Adenovirus-mediated gene transfer in primary murine adipocytes

The transfer of genes into primary murine adipocytes using an adenovirus system has been developed. A recombinant adenovirus was constructed (expressing green fluorescent protein [GFP] under the control of the strong cytomegalovirus [CMV] promoter and a luciferase reporter gene under the control of the weak adipocyte promoter keratinocyte lipid-binding protein [KLBP/FABP5]) and incubated with primary adipocytes from C57BL/6J mice. Analysis of infected cells by confocal microscopy detected GFP expression in both the cytoplasm and nucleus of adipocytes with a 64% efficiency of infection. To demonstrate the applicability of this method in the study of gene regulation, adenovirus-infected adipocytes exhibited significant levels of luciferase activity even from a weak promoter. TPA treatment of infected adipocytes increased luciferase activity, consistent with previous studies indicating that the KLBP/FABP5 gene is up-regulated by phorbol esters.These results provide an efficient, convenient, and sensitive method to transiently infect primary murine adipocytes, facilitating protein expression or permitting analysis of reporter gene activity from both viral and endogenous promoters.     

SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming

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Adenovirus-mediated gene transfer to nonparenchymal cells in normal and injured liver

Adenovirus-mediated gene transfer has become an important tool with which to introduce genetic material into cells. Available data emphasize efficient adenoviral transduction of parenchymal liver cells (i.e., hepatocytes) in both in vitro and in vivo model systems, typically in normal cells. The aim of this study was to evaluate gene transfer to nonparenchymal (and parenchymal) cells of the normal and injured rat liver. Hepatocytes, stellate cells, and endothelial cells were isolated by standard methods. Liver injury was induced by bile duct ligation or carbon tetrachloride administration. Cells were transduced in vitro with an adenovirus encoding beta-galactosidase (Ad.beta-gal) over a range of viral titers, and transduced cells were identified by detection of X-gal. In vivo transduction efficiency was studied in normal and injured livers using cell isolation techniques. Nonparenchymal cells were transduced with greater frequency than hepatocytes at all adenoviral titers tested, both in vitro and in vivo. After liver injury, adenoviral transduction was reduced for all liver cell types compared with that for cells from normal livers (at all virus titers). Notably, transduction efficiency remained greater in nonparenchymal cells than in hepatocytes after liver injury. This work implies that, to achieve comparable gene expression in the injured liver, higher adenoviral titers may be required, an important consideration as gene therapy in disease states is considered.     

Adenovirus-mediated gene transfer in olfactory neurons in vivo

We used recombinant adenoviruses as a means of expressing exogenous genes in olfactory neurons in vivo. A replication incompetent adenovirus (type 5, Ad5) carrying the reporter gene lacZ, which codes for the enzyme β-galactosidase (β-Gal), was applied in solution to the olfactory epithelia of rats. The expression of lacZ was controlled by the cytomegalovirus immediate-early promoter/enhancer. β-Gal expression was observed 1 day postinfection and was maximal at 3–10 days, although it could be detected for at least 21 days postinfection. Expression patterns were heterogeneous, ranging from a few percent to over 25% of the cells in different regions of both turbinate and septal epithelium. Staining was stronger in the olfactory versus respiratory epithelia. In olfactory epithelium staining was almost entirely restricted to olfactory neurons. β-Gal staining was also observed in the olfactory axons so that nerve bundles could be traced to their targets in the glomerular layer of the olfactory bulb. Intense staining of some glomeruli was evident as long as 21 days postinfection. There was no evidence of cell loss or tissue damage due to viral infection. These results demonstrate that it is possible to use recombinant Ad5 for expressing foreign genes in olfactory neurons. This technique could be used in olfactory neurons to increase expression levels of olfactory specific genes, including the odor receptor, putative guidance and growth molecules, or elements of the transduction cascade, in order to elucidate their biological functions in vivo. © 1996 John Wiley & Sons, Inc.     

Sperm protein 17 is expressed in human somatic ciliated epithelia.

