Ectopic expression of murine CD47 minimizes macrophage rejection of human hepatocyte xenografts in immunodeficient mice.

UNLABELLED: 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.

HBeAg-positive hepatitis delta: virological patterns and clinical long-term outcome.

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.

Improved immune status corresponds with long-term decline of quantitative serum hepatitis B surface antigen in HBV/HIV co-infected patients.

In HBV/HIV-co-infected individuals, the course of hepatitis B is aggravated, leading to higher morbidity and mortality rates. Increasing evidence suggests an important role for hepatitis B surface antigen (HBsAg) quantification in monitoring treatment efficacy in HBV monoinfection. However, data concerning any HBsAg decline during treatment of HBV/HIV coinfection are limited. Fifty-one HBV/HIV-co-infected patients were retrospectively followed for a mean of 43 months (median 2 years, interquartile range 2 years). Baseline and on-treatment parameters were associated with longitudinal HBsAg levels. At baseline, serum HBsAg levels were comparable between patients on antiretroviral therapy (ART; n=43) and patients without (n=8). Longitudinally, HBsAg decreased in ART patients (-0.20±0.09 log(10) IU/mL/y), but slightly increased in subjects without therapy (0.22±0.26 log(10) IU/mL/y; p<0.001). In 58% of the ART subjects an HBsAg decline >10% was seen during the initial 24 mo. They showed higher baseline CD4 counts (401±42 versus 265±50 cells/µL, p=0.03), and had significantly higher CD4 counts at the last follow-up compared to patients without a decline (506±39 versus 310±51 cells/µL, p=0.01). A significant correlation was found between HBsAg decline from baseline to the last follow-up and the absolute increase of CD4 cells (r=0.44, p=0.003), as well the last CD4 count (r=0.41, p=0.006). This association was strongest in patients with complete suppression of HBV-DNA and HIV-RNA at the last follow-up visit. The highest HBsAg decline (-1.63±0.32 versus -0.43±0.24 log(10) IU/mL, p=0.001), and yearly HBsAg decline (-0.47±0.13 versus -0.19±0.12 log(10) IU/mL, p=0.03), were found in patients with CD4 increases >100 cells/µL at the last follow-up (n=21, 49%). Four cases of HBsAg loss were observed (8%). HBsAg declines steadily in >50% of HBV/HIV patients on ART. Long-term follow-up of HBV/HIV-co-infected patients is needed to identify distinct HBsAg patterns. Increasing CD4 counts indicating the restoration of immune competence in HBV/HIV-co-infected patients is associated with a stronger HBsAg decline.

Correlation between the Elecsys HBsAg II assay and the Architect assay for the quantification of hepatitis B surface antigen (HBsAg) in the serum.

BACKGROUND: 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. OBJECTIVES: Performance comparison of the qualitative Elecsys HBsAg II assay using a quantitative research protocol and an established quantitative HBsAg assay. STUDY DESIGN: 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. RESULTS: There was significant overall correlation between Elecsys and Architect assays (correlation coefficient [r]=0.97; p<0.001). 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. CONCLUSIONS: 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.

Nitrogen Metabolism in Plant Cell Suspension Cultures: II. Role of Organic Acids during Growth on Ammonia.

Tobacco cells (Nicotiana tabacum) are capable of growth on ammonia as a sole nitrogen source only when succinate, malate, fumarate, citrate, alpha-ketoglutarate, glutamate, or pyruvate is added to the growth medium. A ratio between the molar concentrations of ammonia to succinate (as a complementary organic acid) in the growth medium of 1.5 was optimal. Succinate had no effect on the rate of uptake of ammonia from the medium into the cells although it did affect the intracellular concentration of ammonia. However, the changes were not sufficient to explain inhibition of growth as being due to ammonia toxicity. The radioactivity from (14)C-succinate was incorporated into malate, glutamate, and aspartate within 2 minutes.It appears that the role of organic acids is neither connected to ammonium transport nor to relief of ammonia toxicity, but may be related to the need for additional carbon skeletons for synthesis of amino acids.

Nitrogen metabolism in plant cell suspension cultures: I. Effect of amino acids on growth.

Certain amino acids inhibit growth of tobacco (Nicotiana tabacum L. var. xanthi), tomato (Lycopersicon esculentum) carrot (Daucus carota), and soybean (Glycerine max L. co. Mandarin) cell cultures when nitrate or urea are the nitrogen sources but not when ammonia is the nitrogen source. These amino acids also inhibit development of nitrate reductase activity (NADH:nitrate oxidoreductase EC 1.6.6.1) in tobacco and tomato cultures. Threonine, the most inhibitory amino acid, also inhibits nitrate uptake in tobacco cells. Arginine, and some other amino acids, abolish the inhibition effects caused by other amino acids. We suggest that amino acids inhibit assimilation of intracellular ammonium into amino acids in cells grown on nitrate or urea.