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Analysis of IgG Subclass and IgE Response in Allergic Disease Caused by Aspergillus fumigatus by Immunoblotting Techniques
Abstract
Analysis of sera from patients suffering from allergic asthma, extrinsic allergic alveolitis (EAA) due to Aspergillus fumigatus or allergic bronchopulmonary aspergillosis (ABPA) by means of IEF immunoprint and Western blot showed characteristic IgG subclasses and IgE responses. Asthmatic patients revealed specific IgE antibodies. IgG subclass reactivities in asthmatics and healthy blood donors were weak and uncharacteristic. Major allergens recognized by asthmatic patients have isoelectric points (pI) of pI 4.4 and 4.8 and molecular weights of 20, 29, 34, 38 and 45 kD. Patients suffering from EAA typically demonstrated strong IgG subclass reactivities with an emphasis to IgG1 and IgG2, while no specific IgE was found. Various major antigens could be identified. Reaction to a pI 6.0 antigen seemed to be characteristic for EAA. ABPA patients showed strong reactivities of all IgG subclasses and of IgE. An intense IgG4 reaction appeared as a typical marker for ABPA. Several major antigens and allergens could be determined. Especially the IgG4 reactivities to proteins with pI 4.6, 5.0, 5.2, 5.4 and 6.5 and molecular weights of 14, 20, 29, 34, 38 and 45 kD seemed to be a characteristic pattern in ABPA. Furthermore, IgE antibodies to culture filtrate components of 34 and 38 kD appeared to be typical in ABPA.
... Así, en el caso concreto de la ABPA, se ha comprobado que estos pacientes tienen un elevado nivel de anticuerpos específicos de tipo IgG e IgE, lo cual supone un criterio de ayuda para el diagnóstico [46]. Por tanto las técnicas de inmunoblotting con la subsiguiente detección de subclases de IgG y de IgE pueden jugar un importante papel en el serodiagnóstico de enfermedades alérgicas debidas a A. fumigatus [47]. ...
... La detección de antígenos puede ser un método efectivo cuando se disponga de reactivos suficientemente sensibles y específicos. Las investigaciones actuales apuntan en esa dirección [49], si bien se encuentra la dificultad de estandarización de los extractos de A. fumigatus [47]. En este sentido ya hemos comentado a la hora de hablar de la producción de elastasa, su característica de ser inducida, y por tanto, la posibilidad de síntesis in vivo, sin que sea detectada in vitro. ...
... Cette association pourrait être sous-diagnostiquée du fait de caractéristiques cliniques et fonctionnelles parfois comparables. La PHS et l'ABPA ont en commun la mise en jeu d'une réaction d'hypersensibilité de type III, IgG médiée [9]. Une prédisposition génétique et des formes familiales sont décrites dans ces deux pathologies. ...
Hypersensitivity pneumonitis and allergic bronchopulmonary aspergillosis are two forms of lung disease with presumed distinct immunoallergic mechanisms. We report the observation of a 38-year-old French farmer who, for one month, had fever and dyspnoea resistant to antibiotic therapy. A diagnosis of farmer's lung, and allergic bronchopulmonary aspergillosis was made on clinical, biological, functional and radiological evidence and according to the criteria established. The evolution was favorable with antigenic eviction and corticosteroid therapy. This observation is to our knowledge the 5th case that describes the association of hypersensitivity pneumonitis and allergic bronchopulmonary aspergillosis. It suggests the existence of risk factors and immunoallergic mechanisms common to both diseases and discusses the hypothesis that the same antigen(s) is (are) responsible for them.
Copyright © 2019 SPLF. Published by Elsevier Masson SAS. All rights reserved.
... Diese werden als Auslöser von unspezifischen Symptomen wie rezidivierenden Infektionen der oberen Atemwege, Müdigkeit, Schlafstörungen oder Kopfschmerzen vermutet. (Trompelt et al. 2004). Es wird empfohlen, dass die diagnostische und Therapie-Lösung vom gleichen Hersteller stammen, damit ein vergleichbarer Antigengehalt gewährleistet ist. ...
Asthma and mould exposure in childhood and adolescence The appearance of a mould infestation is seen by the general public as the most relevant of indoor toxic substance problems. The complexity of the problems of mould exposure requires an objectification in the public. An improvement of the medical diagnostic and therapeutic methods can be made through a collective effort by professional associations and industry. Furthermore, the education and further training of doctors related to this subject must be increased. When taking all facts and findings into consideration a sensible and successful clinical diagnostics, prevention and therapy of children and adolescents with asthma and mould allergies is possible. Zusammenfassung Das Auftreten eines Schimmelpilzbefalls wird von der Bevölkerung als das relevanteste Innenraumschadstoffproblem angesehen. Die Proble-matik der Schimmelpilzexposition bedarf einer Versachlichung in der Öffentlichkeit. Durch gemeinsame Anstrengung von Fachgesellschaften und Industrie kann eine Verbesserung der medizinisch-diagnostischen und therapeutischen Methoden erzielt werden. Außerdem muss die Aus-und Fortbildung von Ärzten zu dieser Thematik verstärkt werden. Bei Berücksichtigung aller Fakten und Erkenntnisse ist eine sinnvolle und erfolgreiche Diagnostik, Prävention und Therapie von Kindern und Jugendlichen mit Asthma und Schimmelpilzallergie möglich.
... kDa were identified. Multiple allergenic proteins have been identified in Aspergillus extracts, varying from species to species (Gautam et al. 2007;Samuelsen et al. 1991;Trompelt et al. 1994;Yu et al. 1999). IgEbinding protein profile of A tamarii extract was different from that of A flavus, A fumigatus and A niger. ...
Aspergillus-derived inhalant allergens play an important role in the etiology of allergic respiratory diseases. In the present study, we investigated the allergenic potential of Aspergillus tamarii, quantified its airborne content, identified its major/minor allergens, evaluated heterogeneity of patients' IgE response to its allergens and cross-reactivity of its allergens with other Aspergillus allergens. Skin prick tests with A tamarii extract were performed on 300 patients of bronchial asthma/allergic rhinitis and 20 healthy volunteers. Sixty-six patients (22%) elicited positive cutaneous reactions to A tamarii extract. Only one of the 20 non-allergic healthy volunteer showed a mild positive cutaneous reaction. Allergen-specific IgE levels increased with increase in patients' cutaneous response (0% in negative to 100% in 3+/4+). The skin positivity and allergen-specific IgE levels were significantly higher in patients compared to healthy volunteers (P>0.05). However, no differences were found for these two parameters among patients of bronchial asthma, allergic rhinitis and bronchial asthma with allergic rhinitis. The airborne A tamarii allergen content was highest in February and October. A tamarii extract revealed at least 22 proteins (13.3-120 kDa). Seventeen of these proteins bound patients' IgE with six being major allergens (13.3, 23, 25, 34, 39.5, 43 kDa). Three major allergens (13.3, 34, 43 kDa) were found to cross-react with A flavus and one (34 kDa) with A niger. Our results revealed that A tamarii allergen(s) are present in the air, which might serve as important inhalant allergens in IgE-mediated allergic respiratory diseases.
