Al oh 3

Six female Balb/c mice in each group were sensitized with 100 µg of OVA (Sigma-Aldrich, Steinheim, Germany) formulated with 20 μg of Al(OH)₃ (Sigma-Aldrich) in a total volume of 200 μl by intraperitoneal injection (ip) on day 0, 14 and 21. For preventive groups, mice were treated intraperitoneally with 50 μg of Ts-AE or Ts-MLE prior to the OVA sensitization on days − 21, − 14 and − 7. For therapeutic groups, mice were co-administrated intraperitoneally with 50 μg of Ts-AE or Ts-MLE during the OVA sensitization on day 0, 14 and 21. One week after the last sensitization, all mice were challenged intranasally (in) with 100 μg of OVA in a total volume of 50 μl of sterile PBS under sedation of chloral hydrate (8 µg/20 g) for consecutive three days (day 28, 29 and 30) while mice in therapeutic groups were co-administrated intranasally with 25 μg of Ts-AE or Ts-MLE during the OVA challenge (Fig. 1). One group of mice was OVA-sensitized and challenged without treatment as non-treatment control. Another group of mice without treatment and challenge was used as normal control. Forty-eight hours after the last challenge, all mice were euthanized and the lung tissues were collected for inflammatory assessment, and bronchoalveolar lavage fluid (BALF) and sera were collected for immunological tests. All experiments were repeated once.Fig. 1

Regimen for mouse OVA sensitization and treatment

In a typical synthesis, aluminum hydroxide [Al(OH)₃, Sigma-Aldrich] was dissolved in an aqueous solution of the OSDA in its hydroxide form. Colloidal silica (Ludox AS-40, Aldrich) was then added, and the mixture was maintained under stirring for 20 minutes. If required, a 10% wt solution of NH₄F (Sigma-Aldrich) was added, and the resultant mixture gel was allowed to reach the desired silica to water ratio by evaporation under stirring. The final gel compositions were: SiO₂ : 0.0167–0.033Al₂O₃ : 0.2–0.4OSDA(OH)₂: 0–0.4NH₄F : 3–30H₂O, where OSDA can be one of the dicationic molecules described above.
Finally, the gels were transferred to Teflon lined stainless autoclaves and heated at 150 °C for 10 days. The solids were recovered by filtration, extensively washed with distilled water, and dried at 90 °C overnight. The samples were calcined in air at 550 °C for 4 hours. The resultant solid yields have been calculated based on silica + alumina conversion.

Antibody against C3 complement component was from Calbiochem. Goat anti-Mouse IgG (H + L) Secondary Antibody, Alexa Fluor® 488 conjugate, and Goat anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor® 488 conjugate were from Molecular Probes.
Baby rabbit complement and Guinea Pig complement were from Cederlane Labs. AL(OH)₃ was purchased from Sigma-Aldrich.

Groups of 6 to 12 Balb/c mice were sensitized i.p. on days 0 and 7 with 2 mg/kg chicken egg albumin (OVA; Sigma-Aldrich, A5503) adsorbed on 80 mg/kg aluminium hydroxide (Al(OH)₃; Sigma-Aldrich) in saline. Under i.p. ketamine and xylazine anaesthesia (respectively: Imalgene®, Merial, 50 mg/kg; Rompun®, Bayer, 3.33 mg/kg), sensitized mice were challenged daily on days 18–21 by intranasal (i.n.) instillation of 400 μg/kg OVA in saline, or saline alone for controls (1 mL/kg) [18 (link)]. Control animals were sensitized with OVA and challenged with saline.