Identification of polyamides that enhance adenovirus-mediated gene expression in the urothelium.

Adenovirus-mediated gene therapy of bladder diseases has been limited by the inability to transduce the urothelium successfully using adenoviral vectors. We have sought to identify agents that would increase adenovirus-mediated transgene expression in the bladder. We have utilized a rat model to screen compounds for their ability to enhance viral transgene expression in the rat bladder. Rats received intravesical administration of replication-deficient adenovirus (rAd) formulated in various agents, and transgene expression was evaluated after 48 h by determining the amount of lacZ expression in the luminal epithelium of the bladder. We report the identification of two different polyamides, each capable of dramatically increasing viral transgene expression in the bladder without causing detectable alteration of the umbrella cell layer of the urothelium. We have utilized a carcinogen-induced rat bladder tumor model to demonstrate that these polyamides are also capable of enhancing viral transgene expression in tumor tissue. The identification of these polyamides potentiates the use of adenovirus-mediated gene therapy for the treatment of superficial bladder cancer or other bladder diseases.

Ethanol improves adenovirus-mediated gene transfer and expression to the bladder epithelium of rodents.

OBJECTIVES: Replication deficient adenoviral vectors (rAds) are used as gene delivery systems that can efficiently transduce a variety of tissues and may be appropriate vectors to develop gene therapeutics for many urologic applications. However, the bladder epithelium has been shown to be highly resistant to transgene expression after intracystic administration. A potential explanation for this low gene transfer efficiency may be the protective structure of the urothelium. Since this protective barrier can be disrupted by organic solvents, we assessed whether ethanol co-administration can enhance adenovirus-mediated transgene expression. METHODS: Normal and bladder tumor-bearing rats received a single intracystic administration of rAd encoding beta-galactosidase (rAd-beta gal) or p53 (rAd-p53). rAd was administered in a saline solution or in solutions with increasing concentrations of ethanol. Transgene expression was evaluated in the bladder tissues. RESULTS: A dramatic increase in urothelial beta-galactosidase transgene expression was achieved by rAd-beta gal administered in a 22% ethanol solution. Transgene expression was enhanced in normal urothelium and in superficial bladder tumors. p53 transgene expression was similarly enhanced. CONCLUSIONS: Co-administration of 22% ethanol enhanced local rAd-mediated transgene expression in the normal and neoplastic bladder epithelium in rodents. Improvement of rAd-mediated transgene expression is progress toward local gene delivery to the urothelium and may enable local gene therapy for superficial bladder cancer or other bladder diseases.

c-Fos induction in the rat nucleus of the solitary tract by intraoral quinine infusion depends on prior contingent pairing of quinine and lithium chloride.

Intraoral infusions of sucrose or saccharin induce c-Fos-like immunoreactivity (c-FLI) in the intermediate nucleus of the solitary tract (iNTS) of rats after acquisition of a conditioned taste aversion (CTA). The induction of c-FLI in the iNTS may be a consequence of the shift in behavioral response from ingestive to aversive behaviors that characterize acquisition and expression of a CTA. To test this hypothesis, rats were intraorally infused with 0.3 mM quinine sulfate, an aversive taste, 1. prior to conditioning, 2. after 3 noncontingent (unpaired) infusions of quinine and toxic lithium chloride (LiCl) injections, 3. after conditioning with 3 contingent pairings of quinine and LiCl, and 4. after extinction with repeated unpaired infusions of quinine. Intraoral infusions of quinine induced c-FLI in the iNTS only after acquisition of a CTA against quinine; quinine failed to induce c-FLI in the iNTS of unconditioned, noncontingently treated, or extinguished rats. The pattern of c-FLI in the iNTS induced by expression of a CTA against quinine was quantitatively and anatomically similar to that elicited by sucrose in rats expressing a CTA against sucrose. We conclude that aversive responses per se are not sufficient to induce c-FLI in the iNTS. Furthermore, contingent pairing of quinine and LiCl does not cause a shift in behavioral response from palatable, ingestive behaviors to aversive behaviors as in acquisition of a CTA against sucrose. Thus, we also conclude that a shift in behavior from ingestive to aversive responses is not required for increased c-FLI expression in the iNTS during CTA expression. Therefore, the induction of c-FLI in the iNTS during expression of a CTA may be correlated with neuronal processes specific to acquisition and expression of a CTA.

c-Fos induction in the rat nucleus of the solitary tract correlates with the retention and forgetting of a conditioned taste aversion.

Recently, we have described a potential neuronal correlate of the behavioral expression of a conditioned taste aversion (CTA) against sucrose at the level of c-Fos expression. Intraoral infusions of sucrose induce c-Fos-like immunoreactivity (c-FLI) in the intermediate nucleus of the solitary tract (iNTS) after a CTA has been acquired for sucrose. Sucrose infusions do not induce c-FLI in the iNTS of unconditioned rats or in conditioned rats after extinction of the CTA. Here, we describe persistence of altered responsiveness of the iNTS in rats with CTAs against sucrose by intraorally infusing sucrose 2 days, 3 months, or 6 months after acquisition of the CTA. Sucrose infusions induced c-FLI in the iNTS 6 months after conditioning. The behavioral expression of the CTA was attenuated at 6 months but not at 3 months; the number of c-FLI positive cells in the iNTS was proportional to the magnitude of the expression of the CTA. This evidence strengthens our hypothesis that c-FLI in the iNTS is a neuronal correlate of the expression of a CTA.

Increased c-fos expression in nucleus of the solitary tract correlated with conditioned taste aversion to sucrose in rats.

The pattern of neuronal activation in the rat nucleus of the solitary tract (NTS) in response to a standard gustatory stimulus was examined using c-Fos-like immunoreactivity (c-FLI) before and after conditioned taste aversion (CTA) formation. While unconditioned oral infusions of sucrose solution did not induce c-FLI in the NTS, after three pairings of sucrose with lithium chloride injections, sucrose induced c-FLI in the medial intermediate NTS 1 h after oral infusion. Extinction of the CTA by repeated infusions of sucrose alone reversed the induction of c-FLI.