Hepatocarcinoma cells constitutively expressing adenoviral E1-genes provide a tumor model for intratumoral replication of E1-deficient adenoviruses

OBJECTIVES:In this study, we investigated the effects of angiotensin II and angiotensin-(1-7) at the nucleus tractus solitarii (nTS) in transgenic rats with a severe deficit in brain angiotensinogen production, TGR(ASrAOGEN) (TGR). METHODS: Angiotensin II (10 pmol), angiotensin-(1-7) (10 pmol) or NaCl (0.9%/50 nl) were microinjected into the nTS of urethane-anaesthetized TGR (n = 36) and Sprague Dawley (SD) (n = 34) rats. Mean arterial pressure (MAP) and heart rate were measured via a femoral artery catheter and the baroreflex control of heart rate was evaluated after increases in MAP induced by phenylephrine (baroreflex bradycardia). RESULTS: Angiotensin II microinjections into the nTS of the TGR induced a higher decrease in MAP and heart rate (-37 +/- 5 mmHg and -69 +/- 12 b.p.m., respectively) in comparison to SD rats (-18 +/- 1 mmHg and -43 +/- 5 b.p.m., respectively). In contrast, changes after angiotensin-(1-7) microinjections into the nTS of TGR (-6 +/- 1 mmHg and -13 +/- 4 b.p.m.) were significantly smaller than that induced in SD (-11 +/- 2 mmHg and -24 +/- 6 b.p.m.). The baseline baroreflex sensitivity to phenylephrine of TGR was accentuated in comparison to SD rats (0.70 +/- 0.06 versus 0.44 +/- 0.03 ms/mmHg). Angiotensin II microinjection into the nTS produced similar attenuation in the baroreflex bradycardia in both SD (0.28 +/- 0.07 versus 0.5 +/- 0.07 ms/mmHg, before injection) and TGR (0.44 +/- 0.1 versus 0.82 +/- 0.1 ms/mmHg, before injection). In contrast, angiotensin-(1-7) microinjection elicited a facilitation of the baroreflex bradycardia in SD (0.68 +/- 0.12 versus 0.41 +/- 0.03 ms/mmHg, before injection), while in TGR, angiotensin-(1-7) induced an attenuation of baroreflex bradycardia (0.34 +/- 0.07 ms/mmHg versus 0.55 +/- 0.05 ms/mmHg, before injection). CONCLUSIONS: These results indicate that a permanent inhibition of angiotensinogen synthesis in the brain can lead to an increase in the sensitivity of the baroreflex control of heart rate (baroreflex bradycardia) and an increase in angiotensin II responsiveness at the nTS. However, the nTS effect of angiotensin-(1-7) was significantly attenuated in these transgenic rats. These data further indicate that the decrease in brain angiotensins in the transgenic rats may be functionally relevant and support the concept of differential regulatory mechanisms for the effects of the two angiotensin peptides

Titer determination of Ad5 in blood: a cautionary note

Recombinant adenoviruses are presently the most efficient in vivo gene transfer system available. Targeting single organs or large tumors by adenoviral vectors requires an intravascular route of application. During the first pass of viral particles through the vascular bed of the target tissue, virus uptake is not quantitative and undefinite amounts of particles leak into circulation. To determine the amount of leaking particles and to calculate organ-specific uptake (in-/outflow ratio), it is necessary to titrate virus particles directly in blood. In preclinical and clinical trials titration is currently mostly done with blood plasma instead of full blood. However, this technique provides valid results only as long as there is no affinity between adenovirus particles and erythrocytes. In this study we demonstrate that Ad5 particles, as mostly employed for gene therapy, have a strong affinity to human erythrocytes. At 60 min after coincubation of human erythrocytes and Ad5 particles, more than 98% of the particles are attached to the surface of erythrocytes. Therefore, ignoring the amount of red cell bound particles by performing titration in plasma leads to severe miscalculation of organ-specific transfer rates or virus circulation half-life. The biological impact of an increased affinity between virus particles and erythrocytes will be discussed