Businga, J. first CpG-ODN administration between the antigen treatments. The animals in group 4 served as controls and were given phosphate-buffered saline. Allergen-specific serum immunoglobulins and total immunoglobulin E in different groups of animals were measured by enzyme-linked immunosorbent assay, while airway remodeling and cytokine production were studied by immunohistochemistry. The results exhibited that CpG-ODN administration either before (group 2) or between (group 3) antigen treatments resulted in reduced total immunoglobulin E levels and peripheral blood eosinophil numbers compared to allergen-sensitized group 1 animals. Similarly, treatment with CpG-ODN also downregulated inflammatory cell infiltration, goblet cell hyperplasia, and basement membrane thickening compared to antigens from the environment and the proteins and toxins from inhaled fungal spores complicate asthma and result in immunologic lung destruction (11, 12). The disease is characterized by high serum total IgE, amebocyte assay (BioWhittaker, Walkersville, Md.). Induction of allergic airway inflammation. Six- to 8-week-old female BALB/c mice were purchased from Charles River laboratories (Wilmington, Mass.). Four different groups of animals with five mice in each group were used in this study. The immunization protocol is given in Table ?Table1.1. Three intraperitoneal injections were given at 3-day intervals with doses of 100 g of culture filtrate extract in phosphate-buffered saline (PBS) mixed with 1 mg of alum (Sigma). Following the intraperitoneal injections, the animals in all three groups were given three intranasal challenges with 50 g of antigen in PBS. As shown in Table ?Table1,1, the animals belonging to groups 2 and 3 also received injections of CpG-ODN intraperitoneally or intranazsally (50 g in PBS/mouse). The control mice in group 4 received only PBS instead of antigen or CpG. TABLE 1. Immunization schedule for CpG-mediated immune intervention in mice sensitized with antigen antigens (5 g/ml) and kept overnight at 4C. Serum (1:50, vol/vol) and bronchoalveolar lavage fluid (undiluted) were added to the plate and incubated for 3 h at room temperature. The addition of secondary antibody and color development were carried out as described earlier (14). Estimation of total IgE in serum samples. The total IgE in the serum samples was measured by enzyme-linked immunosorbent assay with a rat anti-mouse monoclonal antibody as previously reported (10, 14). The serum IgE levels were expressed as nanograms per milliliter of serum with a MI-136 standard curve. Lung histology. The lungs were infused with 1 ml Casp3 of sterile PBS and fixed in 10% neutral buffered formalin. The tissues were processed and embedded in paraffin, and sections were cut at a thickness of 5 m and stained with hematoxylin and eosin, periodic acid Schiff base, and Trichrome Masson stains and evaluated microscopically (10). Morphological changes evaluated included perivascular and peribronchial inflammation and infiltration of eosinophils, lymphocytes, macrophages, neutrophils, and plasma cells. Perivascular and peribronchial eosinophil infiltration was graded as none to severe on a 0 to 4 scale as described previously (9, 10, 23). Eosinophil staining of the blood. Eosinophils were evaluated in blood samples collected from the heart at the time of sacrifice as described earlier MI-136 (23). EPO staining of lung sections. Lung tissues embedded in tissue freezing medium (Polysciences, Inc.) were cryosectioned at 10 m and fixed onto poly-L-lysine-coated slides. Lung eosinophil MI-136 infiltration was studied by microscopic examination after staining for eosinophil peroxidase with DAB (3,3-diaminobenzidine tetrahydrochloride; Sigma). The sections were incubated initially with 10 mM cyanide buffer and rinsed in PBS, followed by incubation with peroxidase substrate and DAB for 10 min at room temperature. The slides were rinsed thoroughly with water and examined under a light microscope. The number of eosinophil peroxidase (EPO)-positive cells in each MI-136 lung section was counted.
