This is a prospective cohort follow-up study of 40 patients with

suspected CFLD: they were identified and referred by the CF clinic of JAK inhibitors in development the Royal Children’s Hospital (Brisbane, Australia), were enrolled between 1999 and 2004, and were followed until death, transplantation, or survival as of March 2009. This clinic is a major CF referral center (over 250 patients) for Queensland, Australia. The details and progress of all patients were recorded prospectively via a detailed clinical database. Suspected CFLD was defined as two of the following: (1) hepatomegaly (HM) with or without splenomegaly, (2) a persistent (>6-month) elevation of serum alanine aminotransferase (ALT; level > 1.5 × upper limit Z-VAD-FMK concentration of normal), and (3) abnormal liver US findings (abnormal echogenicity or a nodular edge). Those with liver synthetic dysfunction or a history of hepatobiliary surgery were excluded. The study conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the ethics committees of the Royal Children’s Hospital and the Queensland Institute of Medical Research. Informed consent was obtained from parents and, when appropriate, from patients. At enrollment, the following were performed

or determined for all patients: history, physical examination, Δf508 genotype, lung function, serum aminotransferases, liver synthetic function (international normalized ratio and albumin), and liver US as well as upper gastrointestinal endoscopy, serum draw for research, and dual-pass liver biopsy under general anesthesia. Specific note was made of the presence Montelukast Sodium or absence of PHT, which was defined as the occurrence of any of the following: endoscopic esophageal varices

and persistent clinical splenomegaly (palpable spleen below the left costal margin that was confirmed to span outside the normal range for the patient’s age by US) with or without thrombocytopenia (platelet count <150,000). Portal vein thrombosis was excluded by Doppler imaging. When PHT was present, the age of onset was recorded by chart review. During follow-up (up to 12 years), all patients received standard CF pulmonary and nutritional care, all patients with biopsy-confirmed fibrosis were prescribed ursodeoxycholic acid (15 mg/kg/day), and all patients were reviewed at least on a 6-month basis. For the purposes of this study, prospectively recorded follow-up data included clinical progress, occurrence of cystic fibrosis–related diabetes mellitus (CFRD; defined as insulin-dependent diabetes mellitus), survival, solid organ transplantation, forced expiratory volume in 1 second (FEV1), liver aminotransferases, liver synthetic function, and occurrence of PHT (as defined previously).

We found a link between the expression of CTLA-4 and the proapoptotic mediator Bim in HBV-specific CD8. Longitudinal study of a cohort of CHB patients commencing antiviral therapy showed that viral load reduction did not reduce CTLA-4 or Bim levels in antiviral T cells. We therefore explored the potential to manipulate this coinhibitory pathway in vitro to restore expansion of HBV-specific CD8

T cells. ALT, alanine transaminase; CHB, chronic hepatitis B virus infection; CTLA-4, cytotoxic T lymphocyte antigen-4; HBV, hepatitis B virus; OLP, overlapping peptides. The study was approved by the local Ethical Committees selleck kinase inhibitor and written informed consent was obtained from all patients. A total of 86 patients with CHB, three patients with resolved HBV infection, and 23 healthy volunteers participated in the study; there were no significant differences in their demographics (Table 1). All participants were HCV and HIV seronegative and cytomegalovirus (CMV) seropositive. Patients with CHB were stratified by HBV DNA levels above or below 2,000 IU/mL (determined by real-time polymerase chain reaction [PCR]), according to European Association for the Study of the Liver (EASL) guidelines.13 All CHB patients were treatment-naïve at recruitment; a subgroup of seven patients was followed

