Eight-week-old female BALB/c mice (5 per group) were vaccinated with either Qβ-Eot or Qβ-IL-5, or the combination of both without the addition of adjuvant. 50 μg of total protein of each vaccine was XAV-939 mouse injected subcutaneously on days 0, 21 and 35. Mice were subjected to retro-orbital bleeding on days 0, 21, 35 and

45 and sera analyzed by the use of IL-5 and eotaxin-specific ELISA. ELISA plates were coated with mouse rIL-5 or r-eotaxin at a concentration of 5 μg/ml. Plates were blocked then incubated with serially diluted mouse sera. Bound antibodies were detected with enzymatically labeled anti-mouse IgG antibody. As a control, preimmune serum from the same mice was tested. Antibody titers were calculated as the serum dilution which led to a half-maximal of OD450 (OD50%). To induce allergic airway inflammation, female BALB/c mice (5 per group) were injected (i.p.) with 10 μg of OVA (Grade V, Sigma–Aldrich) mixed with 2 mg of alum (Aluminium Hydroxide

Gel Adjuvant, Brenntag Biosector, Denmark). 10 days later, mice were challenged daily with 100 μg of OVA by intranasal administration for 4 days. 24 hours after the last challenge, BAL and lungs were subjected to histology. Mice injected i.p. with OVA Selleckchem PD-1/PD-L1 inhibitor 2 and alum but not challenged intranasaly with OVA served as a negative control for disease induction in these experiments. To assay the activity of r-eotaxin, BALB/c mice (5 per group) were immunized i.p. on days 0 and 3 with 100 μg of OVA mixed with 2 mg

of alum. On day 14, mice were injected with either PBS or 0.5 μg of r-eotaxin i.v. Thirty min after injection, blood samples were collected from each mouse and blood smears were made. The slides were dried in air and stained with Kit RAL 555 (Réactifs RAL) according to manufacture’s protocol (a fast-acting variation of May-Grünwald Giemsa staining). The percentage of else eosinophils was evaluated with a light microscope. In the model of allergic airway inflammation, bronchoalveolar cells were collected in successive lavages (BAL) using 0.5 ml aliquots of PBS with 2% BSA at room temperature until the total volume reaches 1.2 ml. The total number of cells in the BAL was counted with a Coulter Counter (Beckman Coulter, Inc.). Cytospins were performed with Shandon Cytospin apparatus (Thermo Fisher Scientific, Inc.) and stained with Kit RAL 555 (Réactifs RAL) according to the manufacture’s protocol. Differential cell counts were performed with at least 200 leukocytes. Mouse lungs were removed and fixed in 10% PBS buffered formalin. Paraffin sections were stained with Chromotrope 2R to identify eosinophils [29]. For statistical analysis, Student’s t-test was used. p-Values <0.05 were considered significant. Recombinant murine IL-5 with an N-terminal hexa-histidine tag, an enterokinase cleavage site and a linker containing a cysteine residue was expressed and purified.