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Macrophages play an important role in the rejection of xenogeneic cells and therefore represent a major obstacle to generating chimeric mice with human xenografts that are useful tools for basic and preclinical medical research. The signal inhibitory regulatory protein α (SIRPα) receptor is a negative regulator of macrophage phagocytic activity and interacts in a species-specific fashion with its ligand CD47. Furthermore, SIRPα polymorphism in laboratory mouse strains significantly affects the extent of human CD47-mediated toleration of human xenotransplants. Aiming to minimize macrophage activity and thus optimize human cell engraftment in immunodeficient mice, we lentivirally transduced murine CD47 (Cd47) into human liver cells. Human HepG2 liver cells expressing Cd47 were less frequently contacted and phagocytosed by murine RAW264.7 macrophages in vitro than their Cd47-negative counterparts. For the generation of human-mouse chimeric livers in immunodeficient BALB-ΔRAG/γ(c) -uPA (urokinase-type plasminogen activator) mice, freshly thawed cryopreserved human hepatocytes were transduced with a lentiviral expression vector for Cd47 using a refined in vitro transduction protocol immediately before transplantation. In vivo, Cd47-positive human primary hepatocytes were selectively retained following engraftment in immunodeficient mice, leading to at least a doubling of liver repopulation efficiencies. Conclusion: We conclude that ectopic expression of murine Cd47 in human hepatocytes selectively favors engraftment upon transplantation into mice, a finding that should have a profound impact on the generation of robust humanized small animal models. Moreover, dominance of ectopically expressed murine Cd47 over endogenous human CD47 should also widen the spectrum of immunodeficient mouse strains suitable for humanization. (HEPATOLOGY 2012).

Background & Aims Chronic HDV infection is an inflammatory liver disease and liver transplantation (LTX) remains the only curative treatment option for most patients. The hepatitis D virus (HDV) uses HBsAg as its surface protein, however, it is controversial to what extend HDV may be detected independently of HBsAg in blood and liver after LTX. The aims of this study were to investigate kinetics of HDV RNA and HBsAg early after LTX, to apply the data to a mathematical model and to study long-term persistence of HDV after LTX. Methods We retrospectively analyzed 26 patients with chronic hepatitis delta who underwent LTX between 1994 and 2009. Blood samples were obtained every 1–3days during the first 14days after LTX. Data were applied to a mathematical model to study viral kinetics. Available liver biopsy samples were stained for HBV and HDV viral antigens and tested for HBV DNA/cccDNA. Results HBsAg and HDV RNA became negative after a median of 5days (range 1–13) and 4days (range 1–10), respectively. Early HDV RNA and HBsAg decline paralleled almost exactly in all patients; however the mathematical model showed a high variability of virion death. HDAg stained positive in transplanted livers in six patients in the absence of liver HBV DNA/cccDNA, serum-HBsAg, and HDV RNA for up to 19months after LTX. Conclusions HDV RNA and HBsAg decline follow almost identical kinetic patterns within the first days after LTX. Nevertheless, intrahepatic latency of HDAg has to be considered when exploring novel concepts to withdraw HBIG.

Background Hepatitis E virus (HEV) infection takes a clinically silent, self-limited course in the far majority of cases. Chronic hepatitis E has been reported in some cohorts of immunocompromised individuals. The role of HEV infections in patients with autoimmune hepatitis (AIH) is unknown. Methods 969 individuals were tested for anti-HEV antibodies (MP-diagnostics) including 208 patients with AIH, 537 healthy controls, 114 patients with another autoimmune disease, rheumatoid arthritis (RA), and 109 patients with chronic HCV- or HBV-infection (HBV/HCV). Patients with AIH, RA and HBV/HCV were tested for HEV RNA. HEV-specific proliferative T cell responses were investigated using CFSE staining and in vitro stimulation of PBMC with overlapping HEV peptides. Results HEV-antibodies tested more frequently positive in patients with AIH (n = 16; 7.7%) than in healthy controls (n = 11; 2.0%; p = 0.0002), patients with RA (n = 4; 3.5%; p = 0.13) or patients with HBV/HCV infection (n = 2; 2.8%; p = 0.03). HEV-specific T cell responses could be detected in all anti-HEV-positive AIH patients. One AIH patient receiving immunosuppression with cyclosporin and prednisolone and elevated ALT levels had acute hepatitis E but HEV viremia resolved after reducing immunosuppressive medication. None of the RA or HBV/HCV patients tested HEV RNA positive. Conclusions Patients with autoimmune hepatitis but not RA or HBV/HCV patients are more likely to test anti-HEV positive. HEV infection should been ruled out before the diagnosis of AIH is made. Testing for HEV RNA is also recommended in AIH patients not responding to immunosuppressive therapy.