... In humans, lung hypersensitivity to A. fumigatus can occur in different forms (2). The two opposite extremes are asthma with increased serum IgE titers (2) and hypersensitivity pneumonitis with increased serum IgG and low IgE titers (3,4). Clinically, asthma presents as recurrent bouts of dyspnoea due to bronchoconstriction, whereas hypersensitivity pneumonitis is characterized by bouts of dyspnoea accompanied by influenza-like symptoms (e.g., fever, fatigue). ...
We have used interleukin-10 (IL-10) gene knockout mice (IL-10-/-) to examine the role of endogenous IL-10 in allergic lung responses to Aspergillus fumigatus Ag. In vitro restimulated lung cells from sensitized IL-10-/- mice produced exaggerated amounts of IL-4, IL-5, and interferon-gamma (IFN-gamma) compared with wild-type (WT) lung cells. In vivo, the significance of IL-10 in regulating responses to repeated A. fumigatus inhalation was strikingly revealed in IL-10-/- outbred mice that had a 50-60% mortality rate, while mortality was rare in similarly treated WT mice. Furthermore, IL-10-/- outbred mice exhibited exaggerated airway inflammation and heightened levels of IL-5 and IFN-gamma in bronchoalveolar lavage (BAL) fluids. In contrast, the magnitude of the allergic lung response was similar in intranasally (i.n.) sensitized IL-10-/- and wild-type mice from a different strain (C57BL/6). Using a different route of priming (intraperitoneal) followed by one i.n. challenge we found that IL-10-/- C57BL/6 mice had heightened eosinophilic airway inflammation, BAL-IL-5 levels, and numbers of alphabetaT cells in the lung tissues compared with WT mice. We conclude that IL-10 can suppress inflammatory Th2-like lung responses as well as Th1-like responses given the constraints of genetic background and route of priming.
... IgG4 reactivity to P. glabrum was more intense in HP, suggesting that, like in Aspergillus-induced lung disorders, chronic antigen exposure in the cork industry may favour IgG4 production (8,25). Surprisingly, in the asthmatic group, although IgG4 reactivity was weaker (Fig. 1, bottom), the frequency of reactivity to the 33 kDa component was higher (71 vs 33% in HP). ...
We characterized by immunoblotting the antigenicity of the most frequent fungi colonizing cork during its industrial processing, Penicillium glabrum and Chrysonilia sitophila. Penicillium glabrum is the main causative agent of Suberosis, a hypersensitivity pneumonitis of cork workers. Chrysonilia sitophila induces both IgE sensitization and occupational asthma in the wood processing industry.
Serum-specific IgG, IgG4 and IgE to P. glabrum and C. sitophila from nine cork workers with hypersensitivity pneumonitis (HP) and seven with asthma (four with occupational asthma) were analysed by immunoblotting.
Both HP and asthmatic patients' sera showed immunoreactivity to several proteins resolved in the specific immunoblot strips. The frequency of specific IgG recognition to 12-13.5 and 33 kDa proteins of P. glabrum was significantly higher in HP patients. The sera of HP patients had significantly higher specific IgG recognition to 16 and 51-55 kDa proteins of C. sitophila. There was no specific IgE recognition in the sera of HP or asthmatic patients to both fungi.
Different patterns of antibody reactivity to P. glabrum and C. sitophila are seen in cork workers with hypersensitivity pneumonitis or asthma. The 12-13.5 and 33 kDa proteins of P. glabrum and the 16 and 51-55 kDa proteins of C. sitophila may be major antigens in Suberosis.
Allergic bronchopulmonary aspergillosis (ABPA) is a complication of allergic (atopic) asthma.
ABPA extends the pathology from the bronchi into the pulmonary parenchyma.
ABPA has been diagnosed in perhaps somewhat under 1% of asthmatics, but estimates suggest that 450,000 persons with asthma in the U.S. are at high risk for the disease
Unrecognized ABPA can progress to irreversible bronchiectasis and pulmonary fibrosis.
Worsening of asthma, the production of sputum containing small mucus plugs, pulmonary infiltrates, or a sudden rise in peripheral eosinophilia or serum IgE should alert the clinician to the diagnosis of ABPA.
A positive skin test to Aspergillus antigens is a screening test for the diagnosis, but is insufficient for confirmation.
In ABPA, the fungal organism, Aspergillus, acts as an antigen provoking IgE and immune complex responses, rather than acting as an invasive agent.
An intensive course of systemic glucocorticosteroids is the most effective method to reverse the immunological and clinical manifestations.
Recent studies suggest that itraconazole, an anti-Aspergillus oral fungicide, may exert beneficial effects.
ABPA has been broadened to the category of allergic bronchopulmonary mycoses with the inclusion of several other fungi in a small minority of patients.
Introduction:
IgG4 has recently been a subject of great interest in human pathology. No data are available about the characteristics of asthma patients with elevated IgG4 levels.
Population and methods:
An observational study was conducted from January 2006 to March 2015 in a difficult-to-treat population of asthma patients. Twenty-six difficult-to-treat asthma patients with elevated serum IgG4 levels (IgG4/IgG ratio up to 10%) were compared with a control population of 98 difficult-to-treat asthma patients with normal serum IgG4. Blood eosinophilia, total IgE and FeNO were compared between groups to better characterize asthma patients with elevated serum IgG4 levels.
Results:
Median IgG4 concentrations were 1.72 g/l [1.19-2.36] and 0.22 g/l [0.10-0.49] in the elevated IgG4 group and normal Ig4 group, respectively. Median blood eosinophilia was more than three times higher in patients with elevated serum IgG4 levels than in controls (0.75 10(9)/L [IQR 0.54-1.78] vs 0.22 10(9)/L [IQR 0.09-0.54] respectively, p < 0.0001). Total IgE was twice as high (264.5 kUI/l [IQR 166.3-779] vs 126 kUI/l [IQR 26-350] respectively; p < 0.05) and FeNO was nearly twice as high (61 [IQR 41-111] ppb vs 35 [IQR 23-51] ppb, p < 0.001). Allergic broncho-pulmonary aspergillosis (ABPA) and eosinophilic granulomatosis with polyangiitis (EGPA) were observed in the asthma patients with elevated serum IgG4. Ten patients had unexplained increased blood eosinophilia.
Conclusion:
Asthma patients with elevated IgG4 levels have significantly higher blood eosinophilia, total IgE and FeNO. ABPA and EGPA are observed in patients with elevated serum IgG4.
The appearance of a mould infestation is seen by the general public as the most relevant of indoortoxic substance problems. The complexity of the problems of mould exposure requires an objectification in the public. An improvement of the medical diagnostic and therapeutic methods can be made through a collective effort by professional associations and industry. Furthermore, the education and further training of doctors related to this subject must be increased. When taking all facts and findings into consideration a sensible and successful clinical diagnostics, prevention and therapy of children and adolescents with asthma and mould allergies is possible. © ecomed Medizin, Verlagsgruppe Hüthig Jehle Rehm GmbH, Landsberg.
PhD Thesis, Faculty of Medicine, Porto University 2003
Die Identifizierung des fur das Krankheitsbild einer exogen allergischen Alveolitis (EAA) relevanten Allergens stellt im Einzelfall ein Problem dar, und nur uber eine akribische Anamnese gelingt die Eingrenzung der Allergenquelle. Wir berichten uber eine 32-jahrige Patientin, bei der wir eine chronische EAA diagnostiziert haben, die uber die Persistenz von Wellensittichallergen in einem Eigenheim vermittelt wurde, das sie seit zwei Jahren bewohnte und dessen Vorbesitzer uber Jahre Wellensittiche gehalten hatten. Erganzende Untersuchungen an Staubproben aus dem Wohnbereich der Patientin mit dem Nachweis prazipitierender Antikorper wie auch IgG-reaktiver Banden im Immunoblot machten die genannte Atiologie hinreichend wahrscheinlich. Auch ohne Wohnungswechsel konnte innerhalb eines Jahres eine Normalisierung der initial bis auf etwa 50 % des Sollwertes eingeschrankten Vitalkapazitat erreicht werden, was als Ausdruck einer rucklaufigen Allergenlast anzusehen sein durfte. The identification of disease-inducing allergens in hypersensitivity pneumonitis can be very problematic, and only by a thorough analysis of anamnestic data can the source of allergen be identified. We report on a case of a 32-year-old female diagnosed with hypersensitivity pneumonitis caused by inhalation of budgerigar antigen in her home. She had been living there for two years and had never been a birdkeeper at all. The former proprietor of the house was a budgerigar keeper for years. When we detected precipitating antibodies against different antigens including pigeon and budgerigar antigens as well as hay and Aureobasidium pullulans, the source of antigen exposition was not definitely clear. In the serum of our patient we found precipitating antibodies against protein structures extracted from dust samples from the patient's home, which were not detected in the serum of her husband. Using Western blots of budgerigar serum and of the dust sample from the patient's home we could demonstrate an IgG-reactive banding pattern in our patient's serum. The banding pattern against budgerigar serum correlated very closely to that of a control patient, who was a budgerigar keeper with hypersensitivity pneumonitis. The patient's husband reacted neither against budgerigar serum nor against the dust sample, while he and his wife showed double banding at about 9 kDA when their serum was exposed to dust from a home free of birdkeeping. These results point to the fact that the house dust sample of our patient contained budgerigar antigen, leading to an indirect antigen exposure causing hypersensitivity alveolitis. Our patient received a prolonged treatment with corticosteroids, and after about one year, vital capacity of the lungs which was reduced by 50 % at the beginning of treatment, returned to normal. The patient is still living in her home. Although she has been off medication for one year, lung function tests have not deteriorated. This fact points to a reduction of the amount of antigen in the patient's home.
Detection of Aspergillus-specific serum antibodies aids in the diagnosis of ABPA. The antigens of A. fumigatus, the fungus predominantly implicated in ABPA, are highly visible and consist of a heterogeneous mixture of proteins, carbohydrates, and glycoproteins. With recent advancement in bioseparation, hybridoma, and molecular biology techniques, well-characterized and purified antigens of A. fumigatus have been obtained. The use of these standardized antigens may be an aid in increasing the sensitivity and specificity of immunodiagnosis in ABPA.
Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease mediated by an allergic late-phase inflammatory response to Aspergillus fumigatus antigens characterized by markedly elevated allergen specific and total immunoglobulin E (IgE) levels and eosinophilia, and manifested by wheezing, pulmonary infiltrates, and pulmonary bronchiectasis and fibrosis. ABPA afflicts only a small percentage of susceptible atopic individuals with either asthma or cystic fibrosis (CF). ABPA is unique in that it is caused by A. fumigatus, a living fungus growing within the mucosal lumen of the bronchi that releases a multitude of proteins whose biologic function alters the host immune responses in addition to being immunogenic. Elucidation of the immunopathogenesis of ABPA has provided valuable information related to allergen-induced chronic asthma. More recently, immunogenetic studies have further contributed to the understanding of the immunopathogenesis of why certain individuals are susceptible to develop ABPA. Immunologic responses to Aspergillus, as well as to other microorganisms, may be categorized as either T helper (Th)1 or Th2 CD4+ T-cell responses. 61, 110 and 128 Th1 CD4+ T-cell cytokines, such as interferon gamma (IFN-γ), interleukin 2 (IL-2), and tumor necrosis factor (TNF)-β, enhance cytotoxic and macrophage activity (e.g., cellular immunity), whereas Th2 CD4+ T-cell cytokines, such as IL-4, IL-5, IL-10, and IL-13, promote antibody synthesis (e.g., humoral antibody immunity) (Table 1). It is hypothesized that protective host immunity to A. fumigatus is a Th1 CD4+ T-cell response, whereas a Th2 CD4+ T-cell humoral response found in ABPA is nonprotective and leads to a hypersensitivity pulmonary lung disease.
We investigated the relationship between antibody response to the major Paracoccidioides brasiliensis antigen, a 43-kDa glycoprotein, and the two paracoccidioidomycosis (PCM) clinical presentations, the juvenile and the adult forms. Total immunoglobulin G (IgG), IgG isotypes, and IgA anti-gp43 antibodies were determined by enzyme-linked immunosorbent assay in patients'sera. Juvenile PCM patients had higher (P =.003) IgG anti-gp43 levels than adult form patients. IgG1 subclass levels, however, were comparable between the two clinical forms. Patients with the juvenile form had higher (P <.001) IgG4, but lower (P =.03) IgG2 levels than patients with the adult form. The IgG4 isotype, regulated by interleukin 4, was found in all juvenile form patients but in only 12% of the adult form patients. In contrast, high levels of the IgG2 isotype, regulated by interferon-γ, were found in 41% of the adult PCM patients, mainly those with a more benign disease, but in only 12% of the juvenile patients. IgG3 was either absent or detected at low levels. These results demonstrate, for the first time, specific IgG4 antibodies in the humoral immune response of patients with an endemic deep mycosis and suggest that the switch to the IgG subclasses in PCM is regulated by the patients' T-helper subset (Th-1 or Th-2) dominant cytokine profile. A possible role for IgG4 in the immunopathogenesis of the juvenile, more severe form of the disease is discussed. Finally, IgA was found mainly in adult form patients, probably as a result of the chronic mucosal antigenic stimulation characteristic of this form.
Allergic bronchopulmonary aspergillosis (ABPA), which results from hypersensitivity, primarily to Aspergillus, represents a severe complication in patients suffering from asthma or cystic fibrosis (CF). Since early treatment of ABPA
is supposed to prevent long-term damages, ABPA has to be diagnosed promptly. However, this diagnosis is not straightforward
due to clinical and radiological features of ABPA overlapping with those of CF. Despite ABPA specific diagnosis criteria proposed
by the Cystic Fibrosis Foundation in 2003, making a definitive ABPA diagnosis in CF patients remains a challenge. Recent advances
in the immunopathogenesis of ABPA have initiated the development of new serological tests, such as the recently reported detection
of specific IgE to recombinant A. fumigatus allergens, or Thymus- and activation-regulated chemokine (TARC / CCL17), both of which are of value in the diagnosis of APBA.
We review in this paper the serum markers that can advance ABPA diagnosis in CF patients, ranging from the well known criteria
(anti-A. fumigatus IgE, IgG, and precipitins) to the recent biomarkers (IgE towards recombinant A. fumigatus allergens or TARC detection). Taking into account the up-dated physiopathology of ABPA, we discuss their place and their
usefulness, especially TARC, to improve early ABPA detection and monitoring in CF patients.
Dissertação de Doutoramento em Medicina apresentada à Faculdade de Medicina da Universidade do Porto
Due to the increasing popularity of exotic fruits in the Western diet, allergologists are confronted with allergic reactions to substances in these plants. The present report describes an anaphylactic reaction after the consumption of lychee fruit (Litchi sinensis). The atopic patient also suffers from rhinoconjunctivitis due to a sensitization against pollen of the Compositae family, as well as from dyspnoea after eating sunflower seeds. Our goals were to determine crossreactivity between antibodies against lychee fruit and other plants and to characterize the allergen.
Specific IgE against lychee fruits were detected by an EAST assay. The allergen was characterized by immunoblot, immunoblot inhibition and EAST inhibition assays. Broad crossreactivity between lychee fruit and other plants was found and profilin identified as the protein responsible for the patient's complex allergy syndrome.
Lychee fruit contains a significant amount of profilin. Consumption of this exotic fruit can cause severe anaphylactic reactions in patients being sensitized against the plant pan-allergen profilin.
Members of the genus Aspergillus are opportunistic pathogens for cold- and warm-blooded animals. They are associated with an impressive spectrum of diseases in humans, ranging from benign colonization of the lung to life-threatening diseases such as invasive aspergillosis or allergic bronchopulmonary aspergillosis (ABPA). Aspergillus fumigatus is the etiological agent identified in most of the Aspergillus-related human diseases and is therefore of particular clinical relevance. A major problem in the diagnosis of A. fumigatus-related complications arises from the lack of standardized fungal extracts. The allergenicity of commercial A. fumigatus extracts depends on many factors including the strain used, growth conditions, harvesting and extraction procedures hampering the development of diagnostic reagents suitable for the discrimination between A. fumigatus-related diseases. Molecular DNA/RNA technologies and biotechnological procedures allow cloning, characterization and production of large amounts of single, highly pure allergens. The development of phage surface display technologies for cloning cDNAs has speeded up the isolation of candidate cDNAs encoding allergens. Screening of an A. fumigatus cDNA library displayed on the surface of phage M13 allowed characterization and production of a panel of allergens identified as proteins with known biological function or as allergens without sequence homology to known proteins. They belong to two functional categories: secreted and cytoplasmic proteins. Secreted allergens are recognized by serum IgE of A. fumigatus-sensitized individuals with or without ABPA, whereas nonsecreted allergens are exclusively recognized by serum IgE of ABPA patients. The dissection of IgE-mediated immune responses to single A. fumigatus allergens allows a discrimination between ABPA and A. fumigatus sensitization with high specificity and sensitivity demonstrating the power of recombinant allergens for the differential diagnosis of A. fumigatus-related pulmonary complications.
Inhalant exposure to Aspergillus fumigatus (Asp. f.) antigens induces marked inflammatory and immunological alterations in the lungs of horses affected with chronic obstructive pulmonary disease (COPD). In this study we investigated the role of specific allergen(s) present in Asp. f. on systemic and pulmonary IgE and IgG responses in control and COPD-affected horses, using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting techniques. Compared with controls, horses affected with COPD had significantly higher levels of BALF IgE and IgG to somatic Asp. f. antigens as well as to the allergen 1/a (Asp. f. 1/a). Serum levels of IgE and IgG against these antigens did not differ between control and COPD-affected horses. Antigen specific IgE and IgG levels did not correlate between BALF and serum. Scanning of Asp. f. and IgE and IgG blots revealed bands that are recognised by both IgE- and IgG-specific antibodies. Additionally, all horses responded with BALF IgE- and IgG-specific for 93, 35, 31 and 23 kDa allergens, suggesting that these antigens are involved in the induction of airway IgE and IgG responses. These allergens may have the potential to be used as biomarkers for the diagnosis of Asp. f. related exacerbations of equine COPD.
Aspergillus fumigatus is one of the most ubiquitous of the airborne saprophytic fungi. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide. In this review, the focus is on the biology of A. fumigatus and the diseases it causes. Included are discussions of (i) genomic and molecular characterization of the organism, (ii) clinical and laboratory methods available for the diagnosis of aspergillosis in immunocompetent and immunocompromised hosts, (iii) identification of host and fungal factors that play a role in the establishment of the fungus in vivo, and (iv) problems associated with antifungal therapy.
IgG and IgG subclass antibodies to Aspergillus fumigatus (A fumigatus) were measured in a large population of patients with cystic fibrosis to elucidate a putative antibody pattern specific for allergic bronchopulmonary aspergillosis (ABPA).
An ELISA technique using water soluble somatic hyphal (WSSH) A fumigatus antigens and subclass specific monoclonal antibodies was used for cross sectional quantification of IgG and IgG1-4 subclass antibody levels in the serum of 238 patients with cystic fibrosis and 107 healthy controls.
In patients with cystic fibrosis persistently colonised with A fumigatus the subclass antibody levels were significantly increased compared with patients with cystic fibrosis never or rarely colonised (p < 0.001). The group of patients persistently colonised with A fumigatus with ABPA (+Af+ABPA) had significantly increased levels of IgG antibodies to A fumigatus (Af-IgG) (median 69 ELISA units (EU) versus 31) and of subclasses Af-IgG1 (91 versus 27), Af-IgG2 (143 versus 56), and Af-IgG4 antibodies (72 versus 20), but not of IgG3 (17 versus 15), compared with the colonised patients without ABPA (+Af-ABPA). Patients with cystic fibrosis with no or only rare isolates of A fumigatus without ABPA (-Af-ABPA) also had significantly increased subclass antibody levels (Af-IgG1 9 versus 3, Af-IgG2 28 versus 5, Af-IgG4 16 versus 4; p < 0.001) compared with healthy controls. Low, although detectable, levels of antibodies were demonstrated in healthy controls. ABPA seemed to occur independently of Pseudomonas aeruginosa infection. Using diagnostic cut off levels for ABPA, sensitivity and specificity were calculated. The highest specificity was found for IgG4 (88%); sensitivity was between 65% and 73%. The positive predictive values (PPV) were moderate, whereas the negative predictive values (NPV) were high (96% in all subclasses except IgG3 with 94%). PPV increased to 50% if IgG1 as well as IgG2 and IgG4 were included.
In a large number of unselected patients with cystic fibrosis significantly increased levels of Af-specific antibodies belonging to total IgG and all four subclasses were found in all groups of patients compared with healthy controls. In patients persistently colonised with A fumigatus these levels were significantly higher than in non-colonised patients, and the significantly highest levels (with the exception of IgG3) were found in patients with ABPA. Using a sensitive ELISA technique, measurements of IgG and IgG subclass antibodies to A fumigatus might be of importance in the management of ABPA, especially as a screening test to exclude the presence of ABPA; other tests are needed to confirm the diagnosis.
Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df), the major components of house dust, are considered to be etiologic factors of extrinsic asthma. The relationships between immunoglobulin (Ig) G subclass antibodies specific for Dp (or Df) were compared in specific IgE-positive and -negative asthmatic children.
Serum levels of IgG and IgE subclass antibodies specific for Dp and Df were studied in 52 children (age, 3-13 years; mean age, 8.4 years) with asthma using enzyme-linked immunosorbent assays. The skin prick test was also used in diagnosis of the reactivity of allergic disease.
The levels of serum-specific IgG1 and IgG2 to Dp and Df in mite-specific IgE-(or skin-test) positive asthmatic children were significantly higher than those in mite-specific IgE- (or skin test) negative children (p < 0.01). Significant correlations between the level of the specific IgE and IgG1 (r = 0.6067; p = 0.0001) or IgG2 (r = 0.5851; p = 0.0002) to Dp, and IgG1 (r = 0.3823; p = 0.0214) or IgG2 (r = 0.5057; p = 0.0017) to Df were found. The specific IgE level and skin test reactivity showed a high correlation of greater than 96% (50/52).
The levels of mite-specific IgG subclasses were partially compatible to specific IgE levels and skin test reactivity. We conclude that house dust mite allergy is significantly associated with specific IgG1, IgG2 and IgE responses.
Aspergillus species are common airborne fungi that have been identified as causative agents of extrinsic bronchial asthma. More than 10 allergens from A. fumigatus have been recently characterized by cDNA cloning.
The objective of this study is to identify A. fumigatus allergens through immunoblot analysis using sera from asthmatic patients.
IgE-binding components of A. fumigatus and IgE cross-reactivity among allergens of different prevalent airborne fungal species were analysed by immunoblot and immunoblot inhibition, respectively, using sera from asthmatic patients. The N-terminal amino acid sequences of major allergens identified were determined by Edman degradation.
Among two batches (70 and 41 sera) of asthmatic sera tested, 19 (27%) and 14 (34%), respectively, have IgE immunoblot reactivity towards components of A. fumigatus. A 34-kDa protein that reacts with IgE antibodies in 15 (79%) and 11 (79%) of the 19 and 14 positive samples, respectively, may be considered a major allergen of A. fumigatus. The N-terminal amino acid sequences of the 34 kDa major allergen and the 30.5 and 30 kDa IgE-binding components of A. fumigatus showed sequence identity to that of the vacuolar serine proteinase from A. fumigatus. The results from immunoblot inhibition show IgE cross-reactivity among major allergens of A. fumigatus, P. notatum and P. oxalicum.
Results obtained suggest that the 34 kDa major allergen of A. fumigatus may be a vacuolar serine proteinase. There is IgE cross-reactivity among serine proteinase allergens of A. fumigatus, P. notatum and P. oxalicum.
Suberosis is an occupational lung disease of cork workers associated with repeated exposure to mouldy cork dust in the cork industry, usually presenting as an interstitial lung disorder (Extrinsic Allergic Alveolitis). However, Occupational Asthma can also be associated with cork dust exposure and demonstrated by serial peak expiratory flow changes.
To investigate broncho-alveolar inflammation in patients with cork work-related occupational asthma, evaluated by Broncho-alveolar fluid cellular profiles and serial peak expiratory flow (PEF) rates monitoring.
We studied 14 patients with respiratory symptoms associated with occupational exposure in the cork industry. Positive PEF monitoring occurred in 7 cases (Occupational Asthma-OA), and in 7 (Non-occupational asthmatics-NOA) PEF records were negative. There were no differences in age, lung function (FEV1%, RV%), bronchial hyperresponsiveness, years of exposure, and atopy between the two patients groups. However, patients with work-related asthma had higher BAL eosinophil counts than NOA (1.9 +/- 2.6% versus 0.2 +/- 0.3%; p < 0.05, Wilcoxon test).
Cork worker's asthma, demonstrated by work related changes in serial PEF recordings, is associated with eosinophilic lung inflammation as described in other forms of occupational asthma.
Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease mediated by an allergic late-phase inflammatory response to Aspergillus fumigatus antigens. ABPA is characterized by markedly elevated Aspergillus-specific and total IgE levels and eosinophilia, and manifested by wheezing, pulmonary infiltrates and bronchiectasis and fibrosis, which affect asthmatic and cystic fibrosis (CF) patients. In the pathogenesis of ABPA, A. fumigatus proteases play a role in facilitation of antigen transport across the epithelial cell layer by damaging the epithelial integrity and by a direct interaction with epithelial cell surface receptors, resulting in pro-inflammatory cytokine production and corresponding inflammatory responses. In genetically susceptible asthmatic and CF patients, this leads to an allergic inflammatory response to Aspergillus allergens. A genetic susceptibility is HLA-DR restriction demonstrated by increased frequency of HLA-DR2 and/or DR5 and lack HLA-DQ2. IL-4 plays a central role in the development of allergic inflammatory responses. Our group has demonstrated that ABPA patients have increased sensitivity to IL-4 stimulation and skewing of Th2 responses to Aspergillus allergens in ABPA subjects and a Th1 response in non-ABPA subjects. Interestingly, Aspergillus allergens stimulate IL-10 synthesis is both ABPA and non-ABPA subjects.
For the diagnosis of allergic aspergillosis demonstration of specific immune responses to allergens has been accepted as a significant paradigm. Elevated levels of total IgE and Aspergillus fumigatus specific IgE and IgG antibodies are important criteria for diagnosis of ABPA. Although reference antigens or standardized methods are not available, there are a number of relevant recombinant antigens, which have been isolated in recent years. Several techniques have been employed in the demonstration of specific antibodies against antigens of Aspergillus fumigatus in the sera of patients. Of these methods, the widely followed ones are ELISA, radioimmunoassay, Western blot, and agar gel double diffusion. Recently, semi-automated methods have been developed using recombinant allergens to detect circulating antibody against Aspergillus. However, these methods have not been evaluated widely. Here we review the immunodiagnostic methods currently in use for allergic bronchopulmonary aspergillosis.
Because of the difficulties of recognizing allergic bronchopulmonary aspergillosis (ABPA) in the context of cystic fibrosis
(because of overlapping clinical, radiographic, microbiologic, and immunologic features), advances in our understanding of
the pathogenesis of allergic aspergillosis, new possibilities in therapy, and the need for agreed-upon definitions, an international
consensus conference was convened. Areas addressed included fungal biology, immunopathogenesis, insights from animal models,
diagnostic criteria, epidemiology, the use of new immunologic and genetic techniques in diagnosis, imaging modalities, pharmacology,
and treatment approaches. Evidence from the existing literature was graded, and the consensus views were synthesized into
this document and recirculated for affirmation. Virulence factors in Aspergillus that could aggravate these diseases, and particularly immunogenetic factors that could predispose persons to ABPA, were identified.
New information has come from transgenic animals and recombinant fungal and host molecules. Diagnostic criteria that could
provide a framework for monitoring were adopted, and helpful imaging features were identified. New possibilities in therapy
produced plans for managing diverse clinical presentations.
Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease mediated by an allergic late-phase inflammatory response to Aspergillus fumigatus antigens. ABPA is characterized by markedly elevated Aspergillus-specific and total IgE levels and eosinophilia, and manifested by wheezing, pulmonary infiltrates, and bronchiectasis and fibrosis, which afflict asthmatic and cystic fibrosis (CF) patients. We propose that ABPA develops in genetically susceptible CF patients due to HLA-DR2 and DR5 restriction, increased sensitivity to IL-4 stimulation, and increased A. fumigatus allergen-specific Th2 CD4+ T-cell-mediated responses. In addition, A. fumigatus proteases play a role in facilitation of antigen transport across the epithelial cell layer by damaging the epithelial integrity and by a direct interaction with epithelial cell surface receptors, resulting in pro-inflammatory cytokine production and corresponding inflammatory responses.
Penicillium species are prevalent airborne fungi. However, the prevalence of allergic sensitization to Penicillium antigens and the true impact of these ubiquitous fungi on atopic respiratory disorders remain to be determined.
The purpose of this study was to analyze the prevalence of immunoglobulin (Ig)E and IgG antibodies against Penicillium chrysogenum (Pen ch 13), the alkaline serine protease major allergen of P. chrysogenum, in asthmatic patients of different age groups.
Pen ch 13 was purified from a culture medium of P. chrysogenum. The reactivity of IgE and IgG antibodies to Pen ch 13 in the serum samples of 212 asthmatic patients was analyzed by immunoblotting methods.
Sixty-nine (33%) of the 212 sera analyzed showed IgE and/or IgG immunoblot reactivity to Pen ch 13. Significant differences in the prevalence of IgE and/or IgG antibody reactivity to Pen ch 13 were found among eight different age groups of 212 asthmatic patients. The frequency of IgE-binding reactivity to Pen ch 13 increased significantly with the age of the patients. It was 7% for the group less than 10 years old and 42% for the group older than 70 years old. In addition, a significant difference between the prevalence of IgE (7%) and IgG (33%) antibodies against Pen ch 13 in the group aged 10 or less was also found.
Our study demonstrates that IgE and IgG antibodies specific for Pen ch 13 were detected in approximately one-third of the 212 asthmatic patients analyzed. Our results suggest that allergic sensitization to Pen ch 13, and possibly to other airborne Penicillium species, is more common in older asthmatic patients.
Allergic bronchopulmonary aspergillosis (ABPA) is the result of hypersensitivity to Aspergillus antigens in patients with long-standing atopic asthma. In the present study mycelial and culture filtrate antigens from Aspergillus fumigatus cultures isolated from diverse sources were tested against sera of 10 ABPA patients and 10 control individuals by an ELISA methodology. The results indicate higher antibody reactivity against both antigens in the sera of ABPA patients, while culture filtrate antigens also gave non-specific reactivity with control sera. Mycelial extracts, in general, were useful in the diagnosis of ABPA.
Aspergillosis comprises a wide range of clinical conditions, of which the most serious is invasive aspergillosis, which particularly affects immunodeficient individuals. In the present work we present a review centred on three main aspects of this disease: 1) Possibility of differentiation of strains of Aspergillus fumigatus by phenotyphic and genotypic methods, 2) Mechanisms of pathogenicity of this species and especially the relationships of elastase activity, and 3) Laboratory diagnosis of aspergillosis, principaly in terms of determination of circulating antigens and DNA sequences in the blood and urine of patients.
Commercially available preparations of Aspergillus fumigatus, experimental extracts and fractions of A. fumigatus, and extracts of 5 other aspergilli were evaluated in a blind manner for their abilities to detect antibody in 66 serum specimens from patients with aspergilloma, allergic bronchopulmonary and invasive aspergillosis, and unconfirmed aspergillosis. Rocket immunoelectrophoresis was more sensitive than immunodiffusion in 2 dimensions. Although qualitative differences in antigen composition could be detected, commercial preparations compared favorably with the best experimental extracts in detecting positive specimens. Extracts from young, actively-growing myceliums were most effective and produced the largest numbers of precipitin bands. In contrast to all unfractionated preparations, several fractions of A. fumigatus were devoid of substances that react with C-reactive protein of serums, yet were as effective as the best preparations in detecting serums positive for Aspergillus.
Techniques developed for protein blotting have been applied to the characterisation of allergen extracts. Such extracts, used for the diagnosis and therapy of allergic diseases, are highly variable in their content of active components. Protein blotting allows the direct identification of those components in an extract which bind human immunoglobulin E and cause allergic symptoms. It also allows the components to be defined in terms of molecular weight, isoelectric point and the frequency and intensity of IgE-binding by individual allergens. Because of the large number of samples which can be handled in a single experiment, protein blotting of allergens allows the identification of the individual allergen recognition spectra of large numbers of allergic patients. The resolution of components, loss of antigenicity, and aspects of blocking and probing which are important in identifying IgE-binding components are reviewed. A table of allergen sources investigated by protein blotting is included and important sources of allergens, such as pollens, mites and fungi are discussed in detail.
IgE and IgG antibodies against Aspergillus fumigatus were detected by crossed radio immunoelectrophoresis (CRIE) on the sera of seven patients with aspergilloma, six patients with allergic broncho-pulmonary aspergillosis (ABPA) and 25 patients with extrinsic asthma with Aspergillus allergy. IgE-CRIE analysis indicated the presence of A. fumigatus-specific IgE in sera of patients with ABPA and Aspergillus asthma but not of aspergilloma patients. IgG-CRIE showed that both aspergilloma and ABPA patient sera contained high levels of circulating specific IgG antibodies in contrast to sera of Aspergillus asthma patients, which did not show detectable amounts of Aspergillus-specific IgG antibodies. Specific IgE binding could be demonstrated for the major allergens Ag-10 and AG-40 in all ABPA patients, in 80% of Aspergillus asthma patients but not in sera from aspergilloma patients. Specific IgG antibodies directed towards the major allergens could be detected in most of the aspergilloma patients, between 30-70% of the ABPA patients but not in sera from patients with Aspergillus asthma.
Culture filtrate antigens of Aspergillus fumigatus were fractionated by isoelectric focusing using a pH gradient of 4-6.5. Three fractions, namely, 18, 19 and 20 were pooled and subjected to immunochemical analysis. It contained a concanavalin A binding glycoprotein. Antibody raised against this component was used to prepare an affinity column of IgG-Sepharose and was used to purify crude culture filtrate antigens. This component produced three precipitin arcs in crossed immunoelectrophoresis using anti-A. fumigatus rabbit serum. This fraction has an isoelectric point of 6.5 and showed three components in two-dimensional electrophoresis with approximate molecular weights of 20, 40 and 80 kilo daltons. This antigen reacted with patient sera in the biotin-avidin linked immunosorbent assay and showed high levels of anti-A. fumigatus IgG and IgE antibodies in allergic bronchopulmonary aspergillosis and IgG antibodies in aspergilloma. Both controls and Aspergillus skin test positive asthmatics showed only low levels of specific antibodies. Because of the purity of this antigen, its potential use as a standardized antigen in the detection of antibody is discussed.
A specimen of Aspergillus fumigatus was isolated from a patient with acute bronchopulmonary aspergillosis (ABPA). Cultures were allowed to propagate and were separated into spores and mycelium to greater than 95% homogeneity. Extracts of both the spores and mycelium were prepared and used for study by both dot blot analysis and immunoblotting to study the major allergens. By dot blot analysis, 22 of 22 patients with ABPA and none of 10 healthy controls reacted with mycelium when probed for both IgG and IgE anti-Aspergillus reactivity. Similar results were obtained when these extracts were separated on polyacrylamide gel electrophoresis and then probed; this included both IgG and IgE reactivity. Further, the reactivity to preparations of whole extract was absorbed with mycelium but not spores. Several distinct protein bands were detected with IgG, ranging from 30 to 110 kilodaltons (kD). In contrast, the majority of patients with ABPA, when studied for IgE reactivity, reacted only with a 70-kD protein, although 1 out of 22 patients reacted with a 30-kD protein. Subclass analysis of IgG reactivity demonstrated that 90% of reactive sera contained IgG2 and 73% IgG4 reactivity. IgG1 and IgG3 reactivity were present but less frequently detected. The use of immunoblotting will enable the identification of relevant allergens. Further, the demonstration that the allergenicity is located primarily in mycelium will allow more definitive studies of allergen isolation for studies of reactivity and, ultimately, cloning of the 70-kD protein and, finally, epitope mapping.
Individual immunoprint patterns of sodium dodecylsulfate-polyacrylamide gel electrophoresis-separated Aspergillus fumigatus (Af) allergens/antigens were evaluated in 28 patients with allergic bronchopulmonary aspergillosis in stages II to V. It could be demonstrated that active disease (stage III) is characterized by a very strong IgE response against a variety of Af components, whereas the IgE antibody pattern in corticosteroid-treated patients who have demonstrated improvement is much weaker. In patients in remission (stage II), it is minimal. In contrast, many patients in stage V without corticosteroid treatment demonstrated a strong reactivity of IgE antibodies, indicating persisting active disease. The pattern of IgG antibodies with individual Af components resembles, in general, that of IgE antibodies; however, discrimination between different stages and between treated patients is much weaker. Our results indicate that a certain relationship between the different stages of allergic bronchopulmonary aspergillosis and Af immunoprint patterns exists.
Probing of IgE or IgG subclass reactivities on single allergenic components in an extract is tedious and time-consuming under native conditions. Isoelectric focusing immunoblotting combines a powerful, highly resolving native separation method with the specificity and sensitivity of immunological test methods. This method is easy and quick to perform. The potential usefulness of this method is demonstrated by eight examples of a type I or type III allergy analyzing IgE and IgG subclass reactivities and their distribution.
The performance of ELISA to detect IgG and IgM antibodies to Aspergillus fumigatus has been evaluated in strongly precipitin-positive, weakly precipitin-positive and precipitin-negative patient sera, with immunoblot analysis as the confirmatory test. All strongly precipitin-positive sera contained increased IgG titers and showed clearly positive immunoblot patterns. Most of the weakly precipitin-positive sera contained ELISA titers within the normal range established with sera of healthy blood donors and showed normal immunoblot patterns. Increased titers of IgG and/or IgM were measured in one-sixth of the precipitin-negative patient sera. Immunoblot analysis confirmed the presence of antibodies to A. fumigatus in 55% of the precipitin-negative sera with increased antibody titers. ELISAs for A. fumigatus-specific IgG and IgM are sensitive tests for screening of patient sera. However, positive results with ELISA should be confirmed by means of immunoblot analysis.
