CD4+CD25+ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge.

Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4(+)CD25(+) regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8(+), CD4(+) T cells and antibody response. The appearance of a CD4(+) T-cell response correlated with a stronger memory response. The combined CD25(+) inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.

2-(2,3-Dihydro-5-acetoxy-4,6,7-tribenzofuranyl)acetic acid (IRFI 016): a new antioxidant mucoactive drug.

IRFI 016 has demonstrated significant antioxidant activity, inhibiting hepatic lipid peroxidation (rat intoxicated by CCl4) and the formation of gastric lesions by ethanol (rat). This activity proved equal to or better than that exhibited by the most investigated antioxidant/radical scavenger agents (such as BHA, BHT, Vitamin E). The drug markedly increased mucus production (rabbit, mouse) by all the administration routes used (os, i.v. and inhalatory) and proved more active than, or overlapping, the most noted mucoregulatory/mucolytic drugs (sobrerol, bromexine, thiopronine, ambroxol, N-acetylcysteine) which were chosen for comparison. The tracheo-bronchial mucus viscosity was also significantly reduced (bronchitic animals) as was the fucose and total protein content. In the pigeon, IRFI 016 improved mucociliary clearance. Moreover IRFI 016 evidenced anti-inflammatory activity nearly equal to that exhibited by ASA and phenylbutazone (carrageenin oedema, abcesses and inflammatory pain).

Effect of erdosteine and its metabolites on tracheobronchial mucus production and transport.

The effect of erdosteine and its metabolites on tracheobronchial mucus production and transport was studied. Erdosteine showed important muco-regulating action (increase of mucus production), and also influenced muciliary clearance. Erdosteine, after intravenous administration, was more active than the muco-regulating drugs used for comparative purposes (about 4 times as active as N-acetyl-cysteine; about 1.8 times as active as sobrerol and bromexine; and about 1.4 times as active as ambroxol). After oral administration erdosteine showed significant action on mucus production, causing an effect quantitatively the same as that produced by bromexine. Erdosteine and its metabolites also increased TMV in pigeons after intravenous administration. Moreover, erdosteine and its metabolites were significantly active and their acute toxicity was extremely low.

Compared kinetics of 2-(2,3-dihydro-5-acetoxy-4,6,7-trimethylbenzofuranyl) acetic acid (IRFI 016) and its active metabolite 2-(2,3-dihydro-5-hydroxy-4,6,7-trimethylbenzofuranyl) acetic acid (IRFI 005) in plasma and bronchial alveolar liquid in mice.

The concentration of 2-(2,3-dihydro-5-acetoxy-4,6,7-trimethylbenzofuranyl) acetic acid (IRFI 016) and its active metabolite 2-(2,3-dihydro-5-hydroxy-4,6,7-trimethylbenzofuranyl) acetic acid (IRFI 005) in bronchial alveolar liquid (BAL) and plasma of mice were studied. IRFI 016 and its active metabolite IRFI 005 are both present in BAL and plasma after oral administration of IRFI 016. In BAL no delay times were noted, in comparison with plasma, regarding Cmax time nor significant variation of t1/2 and the elimination constant (Kel). IRFI 016, orally administered, is very rapidly absorbed and, both in unaltered form and as its active metabolite, reaches the anatomic site where it carries out its principal pharmacological activity, according to the same kinetic course observed in plasma.

Fructose-1,6-diphosphate as an in vitro and in vivo anti-alcohol agent in the rat.

Fructose-1, 6-diphosphate (FDP) decreases the effect of ethanol on Ca++ entry and inhibits the ethanol-stimulated phosphate efflux in rat heart slices. FDP also inhibits the ethanol-stimulated [36Cl-]-uptake by rat brain microvesicles and affects the isolated GABA-receptor in a way opposite to that of ethanol. The in vivo effects of FDP include a dose-dependent decrease in ethanol-induced gastric ulcers and a decrease in the serum transaminase levels raised by chronic ethanol administration. Other central actions of ethanol such as diuresis, narcosis, dependence and withdrawal symptoms are also counteracted by FDP.

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus.

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance-i.e., 100% of treated animals showing sustained expression after 4 months-was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.