Mold-sensitivity in children with moderate-severe asthma is associated with HLA-DR and HLA-DQ.

BACKGROUND: Several epidemiologic studies in the United States and Europe have linked Alternaria sensitivity to both persistence and severity of asthma. In this study, we examined T cell responses and HLA class II alleles in children with moderate-severe asthma. METHODS: Ninety-six children with moderate-severe asthma were compared to 90 children with mild asthma. HLA class II genotyping was performed to determine HLA allelic frequencies. Th1/Th2 Alternaria-specific T cell cytokine responses were determined by the use of Alternaria-stimulated cultures. HLA class II restriction was examined by inhibition of Alternaria-stimulated lymphoproliferative responses with blocking anti-HLA class II monoclonal antibodies. RESULTS: Children with moderate-severe asthma had significantly increased sensitivities to Aspergillus fumigatus; sensitivities to Alternaria were similar in both moderate-severe and mild asthmatics. The frequency of HLA-DRB1*13 alleles were increased in mold-sensitive moderate-severe asthmatic children. HLA-DRB1*03 tended to be increased in mold-sensitive moderate-severe asthmatics. The frequency of HLA-DQB1*03 alleles was significantly decreased in mold and Alternaria-sensitive moderate-severe asthma. HLA class II blocking monoclonal antibodies demonstrated HLA-DR restriction. Alternaria-stimulated IL-5 and IL-13 synthesis was significantly increased in moderate-severe asthmatics. IL-5 and IL-13 synthesis was significantly increased in Alternaria-stimulated lymphocyte cultures of HLA-DQB1*03- asthmatics compared to HLA-DQB1*03+ asthmatics. CONCLUSIONS: In children with Alternaria-sensitive moderate-severe asthma, there was increased Th2 sensitivity to Alternaria stimulation. This was associated with HLA-DR restriction and with increased frequency of HLA-DRB1*13 and HLA-DRB1*03. There was decreased frequency of HLA-DQB1*03 in Alternaria-sensitive moderate-severe asthma, suggesting HLA-DQB1*03 may be protective of the development of Alternaria-sensitive severe asthma.

Examination of Alternaria alternata mutagenicity and effects of nitrosylation using the Ames Salmonella test.

Molds of the genus Alternaria are common food pathogens responsible for the spoilage of fruits, vegetables, grains, and nuts. Although consumption of Alternaria alternata-contaminated foodstuffs has been implicated in an elevated incidence of esophageal carcinogenesis, the mutagenic potencies of several A. alternata toxins seem unable to account for the levels of activity found using crude mycelial extracts. In this study, the mutagenic effects of nitrosylation were examined with the major Alternaria metabolites Altenuene (ALT), Alternariol (AOH), Alternariol Monomethyl Ether (AME), Altertoxin I (ATX I), Tentoxin (TENT), Tenuazonic Acid (TA), and Radicinin (RAD) using the Ames Salmonella strains TA98 and TA100. In the absence of nitrosylation, ATX I was mutagenic when tested from 1 to 100 microg/plate in TA98 with rat liver S9 for activation, while AOH and ATX I were weakly mutagenic +/- S9 in TA100. Incubation with nitrite generally increased mutagenic potencies with ATX I strongly mutagenic +/- S9 in both TA98 and TA100, while ALT, AOH, AME, and RAD responses were enhanced in TA100 + S9. However, subsequent examination of three extracts made from A. alternata culture broth, acetone-washed mycelia, and the acetone washes showed a different mutagenic response with both broth and acetone washes directly mutagenic in TA98 and TA100 but with a reduced response + S9. The acetone-washed mycelial extract was found to have the lowest mutagenic activity of the three extracts tested. Nitrosylation had little effect on the mutagenicity of any of the extracts. Thus, while nitrosylation increases the mutagenicity of ATX I, and to a lesser extent that of several other Alternaria toxins, the results demonstrate that Alternaria produces a major mutagenic activity with a S. typhimurium response different from that found with the purified toxins. Efforts are currently underway to chemically identify this mutagenic species. Published 2001 Wiley-Liss, Inc.

Molecular cloning of IgE-binding fragments of Alternaria alternata allergens.