Introduction: UDPS allows the detection of minor viral variants during the natural course and antiviral treatment in HBV-infected patients. It could be shown that double infections with two HBV genotypes and minor strains carrying resistance mutations not detected by clonal sequencing could be analyzed. Here we tested a method for analyzing multiple HBV genotypes in a single sample as well as its potential to identify resistance mutations in patients months before viral breakthrough. Patients and methods: A custom HBV sample was constructed by equimolar pooling of PCR amplicons derived from viral nucleic acid extracts from the serum of three different CHB patients. They had been identified as HBV genotypes B and D. Two samples were described as genotype D (one as wild-type, one as carrier of the resistance mutation rtM204V), and one sample as genotype B (wild-type). The genotype D wild-type developed lamivudine (LMV) and adefovir (ADV) resistance later on treatment. UDPS was performed on a GS Flx 454 sequencer (Roche). Sequencing data was analyzed using GenomiX Workbench (CLC Bio). Results: Sequence analysis showed 37,07% of reads mapping to HBV genotype B (subtype adw), 61.99% to genotype D (subtypes w3 and ayw3) while the remaining 0,96% where scattered among other genotypes. Single nucleotide polymorphism (SNP) analysis of the sequences mapped to distinct genotypes clearly discriminated between wild-type and mutated HBV strains. For one patient, previously described as infected with wild-type HBV genotype D, a minor variant (coverage 35, frequency 100%) in the viral quasi species showed mutations at position rtM204 and rtI233, mediating viral resistance to LMV and ADV. Follow-up of this patient revealed a viral breakthrough with a major species resistant to LMV and ADV (rtM204V, rtI233V) 14 months later. Conclusions: UDPS is a feasible method for the identification of HBV viral sequences including genotype and resistance mutations. The detection of minor variants carrying resistance-bearing mutations more than a year prior to viral breakthrough could constitute a new tool for individualizing antiviral treatment, especially with low genetic barrier nucleos(t)ide analogues. Whether the occurrence of minor quasi-species with resistance mutations could also be reflected by serological parameters like serum HBsAg is currently investigated.

Background and Aims The presence of the hepatitis B virus (HBV)-eAg in patients with hepatitis B is associated with higher HBV replication and with an increased risk to develop liver-related clinical endpoints defined as liver related death, liver transplantation, development of hepatocellular carcinoma and hepatic decompensation. The aim of this study was to investigate the role of HBeAg in patients co-infected with the hepatitis D virus (HDV). Methods We studied virological markers of HBV and HDV infection and as well as biochemical and clinical features of liver disease in a cohort of 534 anti-HDV-positive patients. In addition, we compared the clinical long-term outcome of HBeAg-positive HDV-infected patients with HBeAg-negative control patients matched for age, gender and baseline-MELD score. Results HBeAg-positive hepatitis delta was detected in 71 of 534 patients (13.3%). HBeAg positivity was associated with a higher biochemical disease activity and higher HBsAg levels in HDV co-infected patients. Sixty one per cent of the HBeAg-positive HDV-infected patients presented with HBV DNA levels below 2000 IU/ml, at least once during follow-up. Both HBeAg-positive and -negative patients showed a similar severe clinical long-term course with about half of the patients developing a liver-related clinical complication after a median follow-up period of 51 months (range: 9–193 months). Conclusions HBV DNA levels are low in both HBeAg-negative and HBeAg-positive patients suggesting suppressive effects of HDV on HBV irrespective of the phase of HBV infection. The clinical long-term outcome of HBeAg-positive patients is not different to HBeAg-negative patients infected with the HDV.

