Fungal sensitization and positive fungal culture from sputum in children with asthma are associated with reduced lung function and acute asthma attacks respectively.

BACKGROUND: Sensitization to thermotolerant fungi, including filamentous fungi and Candida albicans, is associated with poor lung function in adults with severe asthma. Data in children are lacking. Environmental exposure to fungi is linked with acute severe asthma attacks, but there are few studies reporting the presence of fungi in the airways during asthma attacks. METHODS: We investigated the association between fungal sensitization and/or positive fungal sputum culture and markers of asthma severity in children with chronic and acute asthma. Sensitization was determined using serum-specific IgE and skin prick testing against a panel of five fungi. Fungal culture was focused towards detection of filamentous fungi from sputum samples. RESULTS: We obtained sensitization data and/or sputum from 175 children: 99 with chronic asthma, 39 with acute asthma and 37 controls. 34.1% of children with chronic asthma were sensitized to thermotolerant fungi compared with no children without asthma (p =< 0.001). These children had worse pre-bronchodilator lung function compared with asthmatics without sensitization including a lower FEV1 /FVC ratio (p < .05). The isolation rate of filamentous fungi from sputum was higher in children with acute compared with chronic asthma. CONCLUSIONS: Fungal sensitization is a feature of children with chronic asthma. Children sensitized to thermotolerant fungi have worse lung function, require more courses of systemic corticosteroids and have greater limitation of activities due to asthma. Asthma attacks in children were associated with the presence of filamentous fungi positive sputum culture. Mechanistic studies are required to establish whether fungi contribute directly to the development of acute asthma.

Cytokine Profile of Patients with Allergic Rhinitis Caused by Pollen, Mite, and Microbial Allergen Sensitization.

Allergic rhinitis (AR) is especially prevalent among the population of large cities. Immunologically, the airway epithelium is a region where the population of allergen-presenting cells concentrates. These cells actively express a group of receptors of the innate immune system. A specific cytokine profile is its representation. The study was aimed at evaluating the cytokine profile in patients with seasonal and perennial allergic rhinitis. The cytokine profile of nasal secretion and blood serum of 44 patients with AR was studied. 24 of them had seasonal allergic rhinitis (SAR), and 20 patients suffered from perennial allergic rhinitis (PAR). The patients' age ranged from 4 to 60 years. It was determined in our study that the activation of the GM-CSF production retained in patients with PAR sensitized to mite allergen components (Dermatophagoides pteronyssinus). There was a higher production profile of TNF-α and TSLP in nasal secretion in the patients with perennial allergic rhinitis and additional high sensitization to SEs. Sensitization to mold fungal allergen components significantly increases in patients with seasonal allergic rhinitis. They demonstrated high level of sensitization to the Aspergillus fumigatus component m3. Thus, along with other clinical trials, the study performed would clarify some aspects of molecular pathogenesis of human allergic rhinitis.

Bacterial extract OM-85 BV protects mice against experimental chronic rhinosinusitis.

OBJECTIVES: To investigate the therapeutic effects of OM-85 BV as an adjunctive treatment on experimental chronic rhinosinusitis (CRS) in mice. METHODOLOGY: Female BALB/c mice aged 8-12 weeks were sensitized and administrated by intranasal Aspergillus fumigatis (AF) three times per week for 1 week, 3 weeks, 2 months and 3 months (n = 10 each time point). The mice were randomly and equally assigned to four groups: normal control group, model group, OM-85-BV plus amoxicillin group, and isolated amoxicillin group. Inflammatory changes were determined by hematoxylin-eosin (HE) staining. The expression levels of suppressor of cytokine signaling (SOCS) 1, SOCS3, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in samples were assessed by using real-time PCR (RT-PCR) and Western blotting. RESULTS: There were significantly inflammatory and structural changes between the model and other groups. Compared to the model group, the mRNA expression levels of SOCS1, SOCS3, TNF-α, and IFN-γ were significantly decreased in OM-85-BV plus amoxicillin group and isolated amoxicillin group, along with the protein levels. CONCLUSION: The bacterial extract OM-85 BV is a low-cost alternatively adjunctive drug to treat CRS with simple oral administration, good safety, and few side effects.