longitudinally after commencing lamivudine and adefovir (Table 2). Hepatitis B s-antigen (HBsAg) was quantitated with the Architect assay. Paired peripheral blood and liver biopsy Cell Cycle inhibitor specimens (surplus to diagnostic requirements) were obtained from eight patients with CHB (Table 1). PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation and cultured on anti-CD3 monoclonal antibody (mAb)-coated plates (1 μg/mL) or in medium alone for 16 hours before analysis of CTLA-4 in total CD8 T cells. For detection of intracellular CTLA-4 on virus-specific CD8 T cells ex vivo, cells were stained with human leukocyte antigen A2 (HLA-A2)/c18-27, HLA-A2/e183-191, HLA-A2/e335-343,

and HLA-A2/e348-357 HBV dextramers (Immudex) before stimulation with HBV-specific peptides for 4 hours in the presence of Brefeldin A. CMV-specific CD8 T cells were detected by HLA-A2/NLVPMVAYV pentamers (Proimmune). For functional detection of virus-specific CD8 T cells, cells were stimulated with HBV or control viral peptide and cultured for 10 (-)-p-Bromotetramisole Oxalate days, supplemented with 20 U/mL IL-2 at 0 and 4 days, restimulated with 1 μM peptide for 16 hours in the presence of 1 μg/mL Brefeldin A (Sigma-Aldrich), and identified by intracellular staining for IFN-γ. To examine the effect of blocking inhibitory pathways, purified, NA/LE monoclonal antibodies against CTLA-4 (BD Biosciences), PD-L1, PD-L2 (eBioscience), or control IgG (BD-Biosciences) were added at 5 μg/mL with peptide at onset of culture. Responses were analyzed as described above. Liver sections from biopsies were homogenized and filtered.

Methods:

Organ retrieval Nutlin-3a and cold perfusion were performed in a standardized fashion using University of Wisconsin solution. In addition, blood from the donor was collected as perfusion solution. The perfusion circuit consisted of a single centrifugal pump which circulates perfusate out of the inferior vena cava through an oxygenator / heat exchanger and then split into a pressure-controlled hepatic artery supply and gravity fed portal venous supply via a reservoir. Throughout the perfusion period of up to six hours there was continuous monitoring of haemo-dynamic parameters and blood, bile, liver and bile duct tissue samples were collected. Results: At the time of submission, one liver donated after cardiac death (DCD) and one donated after brain

death (DBD) have been studied. Both livers were meta-bolically active throughout the perfusion period reflected by lactate clearance (peak lactate 9 and 8.16 mmol/L; 0.95 and 2.56 mmol/L at the end of perfusion), urea production (4.4 and 4 mmol/L at start of perfusion; 11 and 7.9 mmol/L at end of perfusion) and bile production. Liver histology obtained at the end of the perfusion period showed no evidence of hepatocellular injury. However, there was extensive biliary injury in the DCD liver as reflected by epithelial cell loss and mural necrosis of both the left and right hepatic duct. Conclusion: This study shows that normothermic oxygenated machine perfusion is feasible from a technical perspective Anti-infection Compound Library concentration Sinomenine in donor liver currently deemed unsuitable for transplant. This continuing study is comparing the results of both DCD and DBD donor livers. The extensive degree of biliary damage in the DCD liver may underline the need to consider biliary tract integrity when assessing graft viability.

Disclosures: Darrell H. Crawford – Advisory Committees or Review Panels: Roche Products Pty Ltd, Bristol Myers Squibb, Gilead Sciences, Novartis, MSD, Abbvie, Jansen; Consulting: Roche Products Pty Ltd; Grant/Research Support: Roche Products Pty Ltd; Speaking and Teaching: Roche Products Pty Ltd, Bristol Myers Squibb, Gilead Sciences, MSD The following people have nothing to disclose: Janske Reiling, David S. Lockwood, Andrew H. Simpson, Catherine M. Campbell, Kim Bridle, Nishreen Santrampurwala, Laurence J. Britton, Cornelis H. Dejong, Jonathan Fawcett Background and Aims: Zinc is an important trace element with catalytic and defensive functions. We assessed the impact of zinc deficiency in patients with end-stage liver disease (ESLD) awaiting liver transplantation. Methods: Serum zinc levels were measured at the time of evaluation for liver transplantation in ESLD patients (n = 265). Patients were dichotomized in two groups: low and normal zinc serum levels. Results: Medium serum zinc levels were 8.59 μmol/l ± 3.1.

