Anoopa Kumar

Isolation and characterization of a recombinant heat shock protein of Aspergillus fumigatus

BACKGROUND Aspergillus fumigatus, a common environmental fungus, is responsible for a number of lung disorders, including allergy and infection, in human beings. For immunodiagnosis of these diseases, standardized, pure, and relevant antigens are not currently available. METHODS A complementary DNA library of A. fumigatus was constructed with messenger RNA isolated from 96-hour-old culture of the organism. Fusion proteins expressed with the cDNA were characterized and evaluated. RESULTS One of the clones, which reacted with both rabbit anti-A. fumigatus serum and a pool of sera from patients with allergic bronchopulmonary aspergillosis, expressed a 65 kd protein of A. fumigatus. The recombinant protein reacted with immunoglobulin E and immunoglobulin G antibodies in the sera from patients with allergic bronchopulmonary aspergillosis. The deduced amino acid sequence of the partially sequenced complementary DNA of the clone is homologous with the hsp 90 family of heat shock proteins in human beings and other organisms. CONCLUSION The immunodominant nature and the homology to human heat shock proteins suggest a possible role for this protein in protective immunity and autoimmunity.

Aspergillus fumigatus antigen induced eosinophilia in mice is abrogated by anti-IL-5 antibody.

A murine model of allergic bronchopulmonary aspergillosis (ABPA), developed by exposure to Aspergillus fumigatus antigens, demonstrated eosinophilia of peripheral blood (PB), bone marrow (BM), and lung. The eosinophilia was abrogated by monoclonal anti‐inter‐leukin‐5 (IL‐5) antibody (TRFK‐5) and not by an isotype control antibody (GL 113). Eosinophils in PB were enumerated from stained smears and their relative increase or decrease in cells from BM and lung was determined by an eosinophil peroxidase (EPO) assay (measured in optical density). Intraperitoneal injection of TRFK‐5 in mice exposed to A. fumigatus antigen produced a significant reduction in eosinophils (PB 6.6 ± 1.14% vs. 3.8 ± 0.8%, P< .01) and EPO production in BM (0.935 ± 0.03 vs. 0.615 ± 0.02, P < .001). A similar reduction in EPO production in the lung (0.691 ± 0.12 vs. 0.495 ± 0.05, not significant) was also reflected in the histopathol‐ogy for the different groups of mice. These findings confirming the role of IL‐5 in eosinophilia, although not surprising, are significant in elucidating the immu‐nopathogenesis of ABPA in the murine model. We conclude that in this model, eosinophilia may be due largely to the Th2 cytokine ‐IL‐5 induced by A. fumigatus antigens.

Latex Antigens Induce IgE and Eosinophils in Mice

Hypersensitivity to latex proteins has been reported with increasing frequency in recent years. Elevated levels of latex specific IgE have been detected in the majority of these patients. Severe anaphylaxis and death resulting from latex exposure has also been reported. Nevertheless, the immune mechanism of latex allergy is not fully understood. In this report, we describe a model of latex allergy developed in mice exposed to latex proteins. Animals exposed to latex proteins demonstrated enhanced levels of total IgE, peripheral blood and lung eosinophilia, and elevated levels of serum IL-4 and IL-5. mRNA transcripts of IL-4 and IL-5, but not IFN-gamma, could be demonstrated in spleen lymphocytes. Antibodies to latex belonging to all IgG subclasses were detected in the sera of mice exposed to latex antigens. The histology of the lung showed non-necrotizing granulomas and extensive interstitial chronic inflammatory infiltrates, particularly around bronchioles and small blood vessels. Although this model of latex allergy demonstrates a heterogeneous immunological response, the CD4-positive Th2 cell-mediated response predominated.

Murine monoclonal antibodies to glycoprotein antigens of Aspergillus fumigatus show cross-reactivity with other fungi.

Six monoclonal antibodies produced against Aspergillus fumigatus were studied for their cross-reactivity against other fungal antigens from related and unrelated organisms. Five of the monoclonal antibodies reacted with glycoprotein antigens as evidenced by their binding to concanavalin A, although one did not react with concanavalin A. The reactivities of the monoclonal antibodies with various antigens were studied by biotin-avidin linked immunosorbent assay and Western blots. The results indicate that two monoclonal antibodies (Asp D1 and Asp C9) react with antigens from aspergilli as well as other fungi including Penicillium notatum and Candida albicans, whereas three of six antibodies (Asp H10, Asp ILB8, and Asp C2B1) react with A. fumigatus antigen only. Hence, these monoclonal antibodies can be used to obtain group specific and species specific antigens for various immunodiagnostic assays.

Characterization of a monoclonal antibody against latex protein associated with latex allergy

Several hybridomas were produced against antigens extracted from the latex sap of Hevea brasiliensis. One of the monoclonal antibodies (LA-E3) reacted with antigens demonstrating binding to patient sera on crossed enzyme immunoelectrophoresis. This monoclonal antibody reacted with 2 of 10 glove extracts studied and with both Malaysian and Indian latex plant extracts. The antigens purified with monoclonal antibody affinity chromatography demonstrated specific IgE in the sera of patients with latex allergy as determined by ELISA. This monoclonal antibody can thus be utilized to obtain reliable antigens useful in the diagnosis of latex sensitivity, although additional antigens will likely be necessary to enhance sensitivity and specificity.

Monoclonal antibodies bind identically to both spores and hyphae of Aspergillus fumigatus

Immunoelectron microscopy (IEM) was used to determine the binding of six monoclonal antibodies (MoAbs) produced against Aspergillus fumigatus antigens present on or within the conidia and hyphae of the fungus. Antigen‐antibody complexes were demonstrated in EM using labelled colloidal gold particles (15 nm). Three out of 6 MoAbs (C9, F12 and H10) reacted only with the cytoplasmic components of A. fumigatus while the remaining three (B12, F6G5 and D6E6) showed reactivity to both cytoplasm and cell wall of the conidia and hyphae. The results indicate that IEM is of considerable value in determining and selecting monoclonal antibodies having specific reactivity with diverse antigenic components.

Monoclonal Antibodies against Farmer’s Lung Antigens having Specific Binding to IgG Antibodies

Hypersensitivity pneumonitis resulting from environmental exposure to Saccharopolyspora rectivirgula (Micropolyspora faeni) among farmers has been well recognized. The diagnosis of the disease depends on demonstration of circulating antibodies against S. rectivirgula. However, dependable pure antigens are not available for serodiagnosis. In the present study we have employed hybridoma technology to obtain monoclonal antibodies against S. rectivirgula antigens. These monoclonal antibodies were employed to purify antigens through affinity chromatography. When tested in ELISA, high levels of antibodies were demonstrated against these antigens in farmers lung patient sera compared to exposed but asymptomatic individuals from the same household. In Western blots patient sera reacted with components of crude antigens with molecular masses of 28, 35, 60, 65 and 68 kD and 4 components above 100 kD, while the monoclonal antibodies reacted only with the 60-kD protein. These purified antigens can be used as reliable reagents in the specific diagnosis of farmers lung disease.