Adenovirus-mediated transduction of intestinal cells in vivo.

The intestinal tract has many features that make it an attractive target for therapeutic gene transfer. In this study, replication-defective adenoviral vectors were used to explore parameters that may be important in administering gene therapy vectors to the intestine. After surgically accessing the intestine, an E1-, E3-deleted adenoviral vector encoding beta-galactosidase (beta-Gal) was directly injected into various regions of the small and large intestine of rats and rabbits. Significant transduction of the tissue was observed and histochemical staining was used to identify enterocytes as the primary targets of gene transfer. Expression of beta-Gal did not differ substantially when the virus was administered to the duodenum, ileum, or colon. When the vector was directly administered to segments of the distal ileum containing a Peyer's patch, transgene expression was approximately 10-fold higher than in segments lacking a Peyer's patch. In the Peyer's patches, a high level of expression was localized to epithelial cells, potentially M cells, overlying the lymphoid follicle domes. Transduction of these cells could have application in DNA-mediated oral vaccination. Administration of an adenoviral vector encoding a secreted alkaline phosphatase to the lumen resulted in expression and secretion of this gene product into the circulation. This finding demonstrates the potential of enterocytes to serve as heterotopic sites for the synthesis of heterologous gene products that would be secreted into the lumen of the intestinal tract or into the bloodstream.

Central administration of morphine inhibits brain and liver ornithine decarboxylase activity in neonatal rats: involvement of transcription- and non-transcription-dependent mechanisms.

This study examined whether the developmental deficits usually observed in infants born to opiate addicted mothers could involve effects on ornithine decarboxylase, a growth-controlling enzyme. Intracerebroventricular (i.c.v.) injection of a single dose of morphine (2 microg) to 6-day-old rats markedly decreased basal brain and liver ornithine decarboxylase activity as well as the increases in hepatic ornithine decarboxylase activity produced by subcutaneously (s.c.) administered insulin, an important trophic hormone. Centrally applied morphine acts supraspinally to downregulate peripheral ornithine decarboxylase activity, since s.c. administration of the same dose as used i.c.v. decreased neither basal liver ornithine decarboxylase levels nor tissue responsiveness to insulin. This does not imply that the opiate is unable to affect ornithine decarboxylase when applied systemically. In fact, a robust inhibition of both basal and induced liver ornithine decarboxylase activity was obtained in rat pups given 20 microg of morphine s.c. This larger dose is able to trigger the hepatic ornithine decarboxylase effects presumably by stimulating opiate receptors located at central sites after crossing the blood-brain barrier and penetrating into the brain. Concomitant administration of naloxone plus morphine i.c.v. prevented morphine from downregulating ornithine decarboxylase activity, confirming the participation of supraspinal opioid receptors in morphine ornithine decarboxylase actions. Finally, as was the case for insulin induced stimulation of ornithine decarboxylase activity, i.c.v. injection of morphine markedly diminished insulin induced stimulation of hepatic ornithine decarboxylase mRNA accumulation. In turn, contrary to the inhibition of basal ornithine decarboxylase activity, morphine did not lower basal hepatic ornithine decarboxylase mRNA levels when given alone. Thus, CNS morphine can apparently suppress tissue ornithine decarboxylase expression through both transcriptional and posttranscriptional mechanisms. The evidence obtained suggest that postnatal exposure to opiate drugs might detrimentally affect development by altering normal tissue ornithine decarboxylase ontogeny.

Rapid down-regulation of GABAA receptors in the gerbil hippocampus following transient cerebral ischemia.

During transient cerebral ischemia, there is a temporary and robust accumulation of extracellular GABA in the hippocampus. We examined whether the acute exposure of GABAA/benzodiazepine receptors to high concentrations of GABA early after ischemia results in receptor down-regulation as observed in vitro. Gerbils were killed 30 and 60 min following a 5-min bilateral carotid occlusion, and their brains were prepared for receptor autoradiography. The hydrophilic, GABAA receptor antagonist [3H]SR-95531 and the hydrophobic benzodiazepine agonist [3H]flunitrazepam were used to distinguish between cell surface and internalized receptors. Ischemia significantly decreased [3H]SR-95531 binding in hippocampal areas CA1 and CA3 and in the dentate gyrus 30 min after ischemia. Scatchard analysis in area CA1 revealed that ischemia decreased the Bmax as low as 44%. The affinity of the remaining sites was increased substantially (72% decrease in KD). As expected, there were no changes in the binding of [3H]flunitrazepam to hippocampus in the early postischemic period because the benzodiazepine could bind to both internalized receptors and those on the cell surface. We hypothesize that prolonged exposure (approximately 30-45 min) of GABAA receptors to high concentrations of synaptic GABA in vivo causes receptor down-regulation, perhaps via receptor internalization.