IgE antibody to Aspergillus fumigatus recombinant allergens in cystic fibrosis patients with allergic bronchopulmonary aspergillosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) is characterized by a heightened Th2 CD4+ T-cell response to Aspergillus fumigatus (Af) allergens and a hyper-immunoglobulin E (IgE) state compared with cystic fibrosis patients without ABPA. The IgE serologic differentiation of ABPA from atopic CF patients can be difficult. We propose as the reactivity with purified antigens varies qualitatively and quantitatively and that the antibody response is more specific than with crude Af antigen extract, the IgE responses to purified recombinant Af allergens may differentiate ABPA from atopic CF patients. METHODS: Serum IgE reactivity to seven recombinant purified allergens and to a crude extract of Af was measured in 15 ABPA, in 23 Af skin test positive (ST+), and in 19 Af skin test negative (ST-) CF patients. Four of the ABPA CF patients were studied before and after developing ABPA. Nine ABPA patients were studied during flares and remissions of ABPA. RESULTS: Allergic bronchopulmonary aspergillosis patients had significantly increased IgE reactivity to Asp f2, f3, f4, f6, and f16 compared with the Af ST+ and ST- non-ABPA CF patients. In the ABPA patients studied before and after developing ABPA, IgE reactivity also increased to Asp f2, f3, f4, and f6, and to the crude extract. In ABPA CF patients, IgE reactivity to Asp f1, f2, f3, and f6 significantly increased during periods of ABPA flares compared with periods of remission. Analysis of the receiver operating curve demonstrated that IgE reactivity to Asp f3 and f4 gave the best sensitivity and specificity and were better than IgE reactivity to a crude extract of Aspergillus. Furthermore, in ABPA patients studied during periods of remission the IgE reactivity to Asp f3 and f4 remained significantly elevated compared with Af ST+ non-ABPA patients. The IgE responses when considered either to be positive or negative to Asp f3 and f4 significantly differentiated ABPA from Af ST+ and ST- non-ABPA CF patients. In contrast, IgE reactivity was considered positive to the crude extract in 89% of ABPA, 61% of Af ST+, and 0% of Af ST- non-ABPA CF patients. CONCLUSIONS: Immunoglobulin E reactivity to a panel of purified Af allergens, especially to Asp f3 and f4, differentiates ABPA from atopic Af ST+ non-ABPA CF patients. Serial determinations of IgE reactivity to individual purified Aspergillus antigens, especially Asp f3, demonstrates that increases in IgE reactivity may provide improved distinction between stages of flares and remission compared with changes in IgE reactivity to a crude Aspergillus extract.

Evidence for the involvement of two different MHC class II regions in susceptibility or protection in allergic bronchopulmonary aspergillosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a disease with uncertain pathology. Studies have suggested a pathogenic role for T(H)2 cells. Previously, we demonstrated, in a small group of patients, that T(H)2 reactivity to a major Aspergillus fumigatus antigen was restricted by HLA-DR2 or HLA-DR5 alleles. OBJECTIVES: We sought to confirm whether susceptibility to ABPA is exclusively associated with HLA-DR locus and to investigate the involvement of HLA-DQ genes in the development of ABPA. METHODS: Genomic DNA was extracted from patients with ABPA, patients without ABPA but with positive A fumigatus skin test responses and asthma or cystic fibrosis, and healthy control subjects. HLA-DR and HLA-DQ genes were detected by using low-resolution typing; high-resolution typing was done only on HLA-DR2- and HLA-DR5-positive individuals by using sequence-specific primers (PCR-SSP). RESULTS: A significantly higher frequency of HLA-DR2 was observed in patients with ABPA versus those without ABPA (corrected P <.01) or healthy control subjects (corrected P <.01). Genotype analysis revealed that susceptibility to ABPA is associated with HLA-DR2 alleles DRB1*1503 and DRB1*1501 and, to a lesser extent, with the HLA-DR5 allele DRB1*1104. The presence of DR4 or DR7 alleles in non-DR2/5 patients with ABPA suggests that these alleles may also be contributing factors in this disease. Another striking observation was the significantly high frequency of HLA-DQ2 in patients without ABPA (67. 4%) compared with patients with ABPA (20.5%) and normal control subjects (37.7%), suggesting that these alleles may confer protection in the population without ABPA. CONCLUSION: These genetic studies suggest that HLA-DR molecules DR2, DR5, and possibly DR4 or DR7 contribute to susceptibility while HLA-DQ2 contributes to resistance and that a combination of these genetic elements determines the outcome of ABPA in patients with cystic fibrosis and asthma.

The association of HLA-DR alleles and T cell activation with allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease caused by the mold Aspergillus fumigatus. We previously reported that the majority of T cell clones (TCC) isolated from three ABPA patients, and specific for a dominant Ag of A. fumigatus, Asp f 1, were IL-4-producing CD4+ Th2 cells capable of responding to Ag in association with the HLA-DR subtypes DRB1*1501, *1503, and *1601 for HLA-DR2, and DRB1*1101, *1104, and *1202 for HLA-DR5. In the present study we extended the previous findings to determine whether the observed restriction with the HLA-DR2/5 subtypes held importance in a larger patient population. Serotyping revealed that 16 of 18 ABPA patients were either HLA-DR2, HLA-DR5, or both. Compared with a normal control population, the frequencies of HLA-DR2 (50 vs 22.3%) and HLA-DR5 (44.4 vs 19.8%) were significantly increased in these ABPA patients. Genotype analyses of an additional 15 patients identified the same HLA-DR subtypes previously shown functional for Asp f 1 Ag presentation. The relative avidities of Asp f 1 peptides for the purified HLA-DR subtypes, DRB1*1501 (functional) and DRB1*1502 (nonfunctional), were examined to determine whether differential binding to the HLA-DR subtypes explains successful Ag presentation. Similar low binding avidities were detected for both HLA-DR subtypes, indicating that the functionality cannot be simply explained by differences in binding affinities. Thus, the limited number and their role in Ag presentation emphasizes the possibility that the six identified HLA-DR subtypes are important in the pathophysiology of ABPA.

A 12-year longitudinal study of Aspergillus sensitivity in patients with cystic fibrosis.

STUDY OBJECTIVE: The object of the study was to longitudinally follow immune parameters of Aspergillus fumigatus sensitization so as to predict those at risk for developing allergic bronchopulmonary aspergillosis (ABPA). DESIGN: Patients were evaluated for 5 immune parameters (skin test [ST], positive precipitating antibody [PPN], total IgE, IgE anti-A fumigatus antibody [IgE-Af], and IgG anti-A fumigatus antibody [IgG-Af]) at yearly intervals over a 12-year time period. SETTING: Patients were enrolled and evaluated during routine visits to the cystic fibrosis (CF) clinic at Cardinal Glennon Children's Hospital, St. Louis. PATIENTS: One hundred eighteen patients with documented CF participated. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Six patients were diagnosed as having ABPA. In the non-ABPA patient group, 42% had a positive ST, 42% were PPN positive, 54% had IgE-Af, 61% had IgG-Af, and 10% had an IgE greater than 1,000 IU/mL at some point in time. However, on follow-up, 18% lost skin reactivity, 54% lost-PPN, 53% lost IgE-Af, 45% lost IgG-Af, and IgE greater than 1,000 IU/mL declined more than 72% in 64% of patients. These losses were spontaneous, without systemic corticosteroid intervention. CONCLUSIONS: Spontaneous diminution and loss of immune parameters in non-ABPA CF patients prevented us from defining a profile of sensitivity likely to result in ABPA. This variability highlights the importance of obtaining follow-up studies and including clinical symptoms when considering the diagnosis of ABPA in patients with CF.

T cell subsets, epitope mapping, and HLA-restriction in patients with allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease characterized by Aspergillus fumigatus (Af) colonization, IgE and IgG anti-Af antibodies, pulmonary infiltrates, bronchiectasis, and pulmonary fibrosis. Little is known regarding T cell responses and their role in the pathogenesis of ABPA. To examine T cell reactivity to Af antigens, T cell clones (TCC) specific to the Asp f 1 antigen, an 18-kD protein of Af, were established from the peripheral blood of three ABPA patients. The majority of TCC isolated from ABPA patients, and specific for the Asp f 1 allergen of Af, are IL-4 producing CD4+ cells of the Th2 phenotype. Further analysis in this study revealed that the majority of TCC reacted to mainly two epitopes of Asp f 1, while the remaining TCC reacted to three additional "minor" epitopes. Blocking studies using monoclonal antibodies specific for class II HLA-D region gene products showed that most TCC, 19/21, were restricted by HLA-DR molecules, and the remaining two clones by HLA-DP molecules. The use of a panel of HLA-matched and mismatched EBV-transformed B cells as antigen presenting cells revealed that the HLA-DR restriction was mediated exclusively by either the HLA-DR2 or HLA-DR5 alleles. Genotyping of DRB1 gene products showed that class II presentation for most clones was not restricted to a single allele, representing DRB1 gene products of either HLA-DR2 or DR5. These studies offer insight into the cellular and molecular determinants which contribute to the immunopathophysiology of ABPA.

Asp f I CD4+ TH2-like T-cell lines in allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease caused by bronchial colonization with Aspergillus fumigatus (Af) characterized by elevated serum total and Af-specific IgE levels and eosinophilia. In order to examine T-cell reactivity to Af antigens, six T-cell lines were established from the peripheral blood of patients with ABPA to Asp f I, an 18 kd protein purified from Af extracts. The Asp f I-specific T-cell lines, analyzed by flow cytometry, were 100% CD3+ CD4+. Lymphoproliferative responses of the T-cell lines were specific for Asp f I stimulation, 28,999 cpm (stimulation index = 12.2), and showed no response to tetanus toxoid stimulation, 2178 cpm (stimulation index = 1.1) (p < 0.001). Furthermore, Asp f I-stimulated lymphoproliferation was inhibited in two experiments by monoclonal anti-interleukin (IL)-4 antibody in a dose-response fashion, 78% and 84% inhibition at 5% concentration of anti-IL-4. In contrast, anti-IL-2 antibody did not inhibit Asp f I-stimulated proliferation. Asp f I-stimulated T-cell lines synthesized predominantly IL-4 (mean, 21.5 ng/ml) after 48 hours of culture, and nondetectable quantities of interferon-gamma and IL-2. In summary, Asp f I-specific T-cell lines established from patients with ABPA were characterized as being CD4+ TH2-like in their cytokine synthesis pattern, and secreted IL-4 behaved in an autocrine fashion, stimulating proliferation.

Serum anti-Aspergillus fumigatus antibodies by immunoblot and ELISA in cystic fibrosis with allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) occurs with a prevalence of 5% to 15% in patients with cystic fibrosis (CF). Because of the frequent colonization with Aspergillus fumigatus (Af) in CF, the causative agent of ABPA, antibody reactivity to Af proteins is frequently observed, which obscures the diagnosis of ABPA. Patients with CF are also categorized according to the presence of positive skin test responses to Af and/or the presence of positive precipitins. In this study we used ELISA and immunoblot assay to detect IgE and IgG anti-Af antibodies in patients with CF and ABPA (n = 13) compared with other groups of patients with CF: those with positive skin test and positive precipitin results (n = 18), those with positive skin test and negative precipitin results (n = 14), those with negative skin test and positive precipitin results (n = 10), and those with negative skin test and negative precipitin results (n = 35). IgE and IgG anti-Af antibodies were significantly elevated in patients with ABPA as determined by both immunoblot assay (p < 0.01) and ELISA (p < 0.01). However, detection of Af antibodies by ELISA was more sensitive in discriminating patients with CF and ABPA from patients with CF who had positive skin test and positive precipitin results but lacked radiographic and clinical evidence of ABPA. In patients with CF and ABPA the immunoblot assays demonstrated a multitude of IgE, IgG, and IgA antibody responses to Af proteins, which ranged in molecular weight from 14 kd to greater than 106 kd. The level of IgE anti-Af antibody to individual proteins decreased during remissions of ABPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Occupational asthma to the slime mold Dictyostelium discoideum.

Dictyostelium discoideum is a slime mold that exists in a unicellular amoeboid form under certain nutritional conditions. In this form, it produces unique lysosomal enzymes that are valuable in studying cell-to-cell signaling systems. We report on a research microbiologist who developed rhinoconjunctivitis and asthma after release of D. discoideum from a pressurized canister. Immediate skin test reactivity was demonstrated to whole and lysed organisms. Enzyme-linked immunosorbent assay results revealed IgE antibody against D. discoideum whole organism, lysed organism, and lysosomal enzymes with the strongest response being directed toward lysosomal enzymes. Pulmonary function testing showed a decline in forced expiratory volume in 1 second and forced expiratory flow after modified laboratory exposure to D. discoideum. This case represents the first report of occupational rhinoconjunctivitis and asthma from slime mild.

Henoch-Schoenlein purpura due to streptokinase.

The syndrome of Henoch-Schoenlein purpura developed in a 74-year-old woman after receiving streptokinase as thrombolytic therapy for an acute myocardial infarction. Renal biopsy revealed mesangial hypercellularity with deposits of IgA. Skin biopsy also revealed IgA deposition. Immunological studies showed evidence of sensitization to streptokinase. Elevated IgG, IgA, IgM, and IgE antistreptokinase antibodies were detected in the acute serum. Positive immediate skin reactivity to streptokinase was also present. Serum precipitins to streptokinase disappeared when IgA was removed from the serum. Positive staining with biotinylated streptokinase was seen in the skin in the same pattern of distribution as IgA. These findings strongly support the role of streptokinase and IgA in the pathogenesis of Henoch-Schoenlein purpura in this patient. A control group of streptococcal-infected patients showed no immune response to streptokinase. Another control group of streptokinase-treated patients, who had no untoward reaction, had elevated immunoglobulin classes and precipitins to streptokinase. However, the precipitating antibody was IgG and streptokinase skin tests were negative.

Increased incidence of stings in venom-sensitive patients.

We compared the histories of 29 venom-sensitive and 28 control subjects who were selected from our venom referral and general allergy clinics respectively. The variables in the study included insect avoidance knowledge, the number of stings during the previous 2 years, insects involved, and time spent out of doors per week. There was no significant difference between the two groups with respect to age. All venom-sensitive patients were well versed in avoidance techniques while only 3 of 28 controls (11%) claimed such knowledge. Venom-sensitive subjects were stung almost ten times more frequently than control subjects. Wasp stings were the most common, followed by yellow jacket, honey bee, and hornet. The venom-sensitive patients also reported spending a greater amount of time outdoors (x 17.4 hours versus x 11.8, P < .05). An analysis of covariance showed that this difference in outdoor exposure was insufficient to account for the disparity in the number of stings. We conclude that other factors such as intrinsic attractants must be responsible for this phenomenon.

Variability in parameters of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis.

Seventy-nine patients with cystic fibrosis (CF) were evaluated and were followed in a longitudinal, prospective fashion during a 6-year period for the development of immune parameters indicating Aspergillus fumigatus (Af) sensitization and allergic bronchopulmonary aspergillosis (ABPA). Although four patients developed frank ABPA, there was considerable variability in immune parameters in non-ABPA. Twenty-four patients became skin test positive to Af with none losing skin reactivity. Twenty-five patients developed serum precipitins to Af, whereas 12 patients lost their precipitins. Of 15 patients with an elevated total serum IgE of greater than or equal to 2 SD, five demonstrated a marked decline of at least 40%. Three of 16 patients with IgE-Af became negative, whereas eight of 27 patients lost their IgG-Af. None of these patients had received corticosteroid therapy that could have accounted for the findings. Thus, patients with CF frequently lose evidence of Af sensitivity spontaneously without corticosteroid intervention. The diagnosis of ABPA in CF should not be based solely on serology and skin test results, since at any point in time, patients with CF may demonstrate variable responses to Af.

Serum immunoglobulins E and G anti-Aspergillus fumigatus antibody in patients with cystic fibrosis who have allergic bronchopulmonary aspergillosis.

Patients with cystic fibrosis frequently have pulmonary colonization with Aspergillus fumigatus (Af) and develop anti-Af immunoglobulin E (IgE) and IgG antibodies. The diagnosis of allergic bronchopulmonary aspergillosis in subjects with cystic fibrosis is difficult because of the high incidence of Af colonization, with development of humoral antibody responses. In this study, we sequentially measured serum anti-Af IgE (Af-E) and IgG (Af-G) antibodies by ELISA in subjects with cystic fibrosis. In subjects with cystic fibrosis who have allergic bronchopulmonary aspergillosis, Af-E and Af-G antibodies were significantly increased when compared with other groups of patients with cystic fibrosis who had positive skin tests or precipitins to Af (or both) (p less than 0.01, p less than 0.01, respectively). In addition, increased Af-E and Af-G levels were sometimes seen in other groups, especially subjects with cystic fibrosis who had positive Af skin tests or precipitin tests, two of whom later developed criteria diagnostic of allergic bronchopulmonary aspergillosis. Thus, serum Af-E and Af-G levels were quantitatively increased in subjects with cystic fibrosis who had allergic bronchopulmonary aspergillosis and thus adjunctive data in diagnosis. However, it also suggested that subclinical pulmonary inflammation may also occur.

T- and B-cell dysregulation of IgE synthesis in cystic fibrosis patients with allergic bronchopulmonary aspergillosis.

Since Aspergillus fumigatus (Af)-specific and polyclonal serum IgE levels are characteristically elevated in patients with allergic bronchopulmonary aspergillosis (ABPA), we evaluated in vitro regulation of IgE synthesis in cystic fibrosis (CF) patients with ABPA. We studied 11 CF patients with ABPA, 37 patients with positive Af prick skin tests and/or IgG precipitating antibodies (ST/PPT+), and 35 patients with no humoral or skin responses to Aspergillus (ST/PPT-). Mean serum IgE concentration was significantly elevated in CF subjects with ABPA compared to ST/PPT+ and ST/PPT- patients, 2866 vs 303 and 61 IU/ml, respectively (P less than 0.01). In vitro studies demonstrated that ABPA patients' B cells spontaneously synthesized significantly increased amounts of IgE compared to ST/PPT positive and negative subjects, 1980 vs 220 and 13 pg/ml, respectively (P less than 0.01). In addition, preformed B-cell-associated IgE was also significantly elevated in ABPA subjects (P less than 0.01), indicating prior in vivo activation. Supernatant cultures of Af-stimulated T cells from ABPA subjects significantly induced allogeneic B-cell IgE synthesis compared to ST/PPT positive and negative CF subjects, 206 vs 13 and 4 pg/ml, respectively (P less than 0.01). Thus T cells stimulated with Aspergillus antigens secrete cytokines that induce B-cell IgE synthesis in ABPA subjects. B-cell IgE hyperactivity is manifested by in vivo and in vitro increased IgE concentrations. Analyses of T-cell regulation and B-cell IgE synthesis distinguish CF subjects with ABPA from Aspergillus sensitive non-ABPA subjects.

Lack of preventive measures given to patients with stinging insect anaphylaxis in hospital emergency rooms.

A survey of patients receiving venom immunotherapy revealed that at the time of emergency treatment of their anaphylactic reaction, a significant percentage had not received appropriate avoidance instruction, a prescription for epinephrine, or referral to an allergist. Emergency room personnel must be instructed in giving appropriate advice after insect sting anaphylaxis.

A pathologic study of allergic bronchopulmonary aspergillosis.

A lung biopsy specimen was obtained from a 10-year-old boy with cystic fibrosis and allergic bronchopulmonary aspergillosis. Light microscopy revealed a marked inflammatory process that was largely bronchocentric. Infiltrating cells included lymphocytes, plasma cells, monocytes, and numerous eosinophils. Elastin layers were intact in blood vessels and markedly disrupted in bronchioles. By immunofluorescent, major basic protein was demonstrated in eosinophils, was freely deposited outside of eosinophils, especially in the interlobular septum, and was taken up by macrophages. A number of lymphocytes stained positively for IgE. Through an immunoperoxidase stain, septate hyphae of Aspergillus were clearly observed in the lung parenchyma. A significant increase in interleukin-2 positive-staining T cells was observed with an approximate 2:1 ratio of helper to suppressor cells. The use of newer immunohistologic techniques has enabled us to gain additional insights into the pathogenesis of allergic bronchopulmonary aspergillosis.

Participation of cell-mediated immunity in allergic bronchopulmonary aspergillosis.

While IgE and IgG antibodies are thought to play important pathogenetic roles in allergic bronchopulmonary aspergillosis (ABA), the microscopic lung picture would implicate cell-mediated immunity. Patients with ABA do not manifest delayed skin test reaction to Aspergillus fumigatus (AF). This has been attributed to inhibition of lymphokines by histamine locally released as a result of the immediate skin test reaction. We tested 5 ABA patients for delayed hypersensitivity to AF. An attempt was made to ablate the immediate skin test reaction to determine if a delayed reaction might be unmasked. At concentrations of AF previously shown to produce marked immediate skin test reactions, the combination of cimetidine by mouth and chlorpheniramine mixed with AF in the skin test syringe completely eliminated the immediate skin test reaction in all 5 patients. In no case did a delayed reaction appear. Eight ABA patients showed in vitro lymphocyte stimulation responses to AF that were comparable to those of asthmatics and rhinitics with positive immediate skin test reactions to AF and no clinical evidence of ABA. It thus appears that 'peripheral' cell-mediated immune responses, namely delayed skin reactivity and peripheral blood lymphocyte proliferation to AF, are not present in ABA.

Aspergillus fumigatus spore concentration in outside air: Cardiff and St Louis compared.

Intermittent sampling of the atmosphere 3 days/week over a 12-month period using Andersen samplers in Cardiff, Wales, U.K. and St Louis, Missouri, U.S.A., indicated average A. fumigatus spore concentrations of 13.5/m3 in St Louis and 11.3/m3 in Cardiff. Both sites showed seasonal variations with highest concentrations during winter.

The role of monocytes and responder cells in suppression of antigen-specific T cell proliferation in chronic renal failure.

The response of peripheral blood lymphocytes (PBL) to the antigen tetanus toxoid (TT) in patients with chronic renal failure being maintained on hemodialysis was examined. We have found that: (1) the response of patients' unfractionated PBL to TT is markedly suppressed when compared to the response of control PBL; (2) the response of patients' purified T cells and T4+ cells to TT co-cultured with 5% autologous monocytes is suppressed when compared to the response of comparable control cultures; (3) the response of patients' purified T lymphocytes co-cultured with 5% autologous monocytes is significantly enhanced over the response of patients' unfractionated PBL, and (4) the suppressed proliferation of patients' PBL to TT is not reversed by hemodialysis. Thus, the presence of suppressor monocytes and the inability of the responding T cells and accessory monocytes to react to antigen contribute to the suppressed antigen-specific T cell proliferation observed in chronic renal failure patients. These results are relevant to the suppressed cell-mediated immunity observed in uremia.