Results: The mean age of the study patients was 55.4 years, 881 (49.6%) were males, 693 (51.3%) PLX4032 nmr were infected by HCV genotype 1, and 245 (13.8%) had cirrhosis at study entry. There were 1542 (86.7%) patients experienced SVR after receiving treatment. Higher platelet count, lower serum levels of total bilirubin, HCV RNA, and HCV genotype non-1 were independent predictors of achieving SVR. At the 5 years of post-treatment follow-up, there were 49 newly-diagnosed HCC cases (37 with SVR and 12 with non-SVR). The observed 5-year HCC risk was 2.1% for patients with SVR and 4.2% for those with non-SVR, respectively. The cumulative

risk of HCC was significantly higher for the non-SVR patients than the SVR patients (p<0.001). Patients with old ages, male gender, and low levels of hemoglobulin had an increased incidence of HCC. After adjustment for the potential confounders, the patients who

did not achieve SVR had 2.4 folds (95% confidence interval: 1.20-4.94) risk of developing HCC during the follow-up period. Conclusion: Chronic hepatitis C patients receiving peg-interferon plus ribavirin therapy who achieved SVR is associated with a substantial reduction of HCC risk. Patients with CHC infection should be encouraged to receive antiviral therapy. Disclosures: Yong Yuan – Employment: Bristol Myers Squibb Company Ming-Lung Yu – Advisory Committees or Review Panels: Roche, MSD, Abbott, Abbvie, Gilead; Grant/Research Support: Roche, MSD, Abbott, Abbvie; Speaking and Teaching: Roche, MSD, Abbott, Progesterone Abbvie, Gilead Wan-Long GDC-0449 solubility dmso Chuang – Advisory Committees or Review Panels: Gilead, Roche, Abbvie, MSD; Speaking and Teaching: BMS Gilbert J. L’Italien – Employment: bristol myers squibb; Stock Shareholder: bristol myers squibb The following people have nothing to disclose: Mei-Hsuan

Lee, Jia-Horng Kao, Chen-Hua Liu, Sheng-Nan Lu, I-Shyan Sheen, Hwai-I Yang, Chien-Jen Chen Background: Little information is available about early virologic responses for sofosbuvir (SOF)-based regimens in real-world populations with hepatitis C virus (HCV) infection. Methods: All patients starting a SOF-based regimen by 4/12/14 were identified in the VA HCV Clinical Case Registry. Exclusion criteria included: being on a HCV regimen to which SOF was added (n=41), baseline HCV RNA <1000 (n=25) and a non-standard SOF regimen (n=2). Standard regimens included: SOF+pegin-terferon+ribavirin (SPR), SOF+ribavirin (SR) and SOF+sime-previr±ribavirin (SS/R). We assessed 4 week HCV RNA using available results between 2 and 6 weeks after starting SOF in those who received at least 4 weeks of SOF. Advanced liver disease (ALD) was defined as FIB-4 >3.25. Univariate and multivariate analysis including baseline characteristics were performed for undetectable (UD) week 4 HCV RNA. Results: Of 731 patients starting SOF, 663 were included in the analyses.

6 or .9 mg/kg, (3) availability of mean or median National Institutes of Health Stroke Scale (NIHSS) score at presentation, patient demographics (age, gender), and functional outcome assessed at discharge or later, (4) time interval Small Molecule Compound Library between symptom onset and IV and endovascular treatment reported, (5) final recanalization following endovascular treatment reported, and (6) rate of symptomatic intracerebral hemorrhage (sICH) reported. Studies

were included regardless of the modality of endovascular treatment administered (pharmacological and/or mechanical). In the event of overlap of study populations, the smaller study was excluded. Using a predesigned data abstraction form, 2 reviewers (MZM and QAS) independently reviewed all manuscripts and abstracted the following information: (1) study characteristics (year of publication, design, recruitment period); (2) patient characteristics (number of patients, demographic characteristics, NIHSS score at presentation); (3) dose of IV rt-PA given and time from symptom onset to IV rt-PA administration; (4) time from symptom onset to angiography and/or IA treatment; (5) type of IA thrombolytic and mechanical devices used; (6) angiographic recanalization