[Molecular markers: an important tool in the diagnosis, treatment and epidemiology of invasive aspergillosis]. / Marcadores moleculares: una herramienta importante en el diagnóstico, tratamiento y epidemiología de la aspergilosis invasora.

Increase in the incidence of invasive aspergillosis has represented a difficult problem for management of patients with this infection due to its high rate of mortality, limited knowledge concerning its diagnosis, and therapeutic practice. The difficulty in management of patients with aspergillosis initiates with detection of the fungus in the specimens of immunosuppressed patients infected with Aspergillus fumigatus; in addition, difficulty exists in terms of the development of resistance to antifungals as a consequence of their indiscriminate use in prophylactic and therapeutic practice and to ignorance concerning the epidemiological data of aspergillosis. With the aim of resolving these problems, molecular markers is employed at present with specific and accurate results. However, in Mexico, the use of molecular markers has not yet been implemented in the routine of intrahospital laboratories; despite the fact that these molecular markers has been widely referred in the literature, it is necessary for it to validated and standardized to ensure that the results obtained in any laboratory would be reliable and comparable. In the present review, we present an update on the usefulness of molecular markers in accurate identification of A. fumigatus, detection of resistance to antifugal triazoles, and epidemiological studies for establishing the necessary measures for prevention and control of aspergillosis.

El incremento en la incidencia de la aspergilosis invasora representa un grave problema para el tratamiento de pacientes con esta micosis, debido a su elevada tasa de mortalidad por deficiencias diagnósticas y terapéuticas. Éstas se han atribuido a la dificultad para detectar Aspergillus fumigatus, principal agente etiológico de esta micosis, en las muestras biológicas de pacientes inmunosuprimidos, que son los principales afectados por el hongo; además por la resistencia a los antifúngicos como consecuencia del uso incontrolado de éstos, a nivel profiláctico y terapéutico, y el desconocimiento de aspectos epidemiológicos de la aspergilosis. En la actualidad, para superar estas limitaciones se han empleado marcadores moleculares. En México su uso aún no está implementado en la rutina de los laboratorios intrahospitalarios, porque a pesar de que se han reportado ampliamente en la bibliografía, hace falta validarlos y estandarizarlos para asegurar que los resultados que se obtengan en cualquier laboratorio sean confiables y comparables. En este trabajo se presenta una revisión actualizada de la utilidad de los marcadores moleculares en la identificación certera de A. fumigatus en la detección de resistencia a los antifúngicos triazólicos y en estudios epidemiológicos para establecer las medidas necesarias en la prevención y control de la aspergilosis.

Role of growth arrest-specific gene 6 in the development of fungal allergic airway disease in mice.

Growth arrest-specific gene (Gas)6 is a secreted vitamin K-dependent protein with pleiotropic effects via activation of receptor tyrosine kinase Tyro3, Axl, and Mertk receptors, but little is known about its role in allergic airway disease. We investigated the role of Gas6 in the development of fungal allergic airway disease in mice. The immune response was evaluated in Gas6-deficient (Gas6-/-) and wild-type (WT) mice and in recombinant Gas6-treated WT mice during Aspergillus fumigatus-induced allergic airway disease. Gas6 plasma levels were significantly elevated in adult clinical asthma of all severities compared with subjects without asthma. In a murine model of fungal allergic airway disease, increased protein expression of Axl and Mertk were observed in the lung. Airway hyperresponsiveness (AHR), whole lung Th2 cytokine levels, goblet cell metaplasia, and peribronchial fibrosis were ameliorated in Gas6-/- mice compared with WT mice with fungal allergic airway disease. Intranasal Gas6 administration into WT mice had a divergent effect on airway inflammation and AHR. Specifically, a total dose of 2 µg of exogenous Gas6 (i.e., low dose) significantly increased whole lung Th2 cytokine levels and subsequent AHR, whereas a total dose of 7 µg of exogenous Gas6 (i.e., high dose) significantly suppressed Th1 and Th2 cytokines and AHR compared with appropriate control groups. Mechanistically, Gas6 promoted Th2 activation via its highest affinity receptor Axl expressed by myeloid DCs. Intranasal administration of Gas6 consistently exacerbated airway remodeling compared with control WT groups. These results demonstrate that Gas6 enhances several features of fungal allergic airway disease.

