Factors determining the sensitivity and specificity of the spot-blot hybridization technique for the detection of hepatitis B virus DNA in serum were systematically investigated. Methods for pretreatment of serum samples, mode of application of the samples to the transfer membranes, blot treatment and hybridization conditions were all found to affect the sensitivity of the assay. The optimum hybridization procedure was found to be incubation of serum samples with salt, NaOH, formaldehyde and detergent, followed by spot application of the samples. This method specifically detected hepatitis B virus DNA in serum with a sensitivity 5 to 15 times higher than the presently used assay procedures.

DNA, Viral↗

PubMedDate not supplied

Hepatocellular carcinoma in alcoholic liver disease: no evidence for a pathogenetic role of hepatitis B virus infection.

Hepatocellular carcinoma tissues from HBsAg-negative patients with chronic alcoholic liver disease were investigated for the presence of hepatitis B virus DNA. Southern blot analyses of DNA extracted from the hepatocellular carcinomas were negative for hepatitis B virus DNA in all 17 patients examined, at a level of sensitivity of less than 0.01 genome equivalent per cell. Similarly, in liver tissues from another 30 patients with alcoholic cirrhosis without hepatocellular carcinoma, no hepatitis B virus DNA was detectable. We conclude that in our patients there is no molecular evidence for a contribution of hepatitis B virus infection to the development of hepatocellular carcinoma in alcoholic liver disease.

Adult↗

PubMedDate not supplied

Encapsulation of plasmid DNA in PLGA-stearylamine microspheres: a comparison of solvent evaporation and spray-drying methods.

Stearylamine, a positively charged hydrophobic molecule, was tested as a formulation agent for the encapsulation of a model plasmid in PLGA microspheres. The primary objective was to compare the spray-drying and double emulsion solvent evaporation methods and evaluate their suitability for fabricating PLGA-stearylamine plasmid-entrapped microspheres. A luciferase reporter gene plasmid (pGL3-Con) was formulated into microspheres using a 64 kDa PLGA 50:50 polymer blended with stearylamine (SA) at a range of concentrations up to 15%m/m, by the solvent evaporation and spray-drying methods. The microspheres were characterized regarding their size distributions, zeta potentials and morphology by laser diffraction, electrophoretic mobility and scanning electron microscopy (SEM), respectively. Formulated plasmid extracts were assessed for physical damage by agarose gel electrophoresis, and the in vitro biological activity was determined by transfection of a human embryo kidney epithelial (293) cell line. Size distribution analysis showed that SA reduced the median diameters of spray-dried particles from 8.32 to 3.64 microns, with a corresponding reduction in the spread of the distribution, but solvent evaporation microspheres showed an increased median diameter on addition of SA. Concentrations of SA above 10%(m)/(m) resulted in disruption of the smooth morphology of the solvent evaporation particles. There was a SA concentration-dependent tendency in the increase of surface positive charge and resistance to serum nuclease assault and in vitro expression of luciferase protein. These results show that SA and possibly other charged hydrophobic molecules may be useful agents in the formulation of particulate DNA vaccines by both methods.

Amines↗

PubMedDate not supplied