Tanespimycin datasheet rates following endovascular treatment; (7) NIHSS score at 24-48 hours, when available; (8) rates of sICH; and (9) functional outcomes at discharge or later. The primary endpoints assessed in the analysis were partial or complete recanalization, favorable functional outcome, and sICH. Studies sometimes used different definitions of those endpoints (see Table 1). We used a modified Rankin scale score of 0-1 at 1-3 Vildagliptin months after treatment to define favorable outcome. In 2 of the 11 studies, we determined the favorable outcome rates by reviewing individual patient data presented in tables or by acquiring it directly from the authors. In 3 of the 11 studies, favorable outcome was defined by other measures acquired

at discharge or last available follow-up. We repeated the analysis after excluding these 3 studies. Since we did not detect a difference in our results, we decided to include the studies and favorable outcome definitions used. When possible, we used a thrombolysis in myocardial infarction (TIMI) grade of 2-3 flow post-procedure to define partial or complete recanalization. For sICH, we used the definition provided by individual studies. Any disagreements between the 2 data abstractors were reconciled with the mediation of a third investigator (ALG). In order to compare the .6 mg/kg with the .9 mg/kg group, we pooled the demographic and clinical data from single studies: means of means or medians weighted by the sample size, and range of minimum and maximum individual patient data. When study range was not available it was approximated by the 95% confidence interval (CI).

206, 0.301, −0.504, 0.425 respectively, p≤0.001). Finally, ARFI was not significantly correlated with hepatic inflammation level as measured by ALT (p = 0.182) and BMI (p = 0.163). Conclusion: Acoustic radiation force impulse imaging provides reliable and accurate assessment of hepatic fibrosis and is well correlated with the established non-invasive transient elastography. Further strength of this technology EGFR inhibitor resides with absence of influence by hepatic inflammation and BMI, in addition to the ability of providing concurrent conventional ultrasonographic assessment. This combined approach in the hands of trained gastroenterologist may have substantial advantages for delivering efficient

clinical service to these patients. P SUNDARALINGAM, WC TEOH, IB TURNER Department of Gastroenterology, Campbelltown Hospital, NSW University of Western Sydney, Campbelltown, NSW Background: Bacterial infections in the cirrhotic patient are a frequent and leading cause of mortality. Appropriate antibiotic prophylaxis can significantly reduce the incidence of infections BAY 57-1293 solubility dmso in cirrhotics however data on rates of implementation of appropriate prophylaxis is lacking. Saab et al (Journal of Clinical Gastroenterology 2006 Feb;40(2):156–61) suggested that prophylaxis utilization is low. The aim of this study was to determine the rate of prophylactic antibiotic usage in cirrhotic patients

at an outer metropolitan teaching hospital in NSW. Methods: Medical record data of cirrhotic patients admitted to Campbelltown Hospital between April 2011 and March 2013 was collected retrospectively. The data was analyzed to identify patients who were eligible for antibiotic prophylaxis. Specific groups evaluated were: 1) acute upper gastrointestinal haemorrhage 2) spontaneous Vorinostat bacterial peritonitis and 3) high SBP risk (low protein ascites and advanced liver disease). The records of these patients were reviewed to evaluate whether prophylactic antibiotic usage was in accordance with guidelines endorsed by AASLD and EASL. Results: 107 patients with cirrhosis had 193 admissions during the 2-year study period. (1) There were 24 admissions (19 patients) for upper gastrointestinal