Exposure to the hyphomycete Alternaria alternata is recognized as an important risk factor for asthma. IgE immunoblotting has been used to catalogue the number and Mr of allergens in A. alternata extracts, with estimates ranging from 10 to 30, although few are present in nearly all extracts studied. Several A. alternata allergens have been cloned, including a subunit of the major allergen Alt a 1, ribosomal P2 phosphoprotein, aldehyde dehydrogenase and a yeast YCP4 homolog. We have cloned two sequences encoding IgE-binding fragments of allergens from an A. alternata lambda gt11 cDNA library using pooled atopic sera from A. alternata-sensitive individuals. One is homologous to a region near the C-terminus of hsp70 from Cladosporium herbarum; a near-complete isoallergen variant of A. alternata ribosomal P2 protein was also cloned. Their lacZ fusion proteins had reactivities of 5 and 14%, respectively, with individual atopic sera, indicating that the corresponding allergens are both minor. This study describes one new A. alternata allergen candidate and implicates ribosomal P2 protein as an allergen thtat is stable between independently isolated clones.

Isolation and expression of a cDNA clone encoding an Alternaria alternata Alt a 1 subunit.

Alternaria alternata is recognized as an important source of fungal aeroallergens. Alt a 1, the major allergen of this mold, is a dimer of disulfide-linked subunits that migrate in SDS-PAGE under reducing conditions at apparent M(r)s of 14,500 and 16,000. IgE antibodies to this protein are present in the sera of >90% of A. alternata-sensitive individuals. Previous studies from this laboratory showed that the N-termini twenty amino acids of the purified subunits are nearly identical. We now report the isolation of clones from an A. alternata (strain 34-016) cDNA library constructed in lambda(gt)11, using rabbit IgG antiserum against partially purified Alt a 1. One of nineteen clones selected from screens totalling 305,000 pfu (rb51) was sequenced, and determined to harbor an insert of 660 bp. An in-frame open reading frame within the cloned insert encodes a peptide of M(r) 16,960 that bears no significant homology to known allergens or proteins. The size of the rb51 transcript was determined to be approximately 0.7 kb by Northern analysis of A. alternata total RNA. The largely hydrophobic N-terminal region of the peptide contains an alpha-helical domain and other features characteristic of membrane targeting or secretory signals. The peptide sequence downstream of this region matches previously sequenced Alt a 1 N-terminal from two independent sources at 17 of 20, and 24 of 26 positions. Recombinant Alt a 1 expressed as a secreted protein in Pichia pastoris exists as a dimer in conditioned medium, as shown by immunoblotting under nonreducing conditions. Recombinant Alt a 1, like the natural allergen in A. alternata extracts, is also reactive with serum IgE from A. alternata-sensitive individuals.

An allergenic polypeptide representing a variable region of hsp 70 cloned from a cDNA library of Cladosporium herbarum.

BACKGROUND: Extracts of Cladosporium herbarum, a major source of fungal aeroallergens, exhibit a complex profile of IgE-binding proteins. Yields of conventionally purified allergens from this mold have been insufficient to permit further molecular analyses. OBJECTIVE: To enhance and simplify the purification of allergens from C. herbarum, we have sought to use recombinant DNA techniques to clone, identify and bacterially express immunoselected C. herbarum allergens. METHODS: We constructed a cDNA library in lambda ZAP II using mRNA isolated from C. herbarum. From this library, phage clones encoding a new allergen were immunoselected using pooled human atopic IgE. The cloned cDNA was excised from the phage vector as a recombinant pBluescript II SK-phagemid and sequenced. Expression of the recombinant allergen was carried out in E. coli XL1-blue transformants of the phagemid. Bacterial lysates from cells induced to express the cloned allergen were immunoblotted and probed with individual human atopic IgEs. RESULTS: The cDNA clone encodes a 278 amino acid polypeptide homologous to the C-terminal portion of 70 kDa heat shock protein (hsp 70). The polypeptide possesses features common to other hsps 70, i.e. a similar hydropathic profile and a variable C-terminal region with conserved sequence at the very C-terminus. Binding of the recombinant peptide to IgE from 38% of atopic sera or plasma from individuals allergic to C. herbarum was demonstrated. CONCLUSION: These results indicate that amino acid substitutions are relatively conserved even in the variable C-terminal regions of hsp 70 species. Thus, this study should draw attention to the possibility of induction of anaphylactic responses in a sensitized individual when hsp 70 from any pathogenic species is administered for vaccination.

N-terminus of a major allergen, Alt a I, of Alternaria alternata defined to be an epitope.

A 20mer peptide representing the N-terminus of a major allergen, Alt a I, of Alternaria alternata was synthesized and examined for its antibody binding and antibody induction activities. Alt a I peptide-BSA conjugate reacted with both human IgE and rabbit anti-Alt a I IgG in ELISA, albeit the binding of the peptide to IgