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.

Cheilitis associated with sensitization to penicillium notatum in a clarinetist

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Characterization and pro-inflammatory responses of spore and hyphae samples from various mold species.

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, ß-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo- or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of ß-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential.

[Hypersensitivity pneumonitis related to Penicillium chrysogenum and mesophilic Streptomyces: the usefulness of the Medical Indoor Environment Councelor (MIEC)]. / Pneumopathie d'hypersensibilité à Penicillium chrysogenum et Streptomyces mésophile: intérêt de l'enquête environnementale à domicile.

Hypersensitivity pneumonitis (HP) occurred after organic antigens inhalation at home is rare and the diagnosis is very often difficult. We report the case of a 55-year male patient with allergic asthma since childhood, well controlled with inhaled corticosteroids, twice hospitalized for respiratory distresses. The patient presented fever (39°C), dry cough, rapidly progressive dyspnea, chest pain and crackles. Blood gas analysis found a hypoxemia of 52 mmHg, and CT-scan showed ground glass images in the upper lobes. Respiratory function tests showed severe obstructive syndrome and a decrease of diffusion test. HP was suspected because the symptoms were triggered by domestic environmental. The Medical Indoor Environment Councelor (MIEC) visited the patient's house and camper and performed air and dust samples. Moldy walnuts were found in the camper. The identification of microorganisms present in the air and on the surfaces in the camper was used for serum precipitins research by double diffusion (DD) and electrosyneresis (E) methods. From the 14 antigens tested, serological tests were considered significant for mesophilic Streptomyces (five arcs DD, six arcs E) and Penicillium chrysogenum (one arc DD, four arcs E). After removal from the camper of the objects suspected to be contaminated, the patient's symptoms regressed. This is a typical case of domestic HP to mesophilic Streptomyces and P. chrysogenum. The MIEC's intervention was useful in both diagnosis and treatment.

Isolation of Aspergillus fumigatus from sputum is associated with elevated airborne levels in homes of patients with asthma.

Indoor bioaerosols, such as mold spores, have been associated with respiratory symptoms in patients with asthma; however, dose-response relationships and guidelines on acceptable levels are lacking. Furthermore, a causal link between mold exposure and respiratory infections or asthma remains to be established. The aim of this study was to determine indoor concentrations of Aspergillus fumigatus and a subset of clinically relevant fungi in homes of people with asthma, in relation to markers of airways colonization and sensitization. Air and dust samples were collected from the living room of 58 properties. Fungal concentrations were quantified using mold-specific quantitative PCR and compared with traditional microscopic analysis of air samples. Isolation of A. fumigatus from sputum was associated with higher airborne concentrations of the fungus in patient homes (P = 0.04), and a similar trend was shown with Aspergillus/Penicillium-type concentrations analyzed by microscopy (P = 0.058). No association was found between airborne levels of A. fumigatus and sensitization to this fungus, or dustborne levels of A. fumigatus and either isolation from sputum or sensitization. The results of this study suggest that the home environment should be considered as a potential source of fungal exposure, and elevated home levels may predispose people with asthma to airways colonization.

Development and application of mold antigen-specific enzyme-linked immunosorbent assays (ELISA) to quantify airborne antigen exposure.

The aim of our study was to develop specific enzyme-linked immunosorbent assays (ELISAs) and apply these to assess mold antigen exposure in composting plants. Sandwich ELISAs based on polyclonal antibodies to Aspergillus fumigatus (Af), Penicillium chrysogenum (Pc), and Cladosporium herbarum (Ch) antigens were developed and validated. Reactivity to 18 different mold species was tested. To optimize extraction procedure, inhalable dust samples taken by a parallel sampler were extracted with or without homogenization. In 31 composting plants stationary pumps were installed at 4 sites to collect 124 inhalable dust samples. The newly developed ELISAs were used in addition to an anti ß-1,3-glucan ELISA to quantify mold antigens. The Cladosporium ELISA showed less than 0.04% reactivity to extracts from other fungal genera, while the Af ELISA demonstrated a reactivity of up to 3.6% and the Pc ELISA reacted up to 11% to other mold species. Extraction of parallel sampled filters gave higher antigen amounts with homogenization. The increase was highest for Pc-antigens, followed by Af-antigens, and lowest for Ch-antigens. Mean lower detection limits of homogenized inhalable dust samples were 5 ng/m(3) (Af), 0.6 ng/m(3) (Pc), 0.2 ng/m(3) (Ch), and 0.6 ng/m(3) (ß-1,3-glucan). The ELISAs were able to detect antigens in 43% (Af), 37% (Pc), 94% (Ch), or 100% (ß-1,3-glucan) of the 124 airborne dust samples. Inhalable dust, ß-1,3-glucan, and Af-, Pc-, and Ch-antigen concentrations were significantly correlated. The newly developed mold antigen ELISAs are thus able to measure airborne exposure levels in composting plants and differentiate between distinct fungi genera.