bleeding. Appropriate antibiotic therapy was instituted in 20/24 bleeding episodes. In all but two instances, antibiotics were initiated on the first day of admission and prior to endoscopic intervention. The average duration of antibiotic usage was only 2.9 days (recommended duration 5- 7 days). Infection did not complicate the hospital stay of any patient that received antibiotic prophylaxis; both deaths in this group were a result of uncontrolled bleeding. Of the 4 patients who did not receive antibiotics, 2 died from uncontrolled haemorrhage. Infection did not complicate the hospital stay of the remaining 2 patients. (2) There were only 3 admissions (2 patients) with spontaneous bacterial peritonitis defined by an ascitic PMN count of > 250 cells/mm3.

Administration of 2 × 107 PBMCs twice after suppression of mice NK cells by anti–asialo GM1 antibody21 and macrophages and DCs by liposome-encapsulated clodronate22 before transplantation enabled us to establish a human PBMC

chimerism in uPA-SCID mice. We observed an up to 7% human mononuclear cell chimerism among the liver-resident mononuclear cells of uninfected and HBV-infected mice 2-14 days after the initial injection of PBMC (Fig. 1A; Table 1). Chimerism was most prominent 4 days after initial PBMC administration and almost undetectable by day 14 (Fig. 1A). Histological examination of chimeric mice livers showed extensive human liver cell death, comparable to the massive liver cell death observed in fulminant hepatitis, only in HBV-infected and PBMC-treated mice liver (Fig. 1B). Human hepatocytes were almost completely eliminated and replaced by human albumin-negative mouse hepatocytes at days

Carfilzomib supplier 7 and 14. Consistent with these histological changes, we observed a rapid decline buy RXDX-106 of HSA levels and HBV DNA only in HBV-infected and PBMC-treated mice (Fig. 1C). The decline of mice HSA levels and HBV DNA was also observed in 2 of 3 HBV-infected mice transplanted with PBMCs isolated from healthy blood donors without HBsAg vaccination (Fig. 1D and Supporting Fig. 2). We then analyzed liver-infiltrating cells with flow cytometry. Unexpectedly, we did not detect CD8-positive and tetramer-positive CTLs, as reported previously (Fig. 2A). Instead, we observed substantial numbers of CD3-negative and CD56-positive NK cells (Fig. 2B) and small numbers of pDCs and mDCs (Fig. 2C). The majority of NK cells of HBV-infected mice were FasL positive (Fig. 2D). In contrast, such FasL-positive NK cells were not detected in uninfected mice livers (Table 1; Fig. 2D), suggesting that these NK cells were activated in HBV-infected mice. These activated NK cells and DCs were detectable in mice livers only 4 days after the initial PBMC injection, but were undetectable after 2 and 7 days (Supporting Figs. 3 and 4, respectively).

To confirm the necessity of both DCs and NK cells to complete hepatocyte destruction, we depleted DCs or NK cells with http://www.selleck.co.jp/products/Fludarabine(Fludara).html negative selection using antibody-coated magnetic beads before the administration of PBMC. Depletion of either DCs or NK cells completely abolished the decline of human albumin as well as HBV DNA (Supporting Fig. 5A). However, analysis of liver-infiltrating cells revealed that chimerism with human PBMC was poorly established in these animals, probably the result of the loss or damage of human cells by bound antibodies during separation and/or subsequent incubation in mice (Supporting Fig. 5B; Supporting Table 1). To overcome possible confounding resulting from poor chimerism resulting in poor human hepatocyte degeneration in mice, we attempted to remove DCs from transplanted human PBMCs by alternate means.

