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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 FEV 1 /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., (© 2020 John Wiley & Sons Ltd.)

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, β-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 β-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/cm 2 . 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., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

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., (© 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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 β-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) (β-1,3-glucan). The ELISAs were able to detect antigens in 43% (Af), 37% (Pc), 94% (Ch), or 100% (β-1,3-glucan) of the 124 airborne dust samples. Inhalable dust, β-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.

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.

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., (© 2011 Blackwell Publishing Ltd.)

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., (Copyright © 2010 S. Karger AG, Basel.)

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.

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.

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., (Copyright © 2010 S. Karger AG, Basel.)

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.

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.

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.

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.

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.

The identification of collected airborne unicellular fungal conidia and hyphae using nonviable techniques is subjective and an imprecise process. Similarly, to determine whether an individual is allergic to a particular genus requires a separate immunodiagnostic analysis. This study demonstrates the development of a novel double immunostaining halogen assay, which enables (1) the simultaneous identification of collected airborne fungal conidia and hyphae of Aspergillus fumigatus and Penicillium chrysogenum using monoclonal antibodies and (2) the demonstration of patient-specific allergy to the same particles using human serum IgE. The results demonstrate that when conidia were ungerminated the binding of antibodies was homogeneous and localized in close proximity around the entire conidia for both species. However, when conidia were germinated, the proportion expressing antigen increased (P < 0.0001) for both species and the sites of binding of the two antibodies changed with double immunostaining restricted to the hyphal tips for A. fumigatus, in addition to the sites of germination for P. chrysogenum. The described immunoassay has the potential to identify fungal particles in personal environmental air samples, provided species-specific monoclonal antibodies are available, while simultaneously demonstrating allergic sensitization to the same particles by co-staining the samples with the patient's own serum. Such an immunoassay can use those fungi that the patient is actually exposed to and potentially avoids many problems associated with extract variability based on the performance of current diagnostic techniques for fungal allergy.

Indoor mold has been associated with the development of allergic asthma. Penicillium chrysogenum, a common indoor mold, is known to have several allergens and can induce allergic responses in a mouse model of allergic penicilliosis. Our hypothesis is that soluble components of P. chrysogenum (PCE) can dose-dependently induce responses typical of allergic asthma in BALB/c mice. Mice were exposed to 10, 20, 50, or 70 microg of PCE by involuntary aspiration four times over a 4-week period. Serum and bronchoalveolar lavage fluid (BALF) were collected before (day 0), and at days 1 and 3 following the final exposure. PCE-exposed mice demonstrated dose-dependent increases in: BALF total cell numbers including eosinophil, serum and BALF total IgE levels, BALF IL-5 levels, and increased severity of histopathologic lesions. A single exposure to the highest dose of PCE resulted in edema and cellular damage but not immune responses. Four exposures to Metarhizium anisopliae crude antigen (10 microg, positive control) resulted in equivalent or greater allergic asthma-like responses than those demonstrated by multiple exposures to 50 or 70 microg of PCE. Multiple exposures to 70 microg of PCE showed increased allergen-triggered immediate respiratory responses as well as non-specific airway hyperresponsiveness to methacholine as assessed by barometric whole-body plethysmography. Taken together, repeated pulmonary challenge with P. chrysogenum extract induced dose-dependent allergic asthma-like responses in mice.

Background: Through proteomic and genomic approaches we have previously identified and characterized an alkaline serine protease that is a major allergen (88% frequency of IgE binding) of Penicillium chrysogenum (Pen ch 13)., Objective: The aim of the present study is to identify the linear IgE-binding epitopes of Pen ch 13., Methods: IgE-binding regions were identified by dot-blot immunoassay using 11 phage-displayed peptide fragments spanning the whole molecule of Pen ch 13. The minimal epitope requirements for IgE binding were further defined with overlapping peptides synthesized on derivatized cellulose membranes using SPOTs technology. The critical residues on the immunodominant epitopes were mapped through site-directed mutagenesis. The locations of the IgE epitopes identified were correlated with a three-dimensional structure of Pen ch 13., Results: IgE antibodies in 35 serum samples reacted with at least one of the 11 peptide fragments of Pen ch 13. Peptide f-2n (residues 31-61) showed a high-intensity and the highest frequency (77%) of IgE binding. The frequencies of IgE binding to peptide f-4 (residues 93-133), f-1 (residues 1-37) and f-7 (residues 168-206) were 51%, 34% and 31%, respectively. SPOTs assay narrowed down the region of IgE binding of f-2n to residues 48-55 (GHADFGGR). Three, two and one epitope(s) that are four to nine amino acids in length, within f-4, f-1 and f-7, respectively, were found. Site-directed mutagenesis of Pen ch 13 revealed that substitution of His49 and/or Phe52 on Pen ch 13 with methionine resulted in proteins with drastic loss of IgE binding in seven sera tested. Proteins with amino acid replacements at residues 15-18 (RISS), or at residues 112 (I) and 116 (D) have lower IgE-binding reactivity in one of the two patient's sera tested. Substituting residues 117 (W), 119 (V) and 120 (K) also block most of the IgE binding in one of the two patient's sera tested. In addition, replacing residues 203 (V) and 204 (D) along with a deletion at residue 206 (Y) diminished the IgE binding in two serum samples tested. A model was constructed based on the structure of P. cyclopium subtilisin protease that has >90% (256 out of 283 amino acids) sequence identity with Pen ch 13. The major epitope (GHADFGGR) on Pen ch 13 formed a loop-like structure and was located at the surface of the allergen., Conclusions: Several linear IgE-reactive epitopes and their critical core amino acid residues were identified for the Pen ch 13 allergen. The major linear IgE-binding epitope, 48GHADFGGR55, formed a loop-like structure at the surface of the allergen. Substitution of His49 and/or Phe52 with methionine significantly reduced IgE-binding to Pen ch 13. Mapping of these results on a 3D model of the allergen provides valuable information about the molecular basis of allergenicity for Pen ch 13 and for designing specific immunotherapeutics.

Background: Exposure to moulds in indoor air is thought to induce asthma in susceptible persons. Moulds may contain several potent allergens. However, more importantly, moulds may increase the allergic response to other allergens (adjuvant effect). Previously, we have found that a beta-1,3-glucan from the cell wall of the fungus Sclerotinia sclerotiorum increases the allergic response to the model allergen ovalbumin (OVA) in a mouse model., Objective: In the present study, we wanted to confirm the adjuvant effect of another beta-1,3-glucan, MacroGard (MG) from baker's yeast in this model. More importantly, we wished to explore the putative effects of extracts from the moulds Cladosporium herbarum (CH) and Penicillium chrysogenum (PC) using the very same model as used to explore effects of beta-glucans., Methods: Groups of eight Balb/c mice were injected with OVA alone, OVA+extract or OVA+MG, into one footpad. On day 21, all mice were reinjected with OVA, before exsanguination on day 26. The levels of OVA-specific IgE, IgG1 and IgG2a in serum were measured by ELISA., Results: Compared with OVA alone, OVA+MG, OVA+CH extract and OVA+PC extract increased OVA-specific IgE and IgG1 levels significantly. For all groups, the levels of IgG2a anti-OVA remained similar to those of the OVA-alone group., Conclusions: Our results show that extracts from CH and PC, and the beta-1,3/1,6-glucan from baker's yeast have adjuvant effects on the allergic response in mice.

Background: Recent evidence has shown that viable conidia from the fungus Penicillium chrysogenum induce allergic effects in mice. The present study was conducted to determine the specific allergic dose response of C57BL/6 mice to the protease extract, Pen ch, isolated from viable P. chrysogenum conidia., Methods: Mice were treated with primary intraperitoneal (IP) injections of 10 or 100 microg of Pen ch adsorbed to alum, followed by weekly IP injections of 0.1, 1.0, or 10.0 microg Pen ch with alum for 4 weeks, and with 10.0 microg of Pen ch by intranasal (IN) inoculations the final 2 weeks before killing., Results: Intraperitoneal injections of 10 and 100 microg of Pen ch for 5 weeks followed by 2 weeks of IN instillation of 10 microg induced significant increases of total serum immunoglobulin (Ig)E and IgG(1). Bronchoalveolar lavage cell counts revealed increased numbers of eosinophils and neutrophils. Histopathological examination of lungs detected perivascular inflammation by eosinophils and neutrophils and increased mucous production., Conclusions: The data presented in this study indicate that sensitization to protease allergens released by viable P. chrysogenum conidia in vivo induce a strong allergic inflammatory response in a murine model, which could have implications for people exposed to high levels of conidia of this organism.

In this study, the authors determined the association between serum mold-specific immunoglobulin G (IgG) levels in primary school children (N = 181) with asthma, wheezing, or cough symptoms and exposure to indoor mold in 2 schools, with and without mold damage. Microbial exposure was determined with environmental sampling. Serum IgG antibody concentrations against 24 microbial strains were determined with enzyme-linked immunosorbent assay. Children's microbe-specific IgG levels were often higher in the reference school. There was an association between elevated serum IgG antibody levels to Penicillium notatum and moisture damage in the school. In addition, moisture damage both in school and at home was associated with Penicillium notatum and Eurotium amstelodami IgG levels. These observations comported with microbial findings in the index school. No other positive associations between IgG antibodies and microbial findings for the school buildings were observed. IgG levels in sera of school children did not provide a specific means for assessing the microbial exposure associated with moisture and microbial problems in the school buildings. Thus, IgG analysis cannot be readily suggested as a routine method for the evaluation of these exposures.

Background: Previous evidence by our laboratory has shown that mice inoculated with viable Penicillium Chrysogenum conidia or spores at levels comparable to those found in contaminated buildings induced spore antigen-specific allergic responses. We proposed that mice exposed to low levels of viable P. Chrysogenum conidia would not develop allergic symptoms. We also hypothesized that the symptoms induced by high numbers of conidia were the result of sensitization to allergens released by the conidia., Methods: C57BL/6 and BALB/c mice were exposed to 1 x 10(2) viable P. Chrysogenum conidia by intranasal instillation weekly for a period of 11 weeks. C57BL/6 mice were also sensitized to a viable P. Chrysogenum conidia protease extract by intraperitoneal injections for a period of 6 weeks followed by intranasal challenge with protease extract, viable, or nonviable P. Chrysogenum conidia for 2 weeks., Results: C57BL/6 mice inoculated with low numbers of conidia developed no significant lung inflammation or increased serum immunoglobulins. Mice sensitized to the protease extract and challenged with both protease extract and viable conidia produced significant increases in serum IgE and IgG1. Mice sensitized to and challenged with the protease extract developed significant eosinophilia and mucus hyperproduction as determined by bronchoalveolar lavage and histopathological examination of lung tissue., Conclusions: Mice did not develop allergic symptoms in response to challenge with low levels of P. Chrysogenum conidia. Protease allergens from viable conidia induced specific allergic responses in mice, indicating the importance of P. Chrysogenum conidia in allergic sensitization to the organism., (Copyright 2003 S. Karger AG, Basel)

Background: 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., Objective: 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., Methods: 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., Results: 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., Conclusions: 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.

The mould genus, Penicillium, is a significant source of environmental aero-allergens. A major allergen from Penicillium notatum, Pen n 18, was identified by two-dimensional immunoblotting using monoclonal antibody G11A10, raised against the vacuolar serine protease of Penicillium citrinum, followed by matrix-assisted laser-desorption ionization-time-of-flight MS analysis of the peptide digest. Pen n 18 was then cloned and the amino acid sequence deduced from the cDNA sequence. The cDNA encoded a 494 amino acid protein, considerably larger than mature Pen n 18, the differences being due to the N- and C-terminal prosequences. The deduced amino acid sequence showed extensive similarity with those of vacuolar serine proteases from various fungi. The Pen n 18 coding sequence was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni(2+)-chelate affinity chromatography. On immunoblots, the purified recombinant protein specifically bound IgE from mould-allergic patients, and cross-inhibition assays demonstrated the presence of common IgE-binding epitopes on Pen n 18 and a major allergen of P. citrinum, Pen c 18. When mapping of the allergenic epitopes was performed, at least nine different linear IgE-binding epitopes, located throughout the Pen n 18 protein, were identified. Of these, peptide C12, located in the N-terminal region of the molecule, was recognized by serum from 75% of the patients tested and therefore appears to be an immunodominant IgE-binding epitope.

Background: Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis., Methods: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species--P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13)., Results: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116-125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12-73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner., Conclusions: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.

Objective: To study the relationship between basophil histamine release (HRT) to indoor moulds, indicating specific IgE, and building-related symptoms (BRS), asthma, and hay fever in individuals working in damp and mouldy buildings., Methods: A cross-sectional study was performed among 86 school staff members, who on average had worked 143 months (range: 3-396) in moist buildings with mould growth in the constructions. A questionnaire concerning mucous membrane symptoms, facial skin symptoms, central nervous system symptoms, hay fever, and asthma was fulfilled by the participants, and blood samples were taken. Eight mould species growing on building constructions were identified and cultivated to obtain allergenic materials for testing. The presence in serum of IgE specific to moulds was verified by histamine release test (HRT) based on passive sensitization of basophil leukocytes. The validity of the method was confirmed by parallel testing of patients allergic to grass- and birch pollen and by the shift from positive to negative response after removal of serum IgE and by using sham sensitization., Results: The prevalence of most BRS was between 32% and 62%. Positive HRT, showing serum IgE specific to one or more of the moulds, was observed in 37% of the individuals. The highest frequency of positive HRT was found to Penicillium chrysogenum and then to Aspergillus species, Cladosporium sphaerospermum and Stachybotrys chartarum. A significant association was found between most BRS and positive HRT, whereas no association was observed between positive HRT to moulds and self reported hay fever or asthma., Conclusion: Positive HRT to indoor moulds, showing the presence in serum of IgE specific to the fungi, was found to be related to BRS in individuals working in damp and mouldy buildings. Whether the association is of causal character is a question for further studies. The test may be useful in the evaluation and study of possible mould induced BRS.

Background: A study was undertaken to determine the consequences of long term intranasal instillation of Penicillium chrysogenum propagules in a mouse model., Methods: C57 Black/6 mice were inoculated intranasally each week for six weeks with 10(4) viable and non-viable P chrysogenum conidia. Cytokine levels and cellular responses in these animals were then measured., Results: Compared with controls, mice inoculated intranasally each week for six weeks with 10(4) P chrysogenum conidia (average viability 25%) produced significantly more total serum IgE (mean difference 1823.11, lower and upper 95% confidence intervals (CI) 539.09 to 3107.13), peripheral eosinophils (mean difference 5.11, 95% CI 2.24 to 7.99), and airway eosinophilia (rank difference 11.33, 95% CI 9.0 to 20.0). With the exception of airway neutrophilia (mean difference 20.89, 95% CI 3.72 to 38.06), mice inoculated intranasally with 10(4) non-viable conidia did not show significant changes in total serum IgE, peripheral or airway eosinophils. However, when compared with controls, this group (10(4) non-viable) had a significant increase in total serum IgG(2a) (mean difference 1990.56, 95% CI 790.48 to 3190.63) and bronchoalveolar lavage (BAL) fluid levels of interferon (IFN)-gamma (mean difference 274.72, 95% CI 245.26 to 304.19). In addition, lung lavages from mice inoculated intranasally with 10(4) viable P chrysogenum conidia had significantly increased levels of interleukin (IL)-4 (mean difference 285.28, 95% CI 108.73 to 461.82) and IL-5 (mean difference 16.61, 95% CI 11.23 to 21.99). The IgG(2a)/IgE ratio and the IFN-gamma/IL-4 ratio was lower in the group of mice inoculated intranasally with 10(4) viable conidia than in the 10(4) non-viable conidia group and the controls. When proteins were extracted from P chrysogenum conidia, attached to microtitre plates and incubated with serum from the 10(4) viable group, significant increases in conidia-specific IgE and IgG(1) were observed compared with controls, while serum from the 10(4) non-viable group was similar to controls., Conclusions: These data suggest that long term inhalation of viable P chrysogenum propagules induces type 2 T helper cell mediated (Th2) inflammatory responses such as increases in total and conidia-specific serum IgE and IgG(1), together with BAL fluid levels of IL-4 and IL-5 and peripheral and airway eosinophilia, which are mediators of allergic reactions.

Background: Penicillium species are important causative agents of extrinsic bronchial asthma. However, little is known about the allergens of these ubiquitous fungal species. Objective The object was to analyse the composition, the allergenic cross-reactivity and the N-terminal sequences of allergens from two prevalent airborne Penicillium species, P. oxalicum and P. notatum., Methods: The allergenic composition and the immunoglobulin (Ig)E cross-reactivity were analysed by immunoblot and immunoblot inhibition, respectively, using sera from asthmatic patients. The N-terminal amino acid sequences of major allergens were determined by Edman degradation. Allergens identified were also characterized by immunoblotting using monoclonal antibody (MoAb) PCM39 against the alkaline serine proteinase major allergen of P. citrinum., Results: Among the 70 asthmatic sera tested, 18 (26%) and 17 (24%) had IgE immunoblot reactivity towards components of P. oxalicum and P. notatum, respectively. Major allergens (> 80% frequency of IgE-binding) from both species are the 34 and 30 kDa proteins of P. oxalicum and the 34 and 32 kDa proteins of P. notatum. IgE cross-reactivity among these major allergens and the 33 kDa major allergen of P. citrinum can be detected by immunoblot inhibition studies. The N-terminal amino acid sequences of the 34 kDa allergen of P. oxalicum and of the 32 and the 28 kDa allergens of P. notatum share homology with sequences of the vacuolar serine proteinase from Aspergillus fumigatus. The N-terminal amino acid sequence of the 34 kDa allergen of P. notatum shows sequence homology with that of alkaline serine proteinase from P. citrinum. Results obtained from immunoblotting showed that MoAb PCM39 reacted with the 34, 30 and 16 kDa IgE-binding components of P. oxalicum, and with the 34, 32 and 28 kDa IgE-binding components of P. notatum., Conclusions: Results obtained suggest that the 34 kDa major allergen of P. oxalicum may be a vacuolar serine proteinase. The 34 and the 32 kDa major allergens of P. notatum may be the alkaline and the vacuolar serine proteinases of P. notatum, respectively. The 30 and 16 kDa IgE-binding components of P. oxalicum and the 28 kDa IgE-binding component of P. notatum may be breakdown products of the 34 and the 32 kDa major vacuolar serine proteinase allergens of P. oxalicum and P. notatum, respectively.

Penicillium chrysogenum crude extract was prepared in our laboratory. The protein concentration of this allergen assessed by coomassie blue G250 stain was 44-48 mg/ml. Three protein bands P1, P2a and P2b, were obtained by PAGE. Molecular weights of the bands were 94,000, 43,000 and 25,000 respectively as compared with standard molecular weight markers. Gel filtration through sephadex G200 revealed two peaks. These fractions were monitored for absorbance at 280 nm and combined into 3 pools according to the results of PAGE. Tube 1(T1), T2 and T3 were obtained. The average size of wheals developing after intradermal skin testing showed that T2 induced the strongest reaction. Since the main component of T2 was P2a, it is supposed that P2a of P. chrysogenum extract contains the protein with the highest allergenicity.

The effect of three different size molecular weight species of mycoviral dsRNA on the immune response to sRBC was tested in C57Bl/6 mice. The various dsRNA species were extracted from electrophoresis polyacrylamide-agarose slab gels. Their molecular weights ranged from 1.0 x 10(6) daltons to 3.5 x 10(6) daltons. All three sizes of mycoviral dsRNA significantly (p less than 0.0001) suppressed the hemolytic antibody titer of mice 8 days after immunizations with 15 micrograms dsRNA/mouse and 10(8) sRBC when compared to control mice which received only sRBC. No immune suppression was observed in any of the mice challenged with a second sRBC immunization 60 days after the first inoculations. Hemagglutination titers at this time were typical of a secondary antibody response to sRBC. In conclusion these three molecular weight mycoviral dsRNA species appeared to be potent immunosuppressors when approximately 15 micrograms/mouse were used.

A married couple presented with an illness typical of allergic alveolitis. A careful search of their home revealed a leak in the central heating system with a heavy fungal growth on wet flooring and linoleum. Two species of Penicillium, P chrysogenum and P cyclopium, were isolated from floorboards, linoleum, and settle plates. Antibodies against both these fungi were demonstrated in the serum of both patients by an enzyme linked immunosorbent assay (ELISA). Allergic alveolitis caused by P chrysogenum and P cyclopium has not been described previously.