD3neg CD19neg CD56pos cells distinguished by different levels of NKp46, NKp46mid (15% ±4.8 SD) and NKp46high (11% ±1.2 SD) neither subset expressed NKp44. The chemokine receptor expression of NKp46mid and NKp46high populations was: CCR6 (12% ± 3 vs. 7%± 2.4), CCR9 (20% ± 5.6 vs. 9% ± 0.9), CX3CR1 (18% ± 14 vs. 10% ± 1) CXCR3 (47%±14.4 vs 38%±11.0) and CXCR6 19% ± 4.0 vs. 14% ± 4.6). Both populations expressed IL-18R (42% ± 5.4 vs 7% ± 1.0), IL-23R (19% ± 6.0 vs. 11% ± 2.5), surface receptor CD161 (61% ± 12.1 vs 85% ±4.8) and the integrin receptor CD103 (4% ± 1.35 vs. 16% ± 1.7). The NKp46high population was highly enriched with the activation marker CD69 (77%±18%). NKp46 cells were also shown to express TNF-α (29% ± 7.5), IFN-γ (70% ± 7.0), Granzyme B (23% ± 11.0) and Perforin (23% ±11.1) along with transcription factor Tbet (19% ± 9.1). Conclusion We hereby report novel subsets of liver infiltrating CD56+NKp46 cells, which localise around the portal tract biliary epithelium in the inflamed human liver. These populations have distinct cytokine, chemokine and CD103 expression, which may explain their recruitment, positioning and effector functions in the inflamed hepatic microenvironment. Disclosure of Interest None Declared.

PWE-130 Phenotype And Localisation Of Liver Infiltrating B Cell Subsets In Autoimmune And Inflammatory Liver Diseases

Introduction B cells classically provide humoral immunity in the form of antibody production as part of the adaptive immune response. Regulatory and antigen presenting functions of B cells have been reported before and autoantibodies are associated with autoimmune liver diseases. B cell depletion in animal models of PBC has highlighted the regulatory roles of B cells in ameliorating disease. Some evidence of efficacy of anti-B cell therapy using rituximab in human autoimmune liver diseases further supports a role for B cells. Mature B cells (Bm) subpopulations had been described in Sjogren’s syndrome. However, little is known about the localisation, subsets, phenotype and function of B cells in human liver diseases. Methods In this study we characterised the frequencies of B cell subsets in the blood and liver of patients with inflammatory and autoimmune liver diseases. Results Frequencies of naïve mature BM1 cells were reduced in the liver compared to blood (7.5% ± 2.3 vs. 20.2% ±2.8 p = 0.0022) and IgDnegCD27neg subset was increased in diseased livers compared to diseased blood (22.9% ± 6.8 vs. 6.0% ± 1.1 p = 0.0013). B cells localise close to the bile ducts in PBC and reside around hepatocytes in AIH. Frequencies of regulatory B cells (CD19posCD24hiCD38hi) were significantly reduced in diseased blood vs. control blood (1.8% ± 0.4 vs. 3.6% ± 0.5 p = 0.01) similar to recent observation in acute rheumatoid arthritis. However this population is increased in the diseased liver compared with blood (6.2% ± 0.07 vs. 1.8% ± 0.4 p = 0.007), suggesting enrichment of regulatory B cells within the inflamed liver. Liver infiltrating B cells were capable of IL-10 production. Conclusion We have characterised for the first time the heterogeneity of B cell subsets and presence of regulatory B cells and IL-10 secreting B cells in human diseased livers. We showed that B cells reside close to bile ducts along with other immune cells; thus B cells may play a role in biliary pathology. Disclosure of Interest None Declared.

PTU-123 Use Of Rituximab In Resistant Autoimmune Hepatitis – Birmingham Experience

Introduction Autoimmune hepatitis (AIH) is due to breakdown in immunological self-tolerance. Sustained remission in AIH is crucial to prevent the progression to end stage liver disease.1 Around 9% of the patients are refractory/intolerant to the standard therapy with prednisolone (Pred) ± azathioprine (AZA). High levels of immunoglobulin are typical of AIH and plasma cells are frequently observed in liver histology. Rituximab is an anti-CD20 monoclonal antibody that depletes B-cells and has been used to treat other autoimmune conditions such as systemic lupus erythematosus. However, little has been reported on the role of B cells depletion and its outcome in AIH. The aim of the study was to evaluate the safety and efficacy of rituximab in the treatment of refractory AIH. Methods A retrospective case note review of well-defined and biopsy proven type-1 AIH (simplified scoring >6). 5 patients out of 200 who were intolerant/refractory to standard therapy were given Rituximab and the responses were followed up for 72 weeks. Efficacy was measured by biochemical and immunological parameters (bilirubin, AST, ALT and Immunoglobulin every 12 weeks. The dose of Prednisolone as well as UKELD/MELD score pre and post treatment was also evaluated. Results All 5 patients were female and mean age was 45 (range 35–66 yrs). The rituximab dose used was 1000 mg and the total number of doses received varied between 2 and 4 (Mean 3.2). Three patients had other concomitant autoimmune conditions (endocrine, rheumatological and renal related autoimmune diseases). The mean dose of prednisolone used pre-rituximab was 19mg (±SD 12.57) and this was reduced to 12.5mg (± SD 5.0) post treatment (statistically not significant=NS). There was a slight improvement of IgG pre and post Rituximab treatment (NS), with no improvement in UKELD score. There was an improvement in biochemical profile but this was not statistically significant throughout the observation period. All five patients were alive and rituximab was well tolerated without any serious adverse events. Conclusion Rituximab is well-tolerated and safe to use in resistant AIH. It can cause some biochemical and immunological improvement. Current evidence for its use in AIH patients is not well proven. The study numbers are too small to detect the actual outcome of the therapy. A multicenter larger cohort prospective study with longitudinal immunological, biochemical and histological profile assessment is warranted to assess its efficacy in resistant AIH patients. Reference Czaja AJ. Current and future treatments of AIH. Expert Rev Gastroenterology Hepatol 2009:3: 269–91 Disclosure of Interest None Declared.