Administration of 2 × 107 PBMCs twice after suppression of mice NK cells by anti–asialo GM1 antibody21 and macrophages and DCs by liposome-encapsulated clodronate22 before transplantation enabled us to establish a human PBMC

chimerism in uPA-SCID mice. We observed an up to 7% human mononuclear cell chimerism among the liver-resident mononuclear cells of uninfected and HBV-infected mice 2-14 days after the initial injection of PBMC (Fig. 1A; Table 1). Chimerism was most prominent 4 days after initial PBMC administration and almost undetectable by day 14 (Fig. 1A). Histological examination of chimeric mice livers showed extensive human liver cell death, comparable to the massive liver cell death observed in fulminant hepatitis, only in HBV-infected and PBMC-treated mice liver (Fig. 1B). Human hepatocytes were almost completely eliminated and replaced by human albumin-negative mouse hepatocytes at days

Dabrafenib price 7 and 14. Consistent with these histological changes, we observed a rapid decline Erlotinib datasheet of HSA levels and HBV DNA only in HBV-infected and PBMC-treated mice (Fig. 1C). The decline of mice HSA levels and HBV DNA was also observed in 2 of 3 HBV-infected mice transplanted with PBMCs isolated from healthy blood donors without HBsAg vaccination (Fig. 1D and Supporting Fig. 2). We then analyzed liver-infiltrating cells with flow cytometry. Unexpectedly, we did not detect CD8-positive and tetramer-positive CTLs, as reported previously (Fig. 2A). Instead, we observed substantial numbers of CD3-negative and CD56-positive NK cells (Fig. 2B) and small numbers of pDCs and mDCs (Fig. 2C). The majority of NK cells of HBV-infected mice were FasL positive (Fig. 2D). In contrast, such FasL-positive NK cells were not detected in uninfected mice livers (Table 1; Fig. 2D), suggesting that these NK cells were activated in HBV-infected mice. These activated NK cells and DCs were detectable in mice livers only 4 days after the initial PBMC injection, but were undetectable after 2 and 7 days (Supporting Figs. 3 and 4, respectively).

To confirm the necessity of both DCs and NK cells to complete hepatocyte destruction, we depleted DCs or NK cells with Thymidine kinase negative selection using antibody-coated magnetic beads before the administration of PBMC. Depletion of either DCs or NK cells completely abolished the decline of human albumin as well as HBV DNA (Supporting Fig. 5A). However, analysis of liver-infiltrating cells revealed that chimerism with human PBMC was poorly established in these animals, probably the result of the loss or damage of human cells by bound antibodies during separation and/or subsequent incubation in mice (Supporting Fig. 5B; Supporting Table 1). To overcome possible confounding resulting from poor chimerism resulting in poor human hepatocyte degeneration in mice, we attempted to remove DCs from transplanted human PBMCs by alternate means.

136 ± 0.05 versus 0.09 ± 0.02 (× 106) (P = 0.0262) in CD20-treated mice versus controls and 0.64 ± 0.11 versus 0.27 ± 0.03 (×106) (P = 0.004) in CD79-treated mice versus controls; CD8+ CD62L− CD44+ cells, 0.14 ± 0.06 versus 0.05 ± 0.01 (× 106) (P = 0.0348) in CD20-treated mice versus controls and 0.362 ± 0.06 versus 0.158 ±

0.029 (× 106) (P = 0.007) in CD79-treated mice versus controls. As expected, there was no difference in the phenotypic distribution of mononuclear cells in the spleen (data not shown). ALT, AST, and ALP levels were measured to assess Torin 1 the correlation between serum biochemical values and histopathological abnormalities. As seen in Table 2, mice treated with anti-CD79 produced higher levels of ALT, AST, and ALP compared with that of control mice (P < 0.05). Mice treated with anti-CD20 demonstrated Daporinad mw a significant increase in AST levels only (P < 0.05). The levels of serum inflammatory cytokines in B cell–depleted mice were higher compared with control mice starting at 4 weeks of 2OA immunization (Fig.