It was once believed that sperm protein 17 (Sp17) was expressed exclusively in the testis and that its sole function was to bind to the oocyte during fertilization. However, immunohistochemistry of the human respiratory airways and reproductive systems show that it is abundant in ciliated cells but not in human cells with stereocilia and microvilli. The high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP)-binding motif within this region, suggest that Sp17 plays a regulatory role in a PKA-independent AKAP complex in both male germinal and ciliated somatic cells.     

Incorporation of adeno-associated virus in a calcium phosphate coprecipitate improves gene transfer to airway epithelia in vitro and in vivo

Adeno-associated virus (AAV) is inefficient at infecting differentiated airway epithelia because of a lack of receptors at the apical surface. We hypothesized that incorporation of AAV in a calcium phosphate coprecipitate would circumvent this barrier. Interestingly, coprecipitation of AAV type 2 improved gene transfer to differentiated human airway epithelia in vitro and to the mouse lung in vivo. These results suggest that delivery of AAV as a CaP(i) coprecipitate may significantly enhance its utility for gene transfer to the airway epithelia in vivo.     

Adenovirus-mediated human pancreatic lipase gene transfer to rat bile: gene therapy of fat malabsorption

This study explored the potential of using the gene therapy approach, based on adenovirus-mediated expression of pancreatic lipase in the hepatobiliary tract, to increase lipid digestion in the intestinal lumen and promote lipid absorption through the gastrointestinal tract. Recombinant adenovirus containing the human pancreatic lipase cDNA (AdPL) was shown to transduce and mediate pancreatic lipase biosynthesis in rat IEC-6 epithelial cells in vitro. Retrograde infusion of recombinant adenovirus (3 x 10(8) plaque-forming units) containing the bacterial LacZ gene (AdLacZ) into the bile duct of rats resulted in positive X-gal reaction products in the periportal liver cells 7 days after AdLacZ infusion. A high level of human pancreatic lipase was detected in bile after retrograde bile duct infusion of rats with AdPL but not in the bile of animals infused with AdLacZ. Triglyceride hydrolytic activity in the bile of AdPL-infused rats was equivalent to that present in pancreatic juice. In contrast, serum obtained from these animals did not contain any detectable pancreatic lipase activity. These results suggest that ectopic expression of pancreatic enzymes in the hepatobiliary tract may be an alternative therapeutic strategy for treating fat malabsorption due to pancreatic insufficiency.     

Adenovirus-mediated gene transfer as a tool to study angiogenesis in the chick embryo

Abstract. An adenoviral construct encoding a nuclear-localized beta-galactosidase marker protein was injected into the heart of chick embryos at Hamburger-Hamilton (HH) stage 14-15 (approximately 52-56 h of incubation). Reporter gene expression was determined 48-54 h after injection. Efficient gene transfer into endothelial cells (ECs) of intraembryonic and yolk sac vessels was observed. ECs of vessels in the head region, which undergo massive expansion around the time of injection, were efficiently labeled. However, limb bud vasculature, which starts to develop around stage 16 (HH), carried scarce (wing bud) or no (leg bud) lacZ marker. In contrast, ECs of the allantois, a structure that develops even later (around stage HH 18), expressed lacZ reporter. This observation suggests that EC precursors infected at an earlier time migrated into the allantois. A few non-endothelial cell types were also labeled by the reporter. These results suggest that adenovirus-mediated gene transfer provides a powerful tool to study angiogenesis in the developing chick embryo.     

Adenovirus-mediated transfer of RA538 gene and its antitumor effect

The RA538 cDNA was transferred into human ovarian cancer cell line SK-OV-3 and human melanoma cell line WM-983A by its recombinant adenoviral vector constructed through homologous recombination. It was demonstrated that the recombinant adenovirus could transfer RA538 gene with high efficiency, and could obviously inhibit tumor growth, with the inhibiting rates of 85% and 73% respectively, at the same time greatly repress the colony forming ability of the cells. The therapeutic experiments on transplanted subcutaneous tumor model in nude mice demonstrated that RA538 could significantly inhibit tumor growth. Flow cytometry and DNA fragmentation analysis indicated that RA538 could induce the cell cycle G1 arrest/apoptosis of the tumor cells. The expression of cmyc gene was found pronouncedly reduced by Western blot analysis. These results suggest that the RA538 recombinant adenovirus could be a promising drug in cancer gene therapy.     

Expression of endothelial nitric oxide synthase in ciliated epithelia of rats.

Endothelial nitric oxide synthase (eNOS), originally found in the endothelium of vascular tissue, also exists in other cell types, including ciliated epithelia of airways. The eNOS is ultrastructurally localized to the basal body of the microtubules of the cilia, and nitric oxide (NO) stimulates ciliary beat frequency (CBF). We examined whether the expression of eNOS is present in ciliated cells of other organs. Western blotting analysis revealed that eNOS was expressed in the rat cerebrum, lung, trachea, testis, and oviduct. Immunohistochemical staining showed that eNOS was localized in the ciliated epithelia of airways, oviduct, testis, and ependymal cells of brain in addition to the endothelium and smooth muscle of the vasculature. To confirm the activation of eNOS in the ciliated epithelia, we examined the effect of L-arginine (L-Arg), the substrate of NOS, on the production of nitrite and nitrate (NOx) in the cultured explants of rat trachea. L-Arg (100 microM) increased NOx levels significantly (p<0.05). In explants exposed to inhibitors of NOS, the effect of l-Arg on the production of NOx was blocked. These findings suggest that epithelial NO plays an important role in signal transduction associated with ciliary functions.     

Adenovirus-mediated transfer of RA538 gene and its antitumor effect

The RA538 cDNA was transferred into human ovarian cancer cell line SK-OV-3 and human melanoma cell line WM-983A by its recombinant adenoviral vector constructed through homologous recombination. It was demonstrated that the recombinant adenovirus could transfer RA538 gene with high efficiency, and could obviously inhibit tumor growth, with the inhibiting rates of 85% and 73% respectively, at the same time greatly repress the colony forming ability of the cells. The therapeutic experiments on transplanted subcutaneous tumor model in nude mice demonstrated that RA538 could significantly inhibit tumor growth. Flow cytometry and DNA fragmentation analysis indicated that RA538 could induce the cell cycle G1 arrest/apoptosis of the tumor cells. The expression of c-myc gene was found pronouncedly reduced by Western blot analysis. These results suggest that the RA538 recombinant adenovirus could be a promising drug in cancer gene therapy.     

Severe acute respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The SARS-CoV receptor, human angiotensin 1-converting enzyme 2 (hACE2), was detected in ciliated airway epithelial cells of human airway tissues derived from nasal or tracheobronchial regions, suggesting that SARS-CoV may infect the proximal airways. To assess infectivity in an in vitro model of human ciliated airway epithelia (HAE) derived from nasal and tracheobronchial airway regions, we generated recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF7a/7b) and insertion of the green fluorescent protein (GFP), resulting in SARS-CoV GFP. SARS-CoV GFP replicated to titers similar to those of wild-type viruses in cell lines. SARS-CoV specifically infected HAE via the apical surface and replicated to titers of 10(7) PFU/ml by 48 h postinfection. Polyclonal antisera directed against hACE2 blocked virus infection and replication, suggesting that hACE2 is the primary receptor for SARS-CoV infection of HAE. SARS-CoV structural proteins and virions localized to ciliated epithelial cells. Infection was highly cytolytic, as infected ciliated cells were necrotic and shed over time onto the luminal surface of the epithelium. SARS-CoV GFP also replicated to a lesser extent in ciliated cell cultures derived from hamster or rhesus monkey airways. Efficient SARS-CoV infection of ciliated cells in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.     

Efficient gene transfer to airway epithelium using recombinant Sendai virus

Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.