Surfactant protein D inhibits early airway response in Aspergillus fumigatus-sensitized mice.

BACKGROUND: The surfactant protein SP-D has been reported to reduce bronchial hyper-responsiveness, blood eosinophilia, and T-helper type 2 cytokines in models of allergic asthma. However, little is known about the functional effect of SP-D on the early airway response upon allergen inhalation, which is an important feature of this disease. OBJECTIVE: We investigated whether SP-D is able to reduce the immediate allergen-induced mediator release and the early bronchial obstruction in addition to its effects on airway inflammation and bronchial hyperresponsiveness in an Aspergillus fumigatus mouse asthma model. METHODS: A. fumigatus-sensitized mice were treated with a recombinant fragment of human SP-D or placebo. Lung functions were measured in orotracheally intubated, spontaneously breathing animals using body plethysmography. In addition, passively sensitized precision-cut lung slices (PCLS) were used to determine the effect of SP-D on allergen-induced histamine release. RESULTS: SP-D inhibited the allergen-induced early airway response and reduced airway hyperresponsiveness compared with placebo. Eosinophilia in bronchoalveolar lavage and lung tissue was reduced after SP-D treatment, possibly by reducing eotaxin levels in the lung. Furthermore, SP-D treatment reduced the allergen-induced histamine release from PCLS. CONCLUSION: These data suggest that SP-D not only reduces allergen-induced eosinophilic inflammation and airway hyperresponsiveness but also provides protection against early airway obstruction by inhibition of early mediator release.

Paradoxical increase in circulating Aspergillus antigen during treatment with caspofungin in a patient with pulmonary aspergillosis.

A paradoxical increase in circulating Aspergillus antigen was observed during treatment with caspofungin in a patient with proven invasive aspergillosis. With the exception of treatment with the echinocandin, no other factors were found that might explain this clinical observation, which was supported by experiments done in vitro.

Endotoxin contamination contributes to the pulmonary inflammatory and functional response to Aspergillus fumigatus extract inhalation in heaves horses.

BACKGROUND: Mould extract inhalation challenges have been used extensively in the investigation of heaves. Such challenges have induced pulmonary neutrophilic inflammation and dysfunction, consistent with, but less severe than the natural disease. However, the method of mould extract production is likely to result in endotoxin contamination. OBJECTIVE: To investigate whether insufficient dose delivery was responsible for the shortfall in response to inhaled extract compared with natural disease, and whether endotoxin contamination of mould extract contributed to the pulmonary inflammation and dysfunction. METHODS: We measured the response of six heaves horses following inhalation of saline (placebo) and three doses of Aspergillus fumigatus extract. We then compared the response of six heaves horses to A. fumigatus extract inhalation before and after lipopolysaccharide (LPS) depletion. RESULTS: Inhalation challenge with 0.5, 1.6 and 5 mg of A. fumigatus extract resulted in a significant increase in bronchoalveolar lavage fluid (BALF) neutrophil ratio when compared with saline inhalation. Only 1.6 and 5 mg extract inhalation resulted in significant lung dysfunction compared with saline. There was no significant difference between 1.6 and 5 mg extract inhalation with respect to airway neutrophil numbers or lung function, suggesting a plateau in both measured responses. LPS depletion of 1.6 mg A. fumigatus extract resulted in a significant reduction in airway neutrophil numbers and increase in arterial oxygen tension. There was no significant difference between saline and the LPS-depleted A. fumigatus extract challenges with respect to neutrophil count and lung function. The reduction in airway neutrophil numbers was greater than would be predicted by extrapolation from previously reported soluble LPS dose-response inhalation experiments. CONCLUSION: This study supports a role for other inhalants, in addition to soluble components of A. fumigatus, in the aetiopathogenesis of heaves. Also the amplification in response to LPS when inhaled with A. fumigatus extract, suggests that the role of inhaled endotoxin in the pulmonary inflammation and dysfunction in naturally occurring heaves may currently be underestimated.

A protease-activated pathway underlying Th cell type 2 activation and allergic lung disease.

The respiratory allergens that induce experimental Th cell type 2-dependent allergic lung inflammation may be grouped into two functional classes. One class of allergens, in this study termed type I, requires priming with adjuvants remote from the lung to overcome airway tolerogenic mechanisms that ordinarily preclude allergic responses to inhaled Ags. In contrast, the other, or type II, allergen class requires neither remote priming nor additional adjuvants to overcome airway tolerance and elicit robust allergic lung disease. In this study, we show in an experimental model that diverse type II allergens share in common proteolytic activity that is both necessary and sufficient for overcoming airway tolerance and induction of pulmonary allergic disease. Inactivated protease and protease-free Ag fragments showed no allergenic potency, demonstrating that only active protease acting on endogenous substrates was essential. Furthermore, induction of airway tolerance could be aborted and allergic lung disease established by simply adding purified protease to a type I allergen. Thus, exogenous proteases are common to type II allergens and may be generally required to overcome the innate resistance of the airway to Th cell type 2 activation and allergic inflammation, raising concern for their potential contribution to diseases such as asthma.

Intranasal delivery of a truncated recombinant human SP-D is effective at down-regulating allergic hypersensitivity in mice sensitized to allergens of Aspergillus fumigatus.

C57BL/6 mice were sensitized to Aspergillus fumigatus 1-week culture filtrate, which is rich in the non-glycosylated allergen Asp f1, a major allergen in allergic bronchopulmonary aspergillosis (ABPA). A comparison of the effect of treatment of allergen challenged mice by intranasal administration of a 60-kDa truncated recombinant form of human SP-D (rfhSP-D) or recombinant full length SP-A (rhSP-A) was undertaken. Treatment with rfhSP-D produced significant reduction in IgE, IgG1 and peripheral blood eosinophilia and treatment with rfhSP-D, but not rhSP-A resulted in a significant reduction in airway hyperresponsiveness as measured by whole body plethysmography. Lung histology revealed less peribronchial lymphocytic infiltration in mice treated with rfhSP-D. Intracellular cytokine staining of spleen homogenates showed increases in IL-12 and IFN-gamma and decrease in IL-4. The level of endogenous mouse SP-D was elevated sixfold in the lungs of sensitized mice and was not affected by treatment with rfhSP-D. Taken with our previous studies, with a BALB/c mouse model of ABPA using a 3-week A. fumigatus culture filtrate, the present results show that rfhSP-D can suppress the development of allergic symptoms in sensitized mice independent of genetic background and using a different preparation of A. fumigatus allergens.

Immune response modulation by recombinant peptides expressed in virus-like particles.

Aspergillus fumigatus, a ubiquitous fungus, is implicated in the pathogenesis of a number of clinically different allergic diseases in man, including allergic bronchopulmonary aspergillosis. Peptide-based immunotherapy may offer an alternative treatment strategy for the management of allergic disease. The objective of this study was to alter the allergen-specific immune response using dominant T cell epitopes of a major A. fumigatus allergen, Asp f2, expressed in yeast as virus-like particles (VLP). The T cell epitopes of Asp f2, recognized in mice with an H-2d background, were determined by producing T-cell hybridomas. Two dominant T cell epitopes, aa60--71 and aa235--249, were identified and expressed in a yeast VLP system. To induce tolerance VLP-peptides were injected subcutaneously into mice previously immunized with recombinant Asp f2. The T cell immune response was abrogated totally in 3 weeks following a single injection of VLP but was restored 2 months later following intranasal antigen exposure. T-cell depletion resulted in the reduction of 20-30% of all antigen-specific immunoglobulin classes. Thus, recombinant peptides expressed in the VLP system can be used successfully in the modulation of Asp f2-induced immune response in mice, although a single administration is not sufficient to maintain a state of tolerance for a long period of time.