The development and validation of an assay for the quantification of the P. chrysogenum allergen Pch52.

Penicillium chrysogenum clade 4, is a common mold on damp building materials. A capture ELISA assay for the major allergen from P. chrysogenum Pch52 has been developed and tested in house dust samples and potential cross-reactivity examined. Minimal cross-reactivity with other relevant indoor fungi was observed for the assay following thorough purification of the monoclonal and polyclonal antibodies. The limit of quantification for ELISA analysis of Pch52 in sieved house dust is comparable to other assays of other fungi. The LOQ for Pch52 was 0.31 ng/mL in solution or 110 ng/g dust. The LOQ for Asp f1 and Alt a1 were 2.2 ng/g and 17 ng/g, respectively. These results indicate this assay is suitable for the quantification of Pch52 in sieved house dust.

Transaldolases are novel and immunoglobulin E cross-reacting fungal allergens.

BACKGROUND: Mould-induced atopic respiratory diseases are a worldwide problem. Characterization of fungal allergens is of major clinical importance. OBJECTIVE: We identified a novel transaldolase family allergen of Cladosporium and Penicillium species. METHODS: Fungal allergens were identified by immunoblotting, peptide mass mapping and partial sequencing, cDNA cloning and IgE epitope mapping. RESULTS: A 36.5 kDa IgE-binding component in a partially purified C. cladosporioides preparation was identified. Mass spectrometric analyses suggest that this novel IgE-reacting allergen is a transaldolase. A corresponding full-length 1246 bp cDNA encoding a polypeptide of 325 residues was isolated. The newly identified transaldolase allergen has been designated as Cla c 14.0101. The cDNA encoding the Pencillium chrysogenum transaldolase was isolated by RT-PCR according to the cDNA sequence encoding a P. chrysogenum Wisconsin 54-1255 hypothetical protein. The purified rCla c 14.0101 protein reacted with IgE antibodies in 10 (38%) of 26 Cladosporium cladosporioides-sensitized asthmatic patients. Nine of the 10 rCla c 14.0101-positive sera have IgE binding against the recombinant Penicillium transaldolase (rPen ch 35.0101). Among the eight fungal transaldolase-positive sera tested, three showed IgE binding against the recombinant human transaldolase. To determine cross-reactivity between the Cladosporium and Penicillium fungi, IgE cross-reactivity was detected between these two fungal transaldolase allergens by inhibition assays. Both the N- and the C-terminal fragments of Cla c 14.0101 were recognized by IgE antibodies. The C-terminal IgE-reacting determinant was narrowed down to a region encompassing Thr257 to Ser278 of Cla c 14.0101. It was mapped onto a loop-like structure of a 3D model constructed for Cla c 14.0101. CONCLUSION AND CLINICAL RELEVANCE: We identified transaldolase as a novel and IgE cross-reactive allergen family of C. cladosporioides and P. chrysogenum. In addition, an IgE-reacting fragment (Thr257 to Ser278) was pinpointed to a loop-like structure on Cla c 14.0101. Results obtained provide important information in clinical mould allergy.

Analysis of the serum levels of fungi-specific immunoglobulin E in patients with allergic diseases.

BACKGROUND: Atopic patients are exceptionally sensitive to airborne allergens. Sensitization to fungal allergens may be associated with respiratory allergic disease (RAD) and atopic dermatitis (AD). This study investigates the relationship between sensitization to different fungal allergens and the clinical manifestations of atopic disease. METHODS: We retrospectively reviewed the medical records of atopic patients from the National Taiwan University Hospital from 2004 to 2009. A total of 133 atopic patients were found who were sensitive to at least 1 of the 3 most common fungi (Candida albicans, Aspergillus fumigatus, Penicillium chrysogenum) in Taiwan and were enrolled in the study. These patients were further divided into subgroups of isolated AD, isolated RAD and AD + RAD. The association between sensitization to fungi and allergic disease was analyzed by logistic regression analysis. RESULTS: The sensitization rate to Candida-specific IgE was 81.2%, followed closely by Aspergillus at 69.2% and Penicillium at 63.2%. Isolated AD was the most common diagnosis. The levels of specific IgE antibodies against Aspergillus, Candida and Penicillium were highest in patients with isolated AD. Logistic regression revealed that isolated AD was highly associated with sensitization to Candida [odds ratio (OR) 10.45, 95% confidence interval (CI) 2.37-45.9]. In contrast, sensitization to Penicillium (OR 0.34, 95% CI 0.13-0.87)and Candida (OR 0.30, 95% CI 0.10-0.92) showed a negative association with isolated RAD. CONCLUSIONS: Fungal sensitization is more closely associated with AD than RAD. Our results suggest that specific sensitization to fungal allergens plays a critical role in the pathogenesis of atopic disease.

A comparison of the allergic responses induced by Penicillium chrysogenum and house dust mite extracts in a mouse model.

UNLABELLED: A report by the Institute of Medicine suggested that more research is needed to better understand mold effects on allergic disease, particularly asthma development. We compared the ability of the fungal Penicillium chrysogenum (PCE) and house dust mite (HDM) extracts to induce allergic responses in BALB/c mice. The extracts were administered by intratracheal aspiration (IA) at several doses (0, 2.5, 5, 10, 20, 40, and 80 µg) four times over a 4-week period. Three days after the last IA exposure, serum and bronchoalveolar lavage fluid (BALF) were collected. The relative allergenicity of the extracts was evaluated based on the lowest dose able to induce a significant response compared to control (0 µg) and the robustness of the response. PCE induced the most robust response at the lowest dose for most endpoints examined: BALF total, macrophage, neutrophil, and eosinophil cell counts, and antigen-specific IgE. Taken together, our data suggest that PCE may induce a more robust allergic and inflammatory response at lower doses than HDM. PRACTICAL IMPLICATIONS: Our data suggest that Penicillium chrysogenum is a robust allergen and may be a more potent allergen source than house dust mite (HDM) in this mouse model. Two critical factors in the development of human allergic disease, exposure levels and sensitization thresholds, are unknown for most allergens including molds/fungi. Human exposure levels are not within the scope of this article. However, the data presented suggest a threshold dose for the induction of allergic responsiveness to P. chrysogenum. Additionally, P. chrysogenum as well as other molds may play an important role in asthma development in our society.

Pen ch 13 major fungal allergen decreases CD44 expression in human bronchial epithelial cells.

BACKGROUND: Pen ch 13 is an alkaline serine protease major allergen from Penicilliumchrysogenum. CD44 adhesion molecules play important roles in resolving lung inflammation and repairing epithelial damages during bronchial asthma. The purpose of this study was to investigate the effects of Pen ch 13 on CD44 of human bronchial epithelial cells. METHODS: Cells of the SV40-transformed immortalized bronchial epithelial cell line 16HBE14o- and primary cultures of human bronchial epithelial cells were exposed to purified Pen ch 13. CD44 expression on Pen ch 13-treated cells was analyzed by immunoblot analysis and flow cytometry. The release of soluble CD44 (sCD44) into culture supernatants was determined using human sCD44std ELISA kits. RESULTS: Pen ch 13 (0.01-1.0 µg/ml) dose-dependently down-regulates CD44 expression in 16HBE14o- cells. In addition, the decrease in CD44 expression can be abolished by pre-treating Pen ch 13 with a serine protease inhibitor, phenylmethyl-sulfonyl fluoride. Results from flow-cytometric analysis showed that the population mean fluorescence intensity for CD44 was significantly lower (p < 0.05) in Pen ch 13 (1.0 µg/ml)-treated 16HBE14o- cells (18 ± 4) than that of non-treated control cells (41 ± 7). Furthermore, Pen ch 13 induced increased shedding of sCD44 into the culture media compared with the shedding of non-treated 16HBE14o- and primary bronchial epithelial cells. CONCLUSIONS: Pen ch 13 allergen down-regulated CD44 protein expression in airway epithelial cells. It may contribute to atopic asthma by influencing the resolution of lung inflammation and prolonging the repair response of damaged bronchial epithelial cells.

Characterization of a 52 kDa exoantigen of Penicillium chrysogenum and monoclonal antibodies suitable for its detection.

The indoor clade of Penicillium chrysogenum, the so-called Fleming clade, is the most common species of Penicillium on moldy building materials. In a previous study, we identified a 52 kDa human antigen characteristic of the indoor clade of P. chrysogenum not present in a taxonomically diverse selection of fungi. Further investigations revealed that it is a modestly glycosylated mature protein with a pI 5.3. The protein is apparently identical to a glucoamylase previously reported from an aluminum-tolerant P. chrysogenum mutant. Based on sequence similarity, molecular weight, and pI, it is distinct from a number of other glucoamylases from domesticated strains of Aspergillus oryzae and A. niger used to produce industrial enzymes. Surprisingly, it had not been reported as an allergen. The monoclonal antibodies developed have the potential for use in assays of P. chrysogenum antigens in spores and spore/mycelial fragments in dust.

Using human sera to identify a 52-kDa exoantigen of Penicillium chrysogenum and implications of polyphasic taxonomy of anamorphic ascomycetes in the study of antigenic proteins.

We are interested in isolating and identifying antigenic fungal proteins from species that grow on damp building materials. The indoor clade of Penicillium chrysogenum, the so-called Fleming clade, is the most common species of Penicillium on moldy building materials. We have identified a 52-kDa marker protein for the indoor clade of P. chrysogenum not present in a taxonomically diverse selection of fungi. It is found in high concentrations in protein extracted from the fungus grown on paper-faced gypsum wallboard. During this process, we illuminated the variability in response to patient sera and of strains of the fungus collected over a wide geographic area. From a collection of sera from all over the USA, 25 of the 48 patients reacted to the 52-kDa protein from this prescreened collection of sera. Most strain/antibody combinations had proportionate ELISA response associated with the presence of the target. However, approximately 25% of the strain/patient serum combinations included people who responded to many common allergens from the Penicillia. All the P. chrysogenum strains tested produced the target protein. However, there was considerable variability in patient IgG response to 32-, 30-, and 18-kDa antigens and in their production by the various clade 4 strains. The target protein was not found in spores or culture extracts of a wide selection of relevant fungi. It appears that the previous studies have been conducted on strains of the fungus from the three clades not those associated with the built environment.

Aspergillus fumigatus germ tube growth and not conidia ingestion induces expression of inflammatory mediator genes in the human lung epithelial cell line A549.

Inhalation of conidia is the main cause of invasive pulmonary aspergillosis (IPA) and the respiratory epithelium is the first line of defence. To explore the triggering factor for the inflammatory response to Aspergillus fumigatus, the species mainly responsible for IPA, this study analysed the differential expression of three inflammatory genes in A549 cells after challenge with live and killed conidia. The influence of steroids, one of the main risk factors for developing IPA, was also investigated. Quantification of mRNAs of the inflammatory mediator genes encoding interleukin (IL)-8, tumour necrosis factor (TNF)-alpha and granulocyte-monocyte colony-stimulating factor (GM-CSF) was carried out using real-time PCR. Ingestion rates were studied for the conidia of A. fumigatus and Penicillium chrysogenum using a fluorescence brightener. Similar results were obtained for both species, with ingestion rates ranging from 35 to 40 %. Exposure of A549 cells to live A. fumigatus conidia only induced a four- to fivefold increase in the mRNA levels of the three genes, starting 8 h after the initial contact. Both inactivation of live A. fumigatus conidia and treatment by dexamethasone (10(-7) M) prevented the overexpression of TNF-alpha, IL-8 and GM-CSF. Fungal growth, rather than conidia ingestion, appears to be the main stimulus for the production of inflammatory mediators by epithelial cells, and this production is inhibited by steroid therapy. These results underline the role that the epithelium plays in the innate response against IPA.

Lys89, Lys90, and Phe91 are critical core amino acid residues of the Pen ch 18 major fungal allergen recognized by human IgE antibodies.

A vacuolar serine protease (Pen ch 18) has been identified as a major allergen of Penicillium chrysogenum. The molecular features of antigenic determinant(s) on Pen ch 18 recognized by human IgE antibodies, however, have remained unclear. Here, we show that a dominant IgE epitope on the N-terminally processed Pen ch 18 allergen was narrowed down to residues 83-91. In addition, Lys89, Lys90, and possibly Phe91 were identified as the core residues. Substitution of Lys89, Lys90, or Phe91 with alanine can significantly reduce IgE-binding to Pen ch 18. Immunoblot inhibition confirmed that Lys89 and Phe91 played a significant role in IgE-binding against Pen ch 18. Molecular modeling suggests they are located on a loop-like structure at or near the surface of the major fungal allergen.

Pen ch 13 allergen induces secretion of mediators and degradation of occludin protein of human lung epithelial cells.

BACKGROUND: Alkaline serine proteases from six prevalent airborne Penicillium and Aspergillus species have been identified as a group of major allergens (group 13). After entering human airways, the allergens are in initial contacts with respiratory epithelial cells. The purpose of this study is to investigate interactions between the Pen ch 13 allergen from P. chrysogenum and human lung epithelial cells. METHODS: A549 cells, 16HBE14o- cells and primary cultures of human bronchial epithelial cells (HBEpC) were exposed to purified Pen ch 13 and mediators released into culture supernatants were assayed with enzyme-linked immunosorbent assay (ELISA) kits. Cleavage of occludin in 16HBE14o- cells was analysed by immunofluorescent staining of whole cells and immunoblot analysis of whole cell extracts. Fragments generated by incubating Pen ch 13 and a synthetic peptide carrying the occludin sequence were analysed by mass spectrometry. RESULTS: Pen ch 13 induced productions of prostaglandin-E2 (PGE2), interleukin (IL)-8 and transforming growth factor (TGF)-beta1 by A549 cells, 16HBE14o- cells and primary cultures of HBEpC. The protease activity of Pen ch 13 is needed for the induction of PGE2 IL-8, TGF-beta1 and cyclo-oxygenase (COX)-2 expression. A tight junction protein occludin of 16HBE14o- cells can be cleaved by Pen ch 13 at Gln202 and Gln211 which are within the second extracellular domain of the protein. CONCLUSION: Pen ch 13 may contribute to asthma by damaging the barrier formed by the airway epithelium and stimulating the release of mediators that orchestrate local immune responses and inflammatory process from HBEpC.

[Short communication: prevalence of fungal allergy in patients applied to hospital with symptoms of atopic disease in Kocaeli, Turkey]. / Kisa bildiri: kocaeli bolgesinde atopik hastalik semptomlari ile hastaneye basvuran hastalarda mantar alerji prevalansi.

The aim of this study was to investigate the prevalence of fungal sensitization and to detect the IgE and eosinophilic cationic protein (ECP) levels in the positive cases inhabiting in Kocaeli province (located in Northwest region of Turkey). The results of mixed fungus tests of 1.799 patients (870 male, 929 female) ages between 3-80 years old who were admitted to Kocaeli University Medical Faculty Hospital in the period of January 2002-January 2005, with the symptoms of atopic disease such as upper/lower respiratory tract symptoms, asthma and/or rhinitis and/or eczema, were evaluated retrospectively. Serum IgE and ECP levels of all cases were determined by a commercial enzyme immunoassay (UniCAP System, Pharmacia, Uppsala, Sweden) method. The allergy test performed by using mixed fungal panel (Penicillium notatum, Cladosporium herbarum, Aspergillus fumigatus, Candida albicans, Alternaria alternata, Helminthosporium halodes) was found positive in 68 cases (3.8%). Of those cases 7 (10.2%) were found to be sensitized to Alternaria alternata, 8 (11.7%) to Aspergillus fumigatus, 10 (14.7%) to Penicillium notatum, and 6 (8.8%) were sensitized to more than one fungus. The mean IgE (106.2+/-166.1) and ECP levels (31.7+/-18.3) of fungus sensitized cases were found significantly higher than the non-sensitized subjects (IgE; 77.8+/-95.0, ECP; 22.4+/-16.5) (p<0.001). The prevalence rates of fungal allergy in our region were determined as 1.9% (26/1.341), 4.5% (2/44) and 9.6% (40/414) in pediatric, adolescent and adult populations, respectively. In conclusion, it was determined that the rate of the sensitization against fungi was low in Kocaeli, Turkey and it exhibited a tendency towards increase with age.