Striatal readministration of rAAV vectors reveals an immune response against AAV2 capsids that can be circumvented.

Recombinant adeno-associated virus (rAAV) expresses no viral genes after transduction. In addition, because the brain is relatively immunoprivileged, intracranial rAAV transduction may be immunologically benign due to a lack of antigen presentation. However, preexposure to AAV allows neutralizing antibodies (nAbs) to block brain transduction and rAAV readministration in the brain leads to an inflammatory response in the second-injection site. In this study, we replicate our striatal rAAV2/2-GDNF readministration results and extend this effect to a second transgene, green fluorescent protein (GFP). Unlike rAAV2/2-GDNF readministration, striatal rAAV2/2-GFP readministration leads to a loss of transgene in the second site in the absence of detectable circulating nAbs. In order to determine whether the transgene or the AAV2 capsid is the antigenic stimulus in brain for the immune response in the second site, we readministered rAAV2/2-GFP using two different rAAV serotypes (rAAV2/2 followed by rAAV2/5). In this case, there was no striatal inflammation or transgene loss detected in the second-injection site. In addition, striatal readministration of rAAV2/5-GFP also resulted in no detectable immune response. Furthermore, delaying rAAV2/2 striatal readministration to a 11-week interval abrogated the immune response in the second-injection site. Finally, while striatal readministration of rAAV2/2 leads to significant loss of transgene in the second-injection site, this effect is not due to loss of vector genomes as determined by quantitative real-time PCR. We conclude that intracellular processing of AAV capsids after transduction is the immunogenic antigen and capsid serotypes that are processed more quickly than rAAV2/2 are less immunogenic.

Does reduction of circulating prostaglandin E2 reduce fetal hypothalamic-pituitary-adrenal axis activity?

OBJECTIVE: Placental production of prostaglandin E2 (PGE2) increases in fetal sheep as term approaches. It has been suggested that placental PGE2 may act as a hormone to activate the fetal hypothalamic-pituitary-adrenal (HPA) axis. Alternatively, we have proposed that local generation of prostaglandins in the fetal brain and/or pituitary might play a more prominent role in the stimulation of fetal adrenocorticotropin (ACTH) secretion. We performed the present experiments to test the hypothesis that the elevated concentrations of PGE2 in fetal plasma do not tonically stimulate fetal ACTH secretion. METHODS: We studied chronically catheterized late-gestation fetal sheep. Selective inhibitors of prostaglandin synthase-1 and -2 (PGHS-1 and PGHS-2) were injected intravenously. Fetal blood pressure and heart rate were monitored continuously, and circulating concentrations of PGE2, ACTH, and cortisol were measured by specific immunoassay. RESULTS: Injection of vehicle did not have an effect on circulating levels of PGE2 or ACTH, but it did have a mild stimulatory effect on cortisol. The selective PGHS-2 inhibitor, Nimesulide (Cayman Chemical, Ann Arbor, MI), significantly decreased plasma PGE2 concentrations. The selective PGHS-1 inhibitor, Resveratrol (Cayman Chemical), produced smaller decreases in plasma PGE2 concentrations and significantly increased mean arterial blood pressure. Neither inhibitor significantly altered plasma ACTH or cortisol concentrations. CONCLUSION: These results demonstrate that reduction of circulating PGE2 concentrations in response to intravenous injection of PGHS-1 and PGHS-2 inhibitors does not reduce fetal HPA axis activity. We conclude that PGE2 in late-gestation ovine fetal plasma does not tonically stimulate fetal ACTH secretion.