Reduction of liver macrophage transduction by pseudotyping lentiviral vectors with a fusion envelope from Autographa californica GP64 and Sendai virus F2 domain

<p>Abstract</p> <p>Background</p> <p>Lentiviral vectors are well suited for gene therapy because they can mediate long-term expression in both dividing and nondividing cells. However, lentiviral vectors seem less suitable for liver gene therapy because systemically administered lentiviral vectors are preferentially sequestered by liver macrophages. This results in a reduction of available virus and might also increase the immune response to the vector and vector products.</p> <p>Reduction of macrophage sequestration is therefore essential for efficient lentiviral liver gene therapy.</p> <p>Results</p> <p>Fusions were made of <it>Autographa californica </it>GP64 and the hepatocyte specific Sendai Virus envelope proteins. Lentiviral vectors were produced with either wild type GP64, Sendai-GP64, or both wild type GP64 and Sendai-GP64 and tested <it>in vitro </it>and <it>in vivo </it>for hepatocyte and macrophage gene transfer.</p> <p>Sendai-GP64 pseudotyped vectors showed specific gene transfer to HepG2 hepatoma cells, with no detectable transduction of HeLa cervical carcinoma cells, and a decreased affinity for RAW mouse macrophages. Co-expression of wild type GP64 and Sendai-GP64 resulted in improved viral titers while retaining increased affinity for HepG2 cells.</p> <p>In vivo, the Sendai-GP64 vectors also showed decreased transduction of murine liver macrophages.</p> <p>Conclusion</p> <p>We demonstrate reduced macrophage transduction <it>in vitro </it>and <it>in vivo </it>with GP64/Sendai chimeric envelope proteins.</p

Experimental study on long-term exposure to a biocompatible, hypertonic, pyruvate-buffered dialysis solution

BACKGROUND: Chronic exposure to glucose and glucose degradation products (GDPs) in dialysis solutions is involved in the pathogenesis of peritoneal neoangiogenesis and fibrosis, potentially leading to encapsulating peritoneal sclerosis (EPS). High lactate concentrations may contribute to glucose toxicity by creating a state of pseudohypoxia, which stimulates the formation of various growth factors. OBJECTIVE: To study the effects of long-term peritoneal exposure to a filter-sterilized pyruvate-buffered solution with a combination of 3 osmotic agents (amino acids, glycerol, glucose: PYRAGG) on peritoneal function and morphology. METHODS: Rats were exposed daily for a period of 20 weeks to PYRAGG, or to a conventional heat-sterilized solution (LH), or to a filter-sterilized solution (LF), after which a peritoneal function test was done and peritoneal tissue was obtained. RESULTS: Peritoneal solute and fluid transport characteristics at 20 weeks were similar in all groups. Fibrosis was most pronounced in the LH group compared to the others, suggesting an effect of GDPs. A marked reduction in the number of omental vessels was noted in the PYRAGG group (59% reduction compared to LH). A modest reduction (28%) was found in the LF animals. This points to a marked effect of reduced exposure to glucose. CONCLUSIONS: PYRAGG was more biocompatible than a filter-sterilized glucose/lactate solution because it did not induce marked peritoneal abnormalities after long-term exposure. This did not lead to altered peritoneal transport characteristics. It is likely that further development of PYRAGG-like solutions will decrease the incidence of EP

DLK1, a serum marker for hepatoblastoma in young infants

Hepatoblastoma is a malignant pediatric liver tumor. The currently used diagnostic serum marker for hepatoblastoma, a-fetoprotein (AFP), is not always reliable in infants with hepatoblastoma, due to the physiologically elevated levels of AFP in this age group. In this report, we show that Delta-like 1 homolog (DLK1), a protein highly expressed during fetal development, but almost completely absent after birth, and an established liver-stem cell marker, is a new candidate serum marker of hepatoblastoma, especially in young infants. Pediatr Blood Cancer 2012;59:743745. (c) 2011 Wiley Periodicals, In