Identification of a Hevea brasiliensis latex manganese superoxide dismutase (Hev b 10) as a cross-reactive allergen.

BACKGROUND: Cross-reactive allergens play an increasingly important role in latex allergy in complicating both the diagnosis and time course of allergic symptoms. Manganese superoxide dismutase (MnSOD), a ubiquitous protein of prokaryotic and eukaryotic organisms, was described as a cross-reactive allergen in Aspergillus fumigatus. Little information is available on the importance of this pan-allergen in Hevea brasiliensis latex. The aim of this study was to clone and express MnSOD from H. brasiliensis latex, and to obtain the soluble and immunologically active recombinant allergen for diagnosis of latex allergy and to investigate possible cross-reactivities with the structurally related A. fumigatus and human MnSODs. METHODS: A complementary DNA coding for Hevea latex MnSOD was amplified by PCR. The recombinant protein was produced in Escherichia coli with an N-terminal hexahistidyl tag. Enzymatic activity of the recombinant protein was determined using an enzyme assay for SODs. IgE immunoblotting and IgE inhibition assays were performed to characterize the recombinant allergen and its cross-reactivity. RESULTS: A Hevea latex MnSOD consisting of 206 amino acid residues was cloned and expressed in E. coli. The allergen was designated Hev b 10. The recombinant protein was enzymatically active, indicating the correct folding of the protein. In immunoblots, latex- as well as A. fumigatus-allergic patients revealed IgE binding to recombinant (r)Hev b 10. Cross-reactivity to Asp f 6, the MnSOD from A. fumigatus, and human MnSOD was determined by inhibition of IgE binding to these MnSODs by rHev b 10. CONCLUSIONS: Hev b 10 is a new cross-reactive allergen of H. brasiliensis which belongs to the 'latex-mold' group of latex allergens. Furthermore, it is a candidate for primary sensitization in patients allergic to the pan-allergen MnSOD.

Therapeutic effect of IL-13 immunoneutralization during chronic experimental fungal asthma.

IL-13 and IL-4 are key contributors to the asthmatic phenotype. The temporal role of these cytokines in airway function, inflammation, and remodeling were assessed in a chronic murine model of Asperigillus fumigatus-induced allergic asthma. IL-13 and IL-4 protein levels were significantly elevated by 30 days after conidia challenge in A. fumigatus-sensitized mice. Furthermore, IL-13Ralpha1 mRNA expression was significantly elevated 7 days after conidia challenge and remained elevated until day 21. In contrast, IL-13Ralpha2 mRNA expression, although constitutively expressed in naive lung, was absent in the lungs of A. fumigatus-sensitized mice both before and after conidia challenge. Membrane-bound IL-4R mRNA expression was significantly elevated 7 days after conidia challenge; however, soluble IL-4R mRNA expression was increased 30 days after conidia challenge. Immunoneutralization of IL-13 between days 14 and 30 or days 30 and 38 after fungal sensitization and challenge significantly attenuated airway hyperresponsiveness, collagen deposition, and goblet cell hyperplasia at day 38 after conidia challenge; however, the effects of IL-4 immunoneutralization during the same time periods were not as marked. IFN-gamma and IL-12 release after Aspergillus Ag restimulation was elevated from spleen cells isolated from mice treated with IL-4 anti-serum compared with IL-13 anti-serum or normal rabbit serum-treated mice. This study demonstrates a pronounced therapeutic effect of IL-13-immunoneutralization at extended time points following the induction of chronic asthma. Most importantly, these therapeutic effects were not reversed following cessation of treatment, and IL-13 anti-serum treatment did not alter the systemic immune response to Ag restimulation, unlike IL-4 immunoneutralization. Therefore, IL-13 provides an attractive therapeutic target in allergic asthma.

Acute inhibition of nitric oxide exacerbates airway hyperresponsiveness, eosinophilia and C-C chemokine generation in a murine model of fungal asthma.

OBJECTIVE AND DESIGN: This study examined the role of nitric oxide in changes in airway physiology and inflammation in a murine model of fungal allergy induced by Aspergillus fumigatus (A. fumigatus) by treatment of A. fumigatus-sensitized mice with NG-nitro-L-arginine methyl ester (L-NAME) or D-NAME (8 mg/kg; i.p.). MATERIALS AND METHODS: Female CBA/J mice received A. fumigatus antigen dissolved in incomplete Freund's adjuvant (10 mg/100 ml i.p. and s.c.) followed 2 weeks later by A. fumigatus antigens (20 mg; i.n.) and a subsequent i.t. challenge 4 days later. Airway physiology and inflammation were examined (24 to 72 h) following i.t. challenge. RESULTS: L-NAME-treated mice had lower lung nitrite levels 24 h after A. fumigatus challenge, but higher airway hyperresponsiveness and inflammation compared to D-NAME controls. Airway inflammation in the L-NAME treatment group (72 h) was characterized by a greater bronchoalveolar lavage (BAL), peribronchial eosinophilia and augmented levels of CC chemokines compared to controls. CONCLUSIONS: These findings suggest that nitric oxide is an important modulator of airway hyperresponsiveness, inflammation and C-C chemokine generation during allergic airway responses to A. fumigatus.

Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production.

BACKGROUND: Proteases in extracts of Aspergillus fumigatus cause epithelial cell desquamation and release of proinflammatory cytokines. OBJECTIVE: We sought to assess protease activity in Alternaria alternata, Cladosporium herbarum, and Aspergillus fumigatus extracts and study the ability of these extracts to cause desquamation and release of proinflammatory cytokines from epithelial cells. METHODS: Protease activities of the fungal extracts were quantified. Changes with respect to cell morphology, cell desquamation, and cytokine production (IL-6 and IL-8) were measured in the absence and presence of the fungal extracts in an airway-derived epithelial cell line (A549) and primary epithelial nasal cells. RESULTS: Fungal proteases differentially induced morphologic changes, cell desquamation, and production of IL-6 and IL-8 in a dose- and time-dependent fashion. Alternaria alternata extracts induced cell shrinking and cell desquamation and strongly enhanced the production of IL-6 and IL-8 at higher concentrations. Aspergillus fumigatus extracts caused cell shrinking, cell desquamation, and production of IL-6 and IL-8, even at low concentrations. The Aspergillus fumigatus-derived extract grown on collagen medium induced a strong dose-dependent decline in cytokine production at higher concentrations. Cladosporium herbarum extracts did not induce morphologic changes or cell desquamation but enhanced IL-6 and IL-8 productions at higher concentrations. The dependence of these effects on intact protease activity was shown by their abrogation by protease inhibitors. CONCLUSION: Proteases present in fungal extracts interact with epithelial cells, leading to morphologic changes, cell desquamation, and induction of proinflammatory cytokines. It is proposed that these fungal proteases may activate epithelial cells through a protease-activated receptor type 2-driven mechanism.

Sensitivity to the house dust mite and airway hyperresponsiveness in a young adult population.

BACKGROUND: The pathogenic mechanisms of airway hyperresponsiveness (AHR) in asthma are unknown and only a few studies have examined the importance of sensitivity to antigens in AHR in young adults. OBJECTIVE: We investigated the correlation between AHR and sensitivity to specific antigens, atopy, history of childhood asthma and spirometry in a young adult population. METHODS: Based on the results of interviews with 447 students at our university, 308 non-smoker students were classified into six groups. Group 1 comprised subjects with intermittent mild bronchial asthma; group 2, subjects with history of childhood asthma; group 3, subjects with atopic disease, and a RAST score for Dermatophagoides farinae (Def) of > or = 2; group 4, normal subjects with a RAST score for Def of > or = 2; group 5, subjects with cedar pollinosis; and group 6, normal subjects. We measured AHR to methacholine (MCh), spirometry, immunoglobulin E-radioimmunosorbent test (IgE-RIST), IgE-radioallergosorbent test to six common antigens, eosinophil cationic protein (ECP), and eosinophil count in peripheral blood in each subject. RESULTS: Airway hyperresponsiveness to MCh did not correlate with IgE-RIST, eosinophil count, or ECP. The highest AHR to MCh was present in groups 1 and 2 and lowest in groups 5 and 6. Multiple regression analysis showed that sensitivity to Def was the only factor that significantly influenced AHR to MCh. Airway hyperresponsiveness to MCh of groups with a RAST score for Def of 0/1 was lower than groups with a RAST score of 2 to 6. Airway hyperresponsiveness to MCh did not correlate with the degree of positivity to Def antigen among positive sensitized groups (RAST score 2 to 6). CONCLUSIONS: Sensitivity to mite antigen may be important in the pathogenesis of AHR and Def is a major contributing antigen in young adults in Japan. Once asthma occurs, AHR remains positive for a long time even after the disappearance of asthma-related symptoms.

Hypersensitivity pneumonitis in workers exposed to esparto grass (Stipa tenacissima) fibers.

Esparto grass (Stipa tenacissima), which is commonly found in the Mediterranean countries, has a wide variety of uses. Five stucco makers who had cough, dyspnea, malaise, and fever after exposure to esparto fiber used in their jobs showed a significant decrease in symptoms when they were away from work. Precipitating antibodies against an esparto extract were found in the sera of all patients. Specific IgG antibodies against the esparto extract were also demonstrated in all patient sera, as were IgG antibodies to Aspergillus fumigatus and thermophilic microorganisms (Micropolyspora faeni and Thermoactinomyces vulgaris) by means of an ELISA method. Esparto activity was inhibited in different ranges by the above antigens by inhibition ELISA. Only A. fumigatus could be identified after microbiologic evaluation of the esparto fiber samples. After inhalation challenge tests were performed with esparto extracts, all patients showed significant decreases in forced vital capacity, transfer lung CO, and PaO2 blood gas from baseline values. Fever, chills, malaise, dry cough, tachycardia, tachypnea, and rales on chest auscultation were also observed in all patients. Findings from bronchoalveolar lavage were suggestive of allergic alveolitis. Transbronchial biopsy specimens showed interstitial alveolitis with lymphocyte-macrophage infiltrate and granuloma. Unexposed control subjects did not exhibit reactivity to any of the tests listed above. The dust derived from esparto fibers can cause hypersensitivity pneumonitis in exposed subjects. Organisms such as A. fumigatus and thermophilic actinomyces could be the causative antigens. "Stipatosis" might be an appropriate name for this disorder.

Purification and characterization of a 93 kDa Aspergillus fumigatus antigen with diagnostic potential.

A glycoprotein with an apparent molecular weight of 93 kDa was purified from a water-soluble extract of Aspergillus fumigatus NCPF 2109 by single step affinity chromatography using the mannose-specific snowdrop (Galanthus nivalis) lectin coupled to agarose. The carbohydrate moiety contained only mannose and galactose. Partial sequencing of cyanogen bromide fragments of the antigen yielded two sequences, KQNKP and GEIPMKF?PQL, with no homology to any reported proteins. In a preliminary evaluation of its diagnostic potential the 93 kDa antigen was recognized by the sera of four patients with allergic bronchopulmonary aspergillosis, in addition to a monoclonal antibody raised against a partially purified fraction of the A. fumigatus water-soluble extract.