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. 2000 Oct;47(4):563–570. doi: 10.1136/gut.47.4.563

Assessment of efficiency and safety of adenovirus mediated gene transfer into normal and damaged murine livers

T Nakatani 1, S Kuriyama 1, K Tominaga 1, T Tsujimoto 1, A Mitoro 1, M Yamazaki 1, H Tsujinoue 1, H Yoshiji 1, S Nagao 1, H Fukui 1
PMCID: PMC1728072  PMID: 10986218

Abstract

BACKGROUND—When recombinant adenoviruses are infused directly into the circulation, transgene expression is almost completely restricted to the liver.
AIMS—Efficiency and safety of adenovirus mediated gene transfer into damaged livers were examined in mice with liver cirrhosis or fulminant hepatitis.
METHODS—Liver cirrhosis and fulminant hepatitis were induced by intraperitoneal administration of thioacetamide and D-galactosamine followed by lipopolysaccharide, respectively. Mice were infused with adenoviruses carrying the Escherichia coli β-galactosidase gene, lacZ gene, into the tail vein. Transduction efficiency of the lacZ gene was estimated histochemically by X-gal staining and quantitatively using a chemiluminescent assay. Activation of adenovirus specific T cells and development of neutralising antibodies against adenovirus were also examined.
RESULTS—Histochemical evaluation revealed that approximately 40%, 80%, and 40% of cells in normal, cirrhotic, and fulminant hepatitis livers, respectively, were stained blue using X-gal staining. Quantitative analyses revealed that levels of lacZ expression in cirrhotic livers were approximately 2.5-fold and sixfold greater than those in normal and fulminant hepatitis livers, respectively. Although transgene expression in fulminant hepatitis livers was significantly lower than that in normal livers, marked levels of transgene expression were achieved even in fulminant hepatitis livers. Significant adverse effects of adenoviruses were not observed in damaged livers. There were no significant differences in cellular or humoral immune responses to adenoviruses among animals with normal, cirrhotic, and fulminant hepatitis livers.
CONCLUSIONS—Our results suggest that gene therapy with adenoviruses may be used efficiently and safely, even in patients with severe liver disease.


Keywords: adenovirus; transduction efficiency; safety; liver cirrhosis; fulminant hepatitis

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Figure 1  .

Figure 1  

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Histological analysis of fulminant hepatitis and liver cirrhosis models in mice. Haematoxylin-eosin staining of the liver of mice administered with D-galactosamine and lipopolysaccharide revealed acute severe hepatitis with massive haemorrhagic necrosis of hepatocytes, disarray of the lobules, and marked infiltration of inflammatory cells (A). Azan staining of the liver of mice treated with thioacetamide for 32 weeks revealed an increase and thickening of the fibrotic septa, traversing the lobular parenchyma, and formation of pseudolobuli (B). (Original magnification ×40.)

Figure 2  .

Figure 2  

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LacZ gene expression in the liver by intravenous administration of adenoviruses. Mice with normal, cirrhotic, and fulminant hepatitis livers were infused intravenously with adenoviruses (2×108 pfu/200 µl) carrying the lacZ gene. Animals were killed on day 4 after adenoviral infusion and lacZ gene expression was examined by X-gal staining. Approximately 40%, 80%, and 40% of the whole liver was stained blue in the normal (A), cirrhotic (B), and fulminant hepatitis (C) livers, respectively. (Original magnification ×40.)

Figure 3  .

Figure 3  

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Quantitative estimation of lacZ expression in the liver by intravenous administration of adenoviruses. Mice with normal, cirrhotic, and fulminant hepatitis livers were infused intravenously with adenoviruses (2×108 pfu/200 µl) carrying the lacZ gene. Animals were killed on day 4 after adenoviral infusion and lacZ gene expression was quantitatively estimated using the chemiluminescent reporter gene assay system. Each group consisted of eight animals. *0.01<p<0.05, ***p<0.001.

Figure 4  .

Figure 4  

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Cellular immune responses to adenoviral administration. To examine cellular immune responses to intravenous administration of adenoviruses, splenic cells were collected not only from naive mice that had normal livers and were not given the adenoviral infusion but also from mice with normal, cirrhotic, or fulminant hepatitis livers and given adenoviral infusion. Splenic cells were incubated with or without heat inactivated adenoviruses for five days and pulsed with [3H] thymidine for the last 18 hours of incubation. Each bar represents the mean (SD) of eight animals. There were no significant differences in proliferation of splenic cells between adenovirus stimulated and unstimulated cells in all groups. There were also no significant differences in proliferation among adenovirus stimulated splenic cells collected from naive mice and adenovirus treated mice with normal, cirrhotic, and fulminant hepatitis livers.

Figure 5  .

Figure 5  

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Humoral immune responses to adenoviral administration. To examine the development of neutralising antibodies against adenoviruses, serum samples collected from mice with normal, cirrhotic, and fulminant hepatitis livers were decomplemented, serially diluted, and analysed for neutralising antibodies against adenovirus serotype 5. The titre of neutralising antibody for each sample is expressed as the reciprocal dilution of serum that inhibited adenoviral infection by 50%. Each bar represents the mean (SD) of eight animals. There were no significant differences in titres of neutralising antibodies between groups.

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