Aluminium hydroxide

Twenty BALB/c mice were divided into four groups and treated with PBS, OVA, OVA + L900/2 or OVA + L900/3 (4–6 mice per group). Mice were sensitized by intraperitoneal (i.p.) injections of 10 mg of OVA (Worthington, Lakewood, NJ, USA) or 10 mg of OVA + 60 μg of each polysaccharide adsorbed to 100 μl of aluminium hydroxide (Alum, Serva, Germany) in a final volume of 200 μl, on days 1 and 14. Control mice received 100 μl PBS and 100 μl aluminium hydroxide. On day 7 after the last immunization, the mice were euthanized by cervical dislocation. Blood was collected, and serum samples were stored at −40°C until analysis. Mesenteric lymph nodes (MLN, pooled per group) and spleens were aseptically removed and prepared for cytokine determination. All experiments were repeated twice using four to six mice per group each time.

Allergic airway inflammation was performed as previously described [21 (link)]. Briefly, mice were sensitised three times intraperitonealy (on day 0, 14 and 28) with 1 μg of recombinant Bet v 1 (Biomay, Vienna, Austria) precipitated with 100 μl aluminium hydroxide (Alum, Serva, Heidelberg, Germany). Seven days after the last treatment, mice were challenged intranasally three times (day 35, 36 and 37) with 100 μg of birch pollen extract (Allergon, Välinge, Sweden). 20 μg of Bv1-Protein-FAM or Bv1-Peptide-FAM were applied intranasally at day 40. NALT and lungs were excised 1 or 6 hours later.

Airway inflammation was induced in female 6–8 weeks old BALB/c mice with ovalbumin (OVA; grade V; Sigma-Aldrich, St. Louis, MO) as previously described [17 (link)]. Briefly, mice were intraperitoneally (i.p) sensitized with 10 µg OVA and 65 µg aluminiumhydroxide (alum; SERVA Electrophoresis, Heidelberg, Germany) or PBS/alum in 150 µl on days 0 and 14. On days 21–24, mice were anesthetized with 5% isoflurane (CP-pharma, Burgdorf, Germany) and treated with 100 µg OVA in 30 µl PBS intranasally (i.n.) or 30 µl PBS alone. Thirty minutes prior to each OVA application, mice received i.n. 0.1 µg (group OMVs 0.1 µg/OVA), 1 µg of EcO83-OMVs (group OMVs 1 µg/OVA), or 0.9% NaCl (groups Sham/PBS and Sham/OVA). On day 25, airway hyperresponsiveness was tested using unrestrained whole-body plethysmography (Buxco® Small Animal Whole-body Plethysmography, Data Sciences International; St Paul, MN). Mice were subjected to increasing doses (0–50 mg/mL) of nebulized methacholine (Sigma-Aldrich, St. Louis, MO) and the enhanced pause was measured as an index for airway obstruction. Mice were terminally anesthetized on day 26, spleens and lungs were harvested and bronchoalveolar lavage (BAL) and blood were taken.