Summary. Loss of hepatitis ‘e’ antigen (HBeAg) in patients with HBeAg-positive chronic hepatitis B is associated with improved long-term clinical outcome and is defined as a goal of antiviral treatment by clinical practice guidelines. Recent studies suggest that baseline levels and on-treatment monitoring of HBeAg levels may identify patients most likely to respond to therapy. The aim of this study was the development of a protocol for the quantitative determination of HBeAg using the Elecsys® HBeAg immunoassay. The linear range of the Elecsys® HBeAg immunoassay was established using recombinant HBeAg and five different diluents. The assay was validated against the Paul Ehrlich Institute (PEI) international standard serum. Linearity was demonstrated up to a cut-off index (COI) of 1000, independent of the diluent used. Optimal linearity was obtained using the Elecsys® Universal Diluent. Using the PEI reference standard, conversion factors were established as 4.50 COI for 1 PEIU/mL corresponding to 0.222 PEIU/mL for a COI of 1. Based on the results from these analyses, a simple algorithm for the quantitative measurement of HBeAg using the Elecsys® HBeAg immunoassay was developed. Using a simple algorithm with an initial 1:40 dilution, the Elecsys® HBeAg assay provides robust quantification of serum HBeAg in an easy-to-use and rapid system. The use of a commercially available, standardized diluent improves comparability between laboratories.

Hepatitis B surface antigen (HBsAg) clearance during chronic hepatitis B (CHB) infection is associated with improved long-term clinical outcome, so is considered an important therapeutic goal in CHB. Studies have shown that serum HBsAg quantification during, and at end of, treatment may predict long-term HBsAg loss.Performance comparison of the qualitative Elecsys HBsAg II assay using a quantitative research protocol and an established quantitative HBsAg assay.A dilution algorithm was developed for the Elecsys HBsAg II assay to allow quantification of HBsAg levels; this was used to measure HBsAg levels in a range of samples including sera from patients infected with different HBV genotypes, HBV mutants, and longitudinal samples from patients undergoing antiviral treatment. Results were compared with those from the quantitative Architect HBsAg assay.There was significant overall correlation between Elecsys and Architect assays (correlation coefficient [r]=0.97; p0.001). HBsAg levels measured with both assays correlated well in all phases of infection (r=0.80-0.96), across all genotypes tested (HBV genotype A, r=0.89; HBV genotype D, r=0.97), and in samples with lamivudine-resistant mutations (r=0.94). Bland-Altman analysis showed only minor discordance between assays in different phases of chronic HBV-infection (3.8-5.1%). This strong correlation was also present for sera with lower HBsAg concentrations. On-treatment HBsAg levels were similar when measured with either assay.Using a simple dilution algorithm, the quantitative Elecsys HBsAg II assay reliably determined serum HBsAg levels in a wide range of samples, and showed very high correlation with the Architect HBsAg assay.

Results: The slope of changes in LSMs over time in years is shown in Figure 1. LSMs changes over time (expressed in DkPa/month) showed a slight faster pattern of fibrosis regression at the beginning (first two years) and a slow steady pattern later during a follow-up period of four years. Improvement in fibrosis was seen in 33/55 patients (60%) (drop of −2.06±1.6 in kPa). Importantly, regression of cirrhosis (initial LSM >12kPa) to less than 6.1 kPa was observed in 3/55 (5.4%) patients. Worsening of LSMwas observed in 3 cases due to other liver disease cofactors. The baseline median LSM was 9.2±3.1 kPa. LSMs significantly decreased during the follow-up period after the start of NA treatment (8.4±0.9, 7.8±1.3, 7.3±1.9 and 6.8±1.6 kPa at years 1, 2, 3 and 4, respectively). Conclusions: LSMs changes annually showed an early faster and a later steady pattern of fibrosis regression over time as measured by transient elastography for chronic HBeAg (−) hepatitis treated with NA. Also LSMs showed a slight improvement of fibrosis in the first 4 years. Longer follow-up of these patients by TE is necessary to assess fibrosis regression in chronic hepatitis B patients under antiviral therapy.