concanavalin-a and Aspergillosis--Allergic-Bronchopulmonary

Recently, we have developed a model of airway inflammation in a CFTR knockout mouse utilizing Aspergillus fumigatus crude protein extract (Af-cpe) to mimic allergic bronchopulmonary aspergillosis (ABPA) 1, an unusual IgE-mediated hypersensitivity syndrome seen in up to 15% of cystic fibrosis (CF) patients and rarely elsewhere. We hypothesized that replacement of CFTR via targeted gene delivery to airway epithelium would correct aberrant epithelial cytokine signaling and ameliorate the ABPA phenotype in CFTR-deficient (CFTR 489X - /-, FABP-hCFTR + / +) mice. CFTR knockout mice underwent intra-tracheal (IT) delivery of recombinant adeno-associated virus serotype 5 (rAAV5Delta-264CFTR) or rAAV5-GFP at 2.58 x 10(12) viral genomes/mouse. All mice were then sensitized with two serial injections (200 microg) of crude Af antigen via the intra-peritoneal (IP) route. Untreated mice were sensitized without virus exposure. Challenges were performed 2 weeks after final sensitization, using a 0.25% solution containing Aspergillus fumigatus crude protein extract delivered by inhalation on three consecutive days. The rAAV5Delta-264CFTR-treated mice had lower total serum IgE levels (172513 ng/ml +/- 1312) than rAAV5-GFP controls (26 892 ng/ml +/- 3715) (p = 0.037) and non-treated, sensitized controls (24 816 +/- 4219 ng/ml). Serum IgG1 levels also were lower in mice receiving the CFTR vector. Interestingly, splenocytes from rAAV5Delta-264CFTR-treated mice secreted less IL-13, INFg, TNFa, RANTES and GM-CSF after ConA stimulation. Gene therapy with rAAV5Delta-264CFTR attenuated the hyper-IgE response in this reproducible CF mouse model of ABPA, with systemic effects also evident in the cytokine response of stimulated splenocytes.

Topics: Animals; Aspergillosis, Allergic Bronchopulmonary; Aspergillus fumigatus; Cell Proliferation; Complex Mixtures; Concanavalin A; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Dependovirus; Disease Models, Animal; Fluorescein-5-isothiocyanate; Gene Expression Regulation; Genetic Therapy; Green Fluorescent Proteins; Humans; Immunoglobulin E; Immunologic Factors; Mice; Mice, Inbred CFTR; Mutant Proteins; Mutation; Spleen; Transduction, Genetic; Transgenes

Most cases of allergic bronchopulmonary aspergillosis (ABPA) are caused by the fungus Aspergillus fumigatus. Successful treatment of this disease depends on early diagnosis with the use of well-characterized and relevant antigens/allergens of the organism.. The aim of this study was to purify and characterize relevant proteins from A. fumigatus that could be used in the reliable diagnosis of ABPA.. Monoclonal antibodies were raised against A. fumigatus culture filtrate antigens. A Concanavalin A nonbinding protein fraction was purified with use of one of the monoclonal antibody immunoaffinity columns. The purified protein was analyzed on sodium dodecylsulfate-polyacrylamide gel electrophoresis gel and Western blots. The sensitivity and specificity of the purified protein were evaluated by RAST and ELISA with sera from 25 patients with ABPA, from 10 with allergic asthma, and from 10 normal control subjects.. The 37 kD Concanavalin A nonbinding protein reacted specifically with IgE antibodies in patients with ABPA. Among the 25 patients with ABPA studied, 96% had IgE antibody against the allergen, whereas none of the subjects with allergic asthma who had positive results on the skin prick test or normal control subjects had a reaction. Both RAST and ELISA results exhibited strong correlation with IgE binding. This allergen exhibited N-terminal sequence identity to a recombinant allergen Asp f 2.. A 37 kD protein with complete N-terminal homology to Asp f 2 is a major allergen of A. fumigatus that significantly reacts with IgE antibody in patients with ABPA, but does not elicit reaction in Aspergillus-sensitive subjects with asthma and normal control subjects.

Topics: Allergens; Amino Acid Sequence; Antibodies, Monoclonal; Antibody Specificity; Antigens, Fungal; Aspergillosis, Allergic Bronchopulmonary; Aspergillus fumigatus; Binding Sites, Antibody; Cells, Cultured; Chemical Fractionation; Chromatography, Affinity; Concanavalin A; Enzyme-Linked Immunosorbent Assay; Epitopes; Fungal Proteins; Humans; Immunoglobulin E; Radioallergosorbent Test

Topics: Antibodies, Monoclonal; Antigens, Fungal; Aspergillosis, Allergic Bronchopulmonary; Aspergillus fumigatus; Asthma; Concanavalin A; Humans; Immunoglobulin G

A hybridoma secreting immunoglobulin G1 antibody was produced against Aspergillus fumigatus. In this study we used interleukin-4 as a growth factor to augment the survival and multiplication of immunoglobulin G1-secreting hybridomas. The antigen recognized by this monoclonal antibody is a low-molecular weight concanavalin A-nonbinding component of A. fumigatus. The monoclonal antibody affinity-purified antigen showed specific reactivity with serum from patients with allergic bronchopulmonary aspergillosis, whereas all allergic asthmatic patients with skin-test reactivity to A. fumigatus demonstrated only low levels of antibodies similar to those in normal control subjects. This antigen failed to show cross-reactivity with other fungal antigens. These results indicate that this monoclonal antibody can be used for the purification of specific antigens useful in the immunodiagnosis of allergic bronchopulmonary aspergillosis.

Topics: Animals; Antibodies, Monoclonal; Antigens, Fungal; Aspergillosis, Allergic Bronchopulmonary; Aspergillus fumigatus; Blotting, Western; Concanavalin A; Immunoglobulin G; Mice; Mice, Inbred BALB C

Humoral immune responses in allergic bronchopulmonary aspergillosis (ABPA) have been well studied. However, reports of cell-mediated immune responses in ABPA are conflicting and not well documented, perhaps because well-characterized antigens are not available. In the present study, we assessed the role of peripheral blood mononuclear cells (PBMCs) from patients with ABPA in their ability to respond to both crude and semipurified Aspergillus fumigatus antigens by in vitro proliferation and immunoglobulin E synthesis. Eight patients with ABPA, eight patients with immediate wheal and flare skin reactivity to A. fumigatus, and ten healthy control subjects with nonreactive skin tests were included in this study. Four A. fumigatus antigens were tested in vitro. Antigens included culture filtrate, mycelial extract, concanavalin A-nonbinding, and concanavalin A-binding antigens. There was a wide range of response to each antigen by each group of subjects. However, PBMCs from patients with ABPA showed greater response to the antigens than did those from the healthy control subjects when evaluated by lymphoproliferation (tritiated thymidine uptake) and immunoglobulin E synthesis (isotype-specific enzyme-linked immunosorbent assay). The concanavalin A-nonbinding antigen fraction had the ability to specifically stimulate proliferation of PBMCs from seven of eight patients with ABPA and from four of eight skin-reactive subjects; none of the healthy control subjects responded. Significantly high levels of immunoglobulin E were detected in unstimulated PBMC cultures from five patients with ABPA when compared with those from healthy control subjects. These results indicate that concanavalin A-nonbinding A. fumigatus antigens may be significant in the cellular immune response of ABPA; such results are similar to those from previous humoral studies of the disease.

Topics: Antigens, Fungal; Aspergillosis, Allergic Bronchopulmonary; Aspergillus fumigatus; Concanavalin A; Humans; Immunoglobulin E; Lymphocyte Activation; Molecular Weight; T-Lymphocytes