An antigenic component of Aspergillus fumigatus, which was responsible for inducing an initial early antibody response in rabbits immunized with A. fumigatus germlings, has been identified and partially characterized. By immunofluorescent antibody and ELISA techniques, this antigen was demonstrated to be present on the germling surface, although it was also detected in supernatants of conidia/germlings within 1 hour and was present in all shake and stationary culture-filtrate extracts. By incorporation of a monospecific antiserum to this component in an intermediate gel of the reference self-crossed radioimmunoelectrophoresis pattern for allergic bronchopulmonary aspergillosis sera, it was recognized as Ag 5 and was also demonstrated to bind specific IgE. Further immunochemical analyses have revealed that Ag 5 is relatively heat labile, does not bind to concanavalin A, and has a molecular weight of approximately 35 kd. This rapidly released antigenic/allergenic component may play an important role in the initiation of immunologic responses in patients with allergic bronchopulmonary aspergillosis.
An immunologically relevant antigen fraction was isolated from a cytoplasmic extract of Aspergillus fumigatus mycelium. The fraction was obtained by concanavalin-A affinity chromatography and hydrophobic interaction chromatography. Two protein bands were discernible in polyacrylamide gel electrophoresis while two precipitin peaks were detected in crossed immunoelectrophoresis. When tested against sera from patients with Aspergillus-induced diseases, the fraction showed both IgG and IgE antibody-binding activity. All of the sera studied from patients with allergic bronchopulmonary aspergillosis (ABPA) showed high IgG and IgE antibody titers, while sera from patients with aspergilloma and cystic fibrosis with ABPA demonstrated high IgG titers and only a moderate increase in IgE titers. None of the other groups showed any significant antibody titers against this antigen in their sera. Because of its binding to both IgG and IgE antibodies this fraction was found to be useful in the immunodiagnosis of Aspergillus-induced diseases.
A component of Aspergillus fumigatus, Ag 7, previously identified as a major antigen for patients with allergic bronchopulmonary aspergillosis, has been isolated by gel filtration and further purified by affinity chromatography using monospecific antiserum. The antigen, which binds both specific IgG and IgE antibodies, was shown to be a high molecular weight, 150-200 kD, heat-stable glycoprotein, which binds to concanavalin A, suggesting the presence of alpha-D mannopyrannoside, or alpha-D glucopyrannoside end residues. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) it had a subunit of molecular weight 36 kD (with or without prior reduction), which retained antigenicity and allergenicity when tested with patients' sera.
The allergen composition of moulds (Alternaria alternata and Aspergillus fumigatus) and mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae) was studied by electroblotting. The allergens were separated by SDS-gPAGE and transferred to a nitrocellulose membrane by electroblotting. Specific IgE antibodies directed against mould and mite allergens were utilized as probes to detect the allergens. The bands were localized on the nitrocellulose membrane by means of enzyme- or isotope-labelled antibody. Circulating specific IgE antibodies in mould and mite sensitive patient sera were also analyzed and characterized with respect to their IgE specificity. Under appropriate experimental conditions this electroblotting technique could also be used for quantitation of individual allergens by scanning the isotope- or enzyme-stained nitrocellulose strips.
A total of 22 sera from patients with aspergilloma and allergic bronchopulmonary aspergillosis (ABPA) were examined concomitantly for specific antibody against Aspergillus fumigatus antigen and for their activity in antibody-dependent cell-mediated cytotoxicity (ADCC) against Aspergillus antigen-coated target cells. These sera demonstrated significant precipitin bands in agar gel double diffusion test (78% of ABPA and 75% of aspergilloma sera), while in indirect immunofluorescence studies all sera showed positive reactivity with a titer distribution of 1:40 to 1:160 and 1:40 to 1:320, respectively, for ABPA and aspergilloma sera. In enzyme-linked immunosorbent assay all sera demonstrated titers varying from 1:200 to 1:6400. Several sera also displayed marked cytotoxic reactions against A. fumigatus antigen-coated SB target cells in ADCC assays using normal lymphocytes as effector cells (35% of aspergilloma and 25% of ABPA sera). These findings suggest a role for ADCC activity in patients with Aspergillus infections.
Sera of 60 allergic persons and 20 healthy RAST-negative control persons were examined using immunoblotting to establish the presence of grass pollen-specific antibodies. These antibodies were depicted by an enzymatic, immunoglobulin class-specific indicator system. Seven bands of the timothy pollen extract proved to be major allergens. One of the 4 grass pollen-specific monoclonal antibodies tested (Bo 1) recognized the 7 major allergen bands. The possibility of employing monoclonal antibodies for the isolation of allergens is discussed.
IgG antibodies to three purified Aspergillus fumigatus antigens were determined by enzyme-linked immunosorbent assay (ELISA) in sera from 26 patients with definite pulmonary aspergillosis (group 1), 23 patients with suspected pulmonary aspergillosis (group 2), 8 patients with precipitating antibodies to A. fumigatus of undetermined clinical significance (group 3), and 113 healthy blood donors (group 4). With a 470,000-dalton antigen fraction ELISA values exceeded the upper range of group 4 (0.72) in 92, 91 and 13% of cases in groups 1, 2 and 3, respectively. With a 250,000-dalton antigen fraction the figures were 96, 78 and 13%, respectively (upper range of group 4 = 0.25). With an antigen fraction of 25,000-50,000 daltons the figures were 96, 65 and 13%, respectively (upper range of group 4 = 0.18). In 48 of 49 cases of definite or suspected aspergillosis (groups 1 + 2) ELISA values with at least one antigen exceeded the upper range of controls (group 4). It appears that antibody determination with these three antigen fractions may have a high diagnostic sensitivity.
Aspergillus fumigatus somatic antigen prepared from young hyphae was fractionated by hydrophobic interaction chromatography followed by gel filtration. The antigenic activity of fractions was investigated by enzyme-linked immunosorbent assay, crossed immunoelectrophoresis, and immunodiffusion. By enzyme-linked immunosorbent assay, high levels of IgG antibodies were detected against an antigen fraction of approximate molecular weight 470,000 both in patients with aspergillosis (n = 3) and in healthy controls (n = 3). High levels of antibodies to four fractions of MW 250,000 160,000, 160,000-50,000, and 50,000-25,000 were found only in patients. The MW 250,000 fraction exhibited catalase activity, and the MW 50,000-25,000 fraction had neutral protease activity. These purified aspergillus antigens may be useful in serodiagnosis of aspergillosis.
Three different stages were found during growth of Aspergillus fumigatus as characterized by the changes in pH of the medium: (1) An acidic phase (phase I); (2) a second phase (phase II) during which the pH rises till values around pH = 8.5; (3) a third phase (phase III) during which the pH remains constant or drops slightly. During phase I a fast appearance of certain antigenic components was found that is ascribed to an active process of excretion. Additional antigenic components appeared in the culture medium after lysis of the microorganisms (phases II and III). Lysis of the microorganisms and appearance of antigenic components are dependent on the glucose concentration of the medium.