6A). In particular, serum levels of IFN-γ in the CD20- and CD79-treated mice were significantly higher compared with sera from control mice (P = 0.046 and P = 0.011, respectively). Similarly, serum levels of MCP-1 in CD20- and CD79-treated mice were also significantly higher than sera from control mice (P = 0.0017 and P = 0.042, respectively) at 8 weeks after cholangitis induction. As expected, IL-10, in part secreted by B cells, was lower in B cell–depleted mice (Fig. 6B). We demonstrate herein that acute B cell depletion in mice with otherwise normal immune systems exacerbates cholangitis induction. Liver cell infiltrates from B cell–depleted mice contained increased populations of CD4+ and CD8+ T cells and activated counterparts and exhibited many increased serum levels of IFN-γ and MCP-1, but lower levels

of IL-10, compared with B cell–sufficient mice. These findings should be considered in the context of recent studies on the role of B cells in a genetic animal model of PBC, the dnTGF-βRII mouse.23 Anti-CD20 therapy in young dnTGF-βRII mice attenuates liver damage but exacerbates inflammatory bowel diseases; however, B cell depletion in older mice does not modify the course of liver disease.34 Importantly, double transgenic mice with PBC-like disease and B cell depletion (Igμ−/−dnTGF-βRII mice) developed a more severe form of cholangitis.24 The present study demonstrates that B cell depletion immediately before disease induction in the xenobiotic induced murine model enhances disease severity in the absence of AMAs, suggesting that B cells play a critical regulatory role in the breakdown of tolerance against PDC-E2.

3B). This suggests that T lymphocytes from the nonresponder group are generally compromised in their ability to respond to a specific antigen following major histocompatibility complex–dependent presentation by an antigen-presenting cell. No significant

correlation was found between the inability to respond to the HBc-loaded pDC stimulation and HBV-DNA levels (Fig. 3C), HBs antigen level (Fig. 3D), see more ALT measurements (Fig. 3E), or antiviral treatment (Fig. 3F). In contrast, the presence of HBeAg in the serum appeared to differentiate between responder and nonresponder chronic HBV patients (Fig. 4). The HBc-specific T cell response was much greater in inactive carriers and treated or untreated HBeAg-negative hepatitis INK 128 chemical structure patients than in HBeAg-positive patients (Fig. 4A). After pooling patients according to HBeAg status alone, this difference appeared clearly significant (Fig. 4B). This interesting observation was corroborated by data for two patients in whom HBeAg status changed over a

6-month interval (Fig. 4C,D). One HBeAg-positive patient, unexpectedly capable of responding to pDC stimulation, achieved HBeAg loss followed by HBeAg seroconversion 6 months later. The other patient, initially HBeAg-negative and capable of responding to HBc-loaded pDC stimulation, became unresponsive 6 months later during a transient HBeAg-positive peak. Thus, HBeAg status distinguishes between chronic HBV patients capable of responding, or not, to HBc-loaded pDC

stimulation. To investigate the functionality of HBV-specific T cells generated from responder chronic HBV patients we examined T cell exhaustion and cytotoxic potential. PD1 expression, a marker of T cell exhaustion, was not detected on the HBc-specific T cells elicited by the pDC line (Supporting Fig. 1). The cytotoxic potential of expanded HBV-specific T cells was determined by performing a 51Cr release assay using peptide-loaded HLA-A*0201+ T2 cells as targets. As expected, HBc-specific T cells exhibited a strong cytotoxicity toward T2 cells loaded with HBc peptide but not with an irrelevant peptide, showing the specificity of the HBV-specific T cells function (Fig. 5A). Next, we tested the ability of these specific T cells to lyse a more relevant target, such Histone demethylase as HBV-transfected HLA-A*0201+ hepatocytes. Due to the lack of P3 facilities necessary to perform radioactive experiments with virus-producing cells, a CFSE assay was used. This assay consisted of culturing specific T cells with a mixture of two targets labeled with distinct CFSE intensities. The disappearance of the CFSE pic, as measured via flow cytometry, indicates killing of the corresponding cells. For all patients tested, HBc-specific T cells were able to specifically lyse the HBV-transfected HLA-A*0201+ hepatocyte cell line HepG22.15, but not the HBV-free HepG2 line (Fig. 5B).