Human fetal liver cells for regulated ex vivo erythropoietin gene therapy

Possible risks and lack of donor livers limit application of liver transplantation. Liver cell transplantation is, at this moment, not a feasible alternative because engraftment in the liver is poor. Furthermore, there is also shortage of cells suitable for transplantation. Fetal liver cells are able to proliferate in cell culture and could therefore present an alternative source of cells for transplantation. In this study, we investigated the utility of human fetal liver cells for therapeutic protein delivery. We transplanted human fetal liver cells in immunodeficient mice but were not able to detect engraftment of human hepatocytes. In contrast, transplantation of human adult hepatocytes led to detectable engraftment of hepatocytes in murine liver. Transplantation of fetal liver cells did lead to abundant reconstitution of murine liver with human endothelium, indicating that endothelial cells are the most promising cell type for ex vivo liver cell gene therapy. Human liver endothelial cells were subsequently transduced with a lentiviral autoregulatory erythropoietin expression vector. After transplantation in immunodeficient mice, these cells mediated long-term regulation of murine hematocrits. Our study shows the potential of human liver endothelial cells for long-term regulated gene therapy

Cellular Localization and Biochemical Analysis of Mammalian CDC50A, a Glycosylated beta-subunit for P4 ATPases

CDC50 proteins are beta-subunits for P4 ATPases, which upon heterodimerization form a functional phospholipid translocation complex. Emerging evidence in mouse models and men links mutations in P4 ATPase genes with human disease. This study analyzed the tissue distribution and cellular localization of CDC50A, the most abundant and ubiquitously expressed CDC50 homologue in the mouse. The authors have raised antibodies that detect mouse and human CDC50A and studied CDC50A localization and glycosylation status in mouse liver cells. CDC50A is a terminal-glycosylated glycoprotein and is expressed in hepatocytes and liver sinusoidal endothelial cells, where it resides in detergent-resistant membranes. In pancreas and stomach, CDC50A localized to secretory vesicles, whereas in the kidney, CDC50A localized to the apical region of proximal convoluted tubules of the cortex. In WIF-B9 cells, CDC50A partially costains with the trans-Golgi network. Data suggest that CDC50A is present as a fully glycosylated protein in vivo, which presumes interaction with distinct P4 ATPases. (J Histochem Cytochem 60: 205-218, 2012

Kupffer cells and not liver sinusoidal endothelial cells prevent lentiviral transduction of hepatocytes

Lentiviral vectors can stably transduce dividing and nondividing cells in vivo and are best suited to long-term correction of inherited liver diseases. Intraportal administration of lentiviral vectors expressing green fluorescent protein (Lenti-GFP) in mice resulted in a higher transduction of nonparenchymal cells than hepatocytes (7.32 +/- 3.66% vs 0.22 +/- 0.08%, respectively). Therefore, various treatments were explored to increase lentiviral transduction of hepatocytes. Lenti-GFP was injected into the common bile duct, which led to transduction of biliary epithelium and hepatocytes at low efficiency. Transient removal of the sinusoidal endothelial cell layer by cyclophosphamide to increase accessibility to hepatocytes did not improve hepatocyte transduction (0.42 +/- 0.36%). Inhibition of Kupffer cell function by gadolinium chloride led to a significant decrease in GFP-positive nonparenchymal cells (2.15 +/- 3.14%) and a sevenfold increase in GFP-positive hepatocytes compared to nonpretreated mice (1.48 +/- 2.01%). These findings suggest that sinusoidal endothelial cells do not significantly limit lentiviral transduction of hepatocytes, while Kupffer cells sequester lentiviral particles thereby preventing hepatocyte transduction. Therefore, the use of agents that inhibit Kupffer cell function may be important for lentiviral vector treatment of liver diseas

Phospholipid flippases attenuate LPS-induced TLR4 signaling by mediating endocytic retrieval of Toll-like receptor 4

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the β-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR