Immediate Inflammatory Responses to Adenovirus-Mediated Gene Transfer in Rat Salivary Glands

Abstract

Although replication-deficient adenoviruses can efficiently transfer genes to the salivary glands, the current vectors precipitate an immediate, transient decrease in salivary function. To study the cause of this salivary hypofunction, 10(6)-10(10) plaque-forming units (pfu) of the vector AdCMV beta gal were delivered by retrograde ductal infusion to the submandibular glands (SMGs) of rats. Microscopic analysis of infected glands showed a dose-related, rapidly developing inflammatory response, which at the highest amount of virus was characterized by a predominantly neutrophil-containing infiltrate, focal necrosis, and edema. Moreover, the glands of nude rats developed similar morphologic changes to those of immunocompetent rats. After 3 days, the volume of stimulated saliva secreted from SMGs receiving AdCMV beta gal (6.75 x 10(9) pfu) was approximately 20% that of controls. UV-inactivated virus caused a similar decrease in saliva output. We evaluated to what extent the anti-inflammatory glucocorticoid, dexamethasone, could suppress inflammation and preserve salivary function. Three days after infusion with a high dose of AdCMV beta gal (6.75 x 10(9) pfu), the glands from dexamethasone-treated animals showed markedly less inflammation and no necrosis. Furthermore, there was no significant difference in the average amount of saliva secreted from the infected glands (105 +/- 17 microliters) compared to the control glands (123 +/- 18 microliters). In addition, dexamethasone extended the expression of beta-galactosidase in the SMGs. These results suggest that the adenovirus-mediated acute inflammation in rat SMG is responsible for diminished gland function and transgene expression. Furthermore, we demonstrate a useful role for glucocorticoids in controlling acute inflammation during experimental gene transfer with current adenovirus vectors.

Safety and efficacy of adenovirus-mediated transfer of the human aquaporin-1 cDNA to irradiated parotid glands of non-human primates

“…16 Recombinant adenoviruses were plaque-purified and isolated by equilibrium centrifugation on a CsCl gradient as reported previously. 17 Using these methods, we have been able to show that there are Ͻ1 in 10,000 E1A-positive particles in our vector preparation. 17 All experiments performed herein used a single batch of AdhAQP1 with a particle to plaque-forming unit (PFU) ratio of 273:1 and a final titer of 3.4 ϫ 10 10 PFU/mL.…”

“…17 Using these methods, we have been able to show that there are Ͻ1 in 10,000 E1A-positive particles in our vector preparation. 17 All experiments performed herein used a single batch of AdhAQP1 with a particle to plaque-forming unit (PFU) ratio of 273:1 and a final titer of 3.4 ϫ 10 10 PFU/mL. Virus was stored in dilution buffer (10 mM tris(hydroxymethyl)aminomethane (pH 7.4), 1 mM MgCl 2 , and 10% glycerol) at Ϫ80°C until use.…”

“…This likely represents a secondary effect, reflecting an increase in the polymorphonuclear cells associated with the transient acute inflammatory response to the administration of recombinant adenovirus, as reported previously. 17 Effect of AdhAQP1 on parotid salivary flow rates The response to salivary gland administration of AdhAQP1 was variable (Fig 3), although generally similar in both the right and left glands. The results are depicted as the percent change from the previral timepoint (PRE-V, which is the same as the 19 weeks post-IR timepoint, IRϩ19 week, in Fig 2).…”

Pulmonary inflammation associated with repeated, prenatal exposure to an E1, E3-deleted adenoviral vector in sheep

“…E1, E3-deleted adenovirus vectors inhibit cell proliferation in vitro by promoting apoptosis and slowing the cell cycle 34 and inhibit cell function and precipitate a rapidly developing inflammatory response characterized by infiltrating neutrophils, focal necrosis and edema when injected into the submandibular glands of rats. 35 It is possible that the very young fetuses used in the present were particularly susceptible to the harmful effects of the vectors. The high incidence of mortality may also be related to the Hickman catheter used in group 2 animals (n = 7) that all died in utero.…”

Systemic action of human growth hormone following adenovirus-mediated gene transfer to rat submandibular glands

“…All animals received 1 mg of dexamethasone at the time of gene transfer (intramuscularly) and each day until death. 27 At the end of the experiments, animals were anesthetized and blood samples obtained by cardiac puncture. Serum samples were analyzed as described below.…”