Antibody isotype responses in Aspergillus-induced diseases

The Aspergillus fumigatus-specific immunoglobulin class and subclasses were evaluated in the sera of patients with allergic bronchopulmonary aspergillosis, aspergilloma, patients with asthma and immediate wheal-and-flare skin test reactivity to Aspergillus antigens, and normal controls. A sensitive sandwich enzyme immunoassay using a biotin-avidin amplification system was used to detect the specific antibodies. No difference was demonstrated in the specific antibody levels against A. fumigatus between the normal controls and A. fumigatus skin test-reactive asthmatic subjects except for immunoglobulin IgD and IgG4 isotypes. Allergic bronchopulmonary aspergillosis (ABPA) sera showed significant elevation of A. fumigatus-specific antibodies of all isotypes. On the other hand, aspergilloma sera, when compared with that of asthmatic individuals, showed significant increases of IgA, IgG1, IgG2, and IgG3 antibodies. The specific antibodies belonging to both IgG1 and IgG2 subclasses were elevated in ABPA, the former predominant in 40% of cases and the latter in 60%, while in aspergilloma IgG1 was predominant. Among the initially diagnosed ABPA patients, all specific Ig isotypes were considerably elevated when compared with sera from other patients being treated with prednisone or when compared with sera from patients who became asymptomatic after treatment.     

Murine monoclonal antibodies binding to the specific antigens of Aspergillus fumigatus associated with allergic bronchopulmonary aspergillosis

Four murine monoclonal antibodies, which were produced against Aspergillus fumigatus antigens using hybridoma technology, reacted with different antigenic components of A. fumigatus, and in turn these antigens showed reactivity with the sera from allergic bronchopulmonary aspergillosis (ABPA). All four antibodies were of IgM isotype. These antibodies reacted against eight antigen preparations from three different strains of A. fumigatus by enzyme-linked immunosorbent assay (ELISA). Only two of four antibodies reacted with the antigens in crossed immunoelectrophoresis (CIE), rocket immunoelectrophoresis, and agar gel double diffusion. In western blot analysis it was found that the antigenic components reacting with the monoclonal antibodies were mostly of the low molecular weight components of A. fumigatus antigens. These components also showed binding to both IgG and IgE antibodies in the sera of ABPA patients, but failed to show any reactivity with sera from aspergilloma patients. Hence these antigenic components may be of diagnostic significance and can be isolated using immunoaffinity chromatography.     

The uptake of Aspergillus fumigatus protein by serum IgG antibody from patients with pulmonary aspergillosis.

A method is presented for measuring the uptake of Aspergillus fumigatus protein by IgG antibodies from human serum. The human IgG is first isolated with an anti-IgG conjugated immunosorbent and then incubated with radio-iodinated A. fumigatus protein. A group of twenty-three control sera gave the same background levels as tests without sera. The highest uptake of A. fumigatus protein was given by the sera of patients with aspergilloma, and lower values were obtained with six out of thirteen sera from patient's with asthma and twenty-five out of twenty-eight sera from patients with asthma and pulmonary eosinophilia, i.e. allergic bronchopulmonary aspergillosis. These results were in agreement with precipitin tests, Inhibition studies with unrelated antigens showed that the reactions were specific for the A. fumigatus protein, of which more was bound by test sera than with a crude extract.     

Allergic bronchopulmonary aspergillosis and sensitization to Aspergillus fumigatus in chronic bronchiectasis in adults

Fifty adult subjects for whom a diagnosis of idiopathic bronchiectasis (excluding those secondary to tuberculosis or hypogammaglobulinaemia) had been confirmed previously were investigated by: questionnaire; blood eosinophil count; sputum culture for Aspergillus fumigatus and eosinophil count; chest radiography; skin-prick tests with several aeroallergens and four preparations of A. fumigatus, including a reference extract; measurement of specific IgE antibodies; precipitin testing and self-crossed immunoelectrophoresis with A. fumigatus. Five subjects were possible cases of allergic bronchopulmonary aspergillosis in whom the condition had been previously misdiagnosed or in whom sensitization to A. fumigatus had occurred after the onset of bronchiectasis. These five subjects had positive immediate skin reactions to A. fumigatus and a history of recurrent pneumonias. Four had a previous history of asthma and the others showed increased bronchial responsiveness to inhaled methacholine. At the time of the survey, A. fumigatus grew in the sputum of one out of five subjects. These subjects had increased levels of specific IgE. Two had precipitins by double diffusion and three subjects were positive on self-crossed immunoelectrophoresis. It is concluded that allergic bronchopulmonary aspergillosis or evidence of sensitization to A. fumigatus can be identified in a significant proportion of adult subjects with so-called idiopathic bronchiectasis.     

Recombinant antigens as diagnostic markers for aspergillosis

Eight recombinant proteins and purified galactomannan of Aspergillus fumigatus were tested by enzyme-linked immunosorbent assay to quantify the anti-Aspergillus antibodies in sera of patients with aspergilloma, allergic bronchopulmonary aspergillosis (ABPA), and invasive aspergillosis (IA). In spite of the variability observed in the immune responses of individual patients, quantification of the antibody titers against the 18-kDa ribonuclease (RNU), the 360-kDa catalase (CAT), and the 88-kDa dipeptidylpeptidase V (DPPV) was useful for the diagnosis of aspergilloma and ABPA. Differential diagnosis of ABPA was even possible among cystic fibrosis as well as noncystic fibrosis patients. In the group of immunocompromised patients with IA, no antibody response was mounted in response to the Aspergillus infection in any of the patients. Interestingly, about half of the patients with proven IA came to the hospital with high titers of anti-Aspergillus antibodies, suggesting that they were infected upon entry to the hospital. These results suggest that recombinant RNU, CAT, and DPPV have a great potential in the serodiagnosis of all forms of aspergillosis in the immunocompromised and immunocompetent patient.     

Concanavalin A-nonbinding Aspergillus fumigatus antigen: a major immunogen in allergic bronchopulmonary aspergillosis.

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.     

Quantification of IgG antibodies to Aspergillus fumigatus and pigeon antigens by ImmunoCAP technology: an alternative to the precipitation technique?

BACKGROUND: We evaluated the ImmunoCAP technique for measurement of IgG specific to Aspergillus fumigatus and pigeon antigens. METHODS: We used ImmunoCAP and precipitation technique to measure concentrations of IgG to A. fumigatus or pigeon antigens in sera from 265 patients and 42 controls. We also evaluated linearity, interference, imprecision, concordance, and diagnostic accuracy of the measuring techniques. RESULTS: The precipitation and ImmunoCAP technique showed moderate concordance (kappa, 0.46 for both A. fumigatus and pigeon antibodies). Specific IgG results for A. fumigatus and pigeon were linear (r = 0.98 and 0.97, respectively), with interrun reproducibility rates of 23% and 14% and maximal interference of 36.5% and 8% by lipid and 24% and 21% by hemolysis, respectively. A. fumigatus antibody concentrations were higher in patients with aspergillosis and allergic bronchopulmonary aspergillosis (ABPA) (median, 103 and 70.1 mgA/L, respectively) than in patients with other pulmonary diseases (median, 18.15-33.40 mgA/L). Antibodies to pigeon antigens were high in patients with hypersensitivity pneumonitis (median, 1024 mgA/L) but also in patients with other pulmonary diseases (median, 445 mgA/L). Antibody titers were substantially higher in patients with other pulmonary diseases and contact with pigeons (median, 1060 mgA/L) than in patients without antigen contact (median, 27.35 mgA/L) (P <0.004). CONCLUSIONS: Agreement between the precipitation and ImmunoCAP technique was 86% for A. fumigatus and 70% for pigeon antigens. Highest concentrations of specific IgG to A. fumigatus were found in patients with aspergillosis and ABPA. Our results suggest that antigen contact was the most important variable affecting the presence of antibodies to pigeon antigen.     

Immunological studies in thymectomized and non-thymectomized patients with myasthenia gravis.

Eleven thymectomized and ten non-thymectomized patients with myasthenia gravis, matched with respect to sex, age, duration and severity of the disease were investigated with respect to routine clinical features, electrophysiological examination, HLA-typing, auto-antibodies, lymphocyte subpopulations in peripheral blood, Concanavalin A-induced release of leucocyte migration inhibitory factor (LIF), in vitro lymphocyte activation by mitogens and antigens and response to primary immunization with dinitrochlorobenzene measured in vivo and in vitro. The following conclusions could be drawn. The immune response to external antigens seems to be normal in myasthenia gravis and thymectomy is not followed by general defects in immune competence; at least as investigated by current techniques. The only reduction of responsiveness demonstrable in the thymectomized group was a decreased release of LIF by Concanavalin A-stimulated lymphocytes. Primary immune responses appear to be increased after adult thymectomy, which may be due to a decrease in suppressor T-lymphocyte activity. A hypothesis is formulated that Concanavalin A-induced release of LIF may reflect the competence of suppressor T-lymphocytes in man.     

Cell-Mediated Immunity to Pityrosporum Orbiculare in Tinea Versicolor

Pityrosporum orbiculare, the presumed etiologic agent of tinea versicolor, was cultured in vitro and antigenic extracts prepared from the cultured organisms. Studies with lymphocytes from human cord blood and peripheral blood of guinea pigs demonstrated that such extracts were not mitogenic. Further studies in guinea pigs indicated that the animals could be sensitized by the injection of P. orbiculare extract in Freund's complete adjuvant and that this extract could elicit lymphocyte transformation and delayed skin test responses in sensitized animals. A group of 12 tinea versicolor patients and 15 normal subjects were studied in vitro for cell-mediated immunity to P. orbiculare extract. The majority of the subjects tested in both groups demonstrated positive lymphocyte transformation responses to this extract, as well as to standard mitogens and common microbial antigens. However, lymphocytes from tinea versicolor patients produced significantly less leukocyte migration inhibitory factor activity when stimulated by Candida albicans and P. orbiculare extracts than did lymphocytes from normal subjects. This was also true if only subjects with positive lymphocyte transformation responses to these antigens were considered. Leukocyte migration inhibitory factor responses to streptokinase/streptodornase were not significantly different between the two groups. Therefore, it appears that although both normal subjects and tinea versicolor patients demonstrate prior sensitization to antigens of P. orbiculare, the effector function of lymphocytes from most tinea versicolor patients appears to be impaired in that they produce subnormal amounts of the mediator leukocyte migration inhibitory factor when stimulated with antigenic extracts of this organism.     

Paranasal aspergilloma caused by an albino variant of Aspergillus fumigatus.

An unusual case of aspergilloma in the maxillary sinus caused by an albino variant of Aspergillus fumigatus var ellipticus is presented. Removal of the fungus ball by a left Caldwell-Luc procedure resulted in complete recovery for the patient. This case is the 17th culturally proven case of paranasal aspergilloma caused by A fumigatus, the third case reported from the United States, the first case due to aspergillosis caused by an albino strain of A fumigatus.     

Chronic pulmonary aspergillosis

Aspergillus species causes a variety of pulmonary diseases, invasive pulmonary aspergillosis in severely immunocompromised patietnts, chronic pulmonary aspergillosis (CPA) in patients with chronic lung diseases and allergic bronchopulmonary aspergillosis in patients with hypersensitivity to Aspergillus antigens. There are many species of Aspergillus, however Aspergillus fumigatus is the most commonly encountered species. CPA is usually seen in patients with documented or suspected underlying lung diseases like cystic fibrosis, bronchiectasis, inactive tuberculosis, pulmonary fibrosis, sarcoidosis, previous lung section, of these, previous pulmonary tubercurosis is the most associated condition. Development of new antifungal agents, such as micafungin and voriconazole significantly affect the management and outcome of patients with CPA. This article reviews the clinical features, diagnosis, and treatment of CPA, and that provides recent advances in the CPA-related pathogenic factor.     

Comparison of azoles against aspergilli in vitro and in an experimental model of pulmonary aspergillosis.

Current treatment modalities for bronchopulmonary aspergillosis are not very satisfying. We determined the in vitro activity of recently available azoles against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Subsequently, these agents were evaluated in an animal model of bronchopulmonary aspergillosis using A. fumigatus as test organism. In vitro, detectable activity was only found for itraconazole (all minimal inhibitory concentrations, MICs, less than or equal to 3.2 micrograms/ml). The MICs for SCH39304 were greater than or equal to 12.8 micrograms/ml and greater than or equal to 25.6 micrograms/ml for ketoconazole and fluconazole. In vivo, amphotericin B was the most active agent tested, and SCH39304 was the most active azole in terms of survival and reduction in lung weight, followed by itraconazole. Ketoconazole and fluconazole did not improve survival nor reduce the lung weight of infected animals. We conclude, (1) that in vitro activity of azoles against aspergilli does not always correlate with in vivo activity; (2) that in vivo, SCH39304 was the most active azole tested, followed by itraconazole; (3) that for those agents for which data about effectiveness in human pulmonary aspergillosis are available (amphotericin B, ketoconazole, itraconazole) antifungal activity in our model corresponds to activity as seen in human beings, and (4) that SCH39304 and itraconazole are rational choices for clinical trials in human pulmonary aspergillosis.     

The prevalence of allergic bronchopulmonary aspergillosis in patients with asthma, determined by serologic and radiologic criteria in patients at risk

Allergic bronchopulmonary aspergillosis, a hitherto uncommon but potentially crippling complication of asthma, occurs at an unknown prevalence in the United States. Using both modern serologic criteria complete with a history of asthma and radiologic findings, we were able to make the diagnosis of allergic bronchopulmonary aspergillosis in 28 of 100 consecutive patients with asthma who had immediate cutaneous reactivity to Aspergillus fumigatus and who were seen in an outpatient setting. Problems associated with use of less comprehensive criteria for this diagnosis are discussed.     

Incidence of immediate sensitivity to Aspergillus fumigatus in a North American asthmatic population.

Allergic bronchopulmonary aspergillosis might be less frequent in North America because the incidence of immediate sensitivity by asthmatics to A. fumigatus is less. In order to check this hypothesis, 200 asthmatics were skin tested with two extracts of A. fumigatus which had been shown to produce positive reactions in fifty patients who had allergic aspergillosis. Of the asthmatics, 21.5% reacted to the commercial extract by prick testing and 39% by intradermal testing. Using an extract kindly provided by Professor Pepys, 19.5% reacted to a concentration of 1 mg/ml and 31.5% to 10 mg/ml. By the prick method, 21.5% reacted to both extracts. Specific IgE was measured with one of the extracts and a good correlation (r = 0.48) was found with the size of the prick reaction. The increase in specific IgE was reflected in the increase of total IgE (r = 0.84). The authors conclude that the incidence of immediate sensitivity to A. fumigatus in asthmatic patients in North America is at least equal to that found in the U.K.     

Impact of Aspergillus fumigatus in allergic airway diseases

ABSTRACT: For decades, fungi have been recognized as associated with asthma and other reactive airway diseases. In contrast to type I-mediated allergies caused by pollen, fungi cause a large number of allergic diseases such as allergic bronchopulmonary mycoses, rhinitis, allergic sinusitis and hypersensitivity pneumonitis. Amongst the fungi, Aspergillus fumigatus is the most prevalent cause of severe pulmonary allergic disease, including allergic bronchopulmonary aspergillosis (ABPA), known to be associated with chronic lung injury and deterioration in pulmonary function in people with chronic asthma and cystic fibrosis (CF). The goal of this review is to discuss new understandings of host-pathogen interactions in the genesis of allergic airway diseases caused by A. fumigatus. Host and pathogen related factors that participate in triggering the inflammatory cycle leading to pulmonary exacerbations in ABPA are discussed.     

Diagnosis of allergic bronchopulmonary aspergillosis

Five patients (4 men and 1 woman) afflicted with bronchopulmonary aspergillosis (ABPA) were examined. The main criteria for the diagnosis were a high level of the total IgE, demonstration of specific IgE to Aspergillus fumigatus, positive skin tests with antigens of moldy fungi. All the 4 patients with active process, not given corticosteroids, manifested eosinophilia of the sputum and peripheral blood. The clinical picture of bronchial asthma was recorded in 2 patients. It is desirable that all the patients with protracted and relapsing infiltrative processes in the lungs running with sputum and blood eosinophilia undergo examinations for the presence of ABPA.     

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.     

Cryptogenic pulmonary eosinophilia.

The clinical and immunological features of fifteen cases of cryptogenic pulmonary eosinophilia are reported. There were ten women (mean age 35.4 years) and five men (mean age 42 years). Eight gave a previous history of asthma and seven had none. Thirteen of the fifteen patients had negative skin test to common allergens. Many features of a systemic illness were present in the asthmatic and non-asthmatic groups including anaemia, weight loss, fever and a grossly raised ESR. An absolute polymorphonuclear leucocytosis was frequent as well as the obligatory increase in blood eosinophils used as one of our criteria for inclusion. Hepatomegaly (three cases), splenomegaly (four cases) and hilar node enlargement (one case) were seen in the group without asthma. Evidence of renal involvement or necrotizing vasculitis was notably absent and the response to small doses of corticosteroids was dramatic. Immunologically the striking feature was a disproportionate increase in blood eosinophils compared with only minor elevations in the total serum IgE levels. This stands in contrast to patients with bronchopulmonary aspergillosis and helminth infestation. Studies of cytophilic antibodies using histamine liberation after challenge with antibodies to immunoglobulin sub-classes in six patients showed a marked increase in IgG2 and lesser increases of IgE and IgG3. No evidence of antibodies specific to A. fumigatus was found. The amount of cytophilic antibody was also in contrast to that found in bronchopulmonary aspergillosis.     

Pathogenesis and clinical features of chronic pulmonary aspergillosis – Is it possible to distinguish CNPA and CCPA clinically?

BackgroundThe pathogenesis of chronic pulmonary aspergillosis (CPA) including chronic necrotizing pulmonary aspergillosis (CNPA), chronic cavitary pulmonary aspergillosis (CCPA), and simple aspergilloma (SA) has been poorly investigated. We examined all types of CPA cases with histopathological evidence to clarify the differences in pathogenesis and clinical features.MethodWe searched for cases diagnosed as pulmonary aspergillosis by histopathological examination in Nagasaki University Hospital between 1964 and September 2010. All available clinical information including radiological findings were collected and analyzed.ResultWe found 7, 5, 8, and 7 cases of proven CNPA, probable CNPA, CCPA, and SA, respectively. The radiograph of proven and probable CNPA was initially infiltrates or nodules that progress to form cavities with or without aspergilloma, whereas the radiograph of CCPA showed pre-existed cavities and peri-cavitary infiltrates with or without aspergilloma. The patients with proven and probable CNPA exhibited not only respiratory symptoms but also systemic symptoms and malnutrition. Aspergillus fumigatus was the most frequently isolated Aspergillus species (n = 14), however, Aspergillus niger was the predominant isolated species in proven CNPA cases (n = 4).ConclusionOur data indicate that the cases with chronic infiltration, progressive cavitation, and subsequent aspergilloma formation should be diagnosed as CNPA, and the cases with pre-existed cavities showing peri-cavitary infiltrates with or without aspergilloma would mean CCPA. However, it may be difficult to distinguish the two subtypes if a series of adequate radiography films are not available. We propose the term “chronic progressive pulmonary aspergillosis (CPPA)” for the clinical syndrome including both CNPA and CCPA.     

Sputum itraconazole concentrations in cystic fibrosis patients

Itraconazole diffusion in sputum was studied in 11 cystic fibrosis patients with allergic bronchopulmonary aspergillosis. There was a high interindividual variability in sputum itraconazole concentration and sputum/serum drug concentration ratio. Three children had sputum drug concentrations before oral administration that were lower than the itraconazole MIC at which 90% of Aspergillus fumigatus strains were inhibited, although their serum drug concentrations were within the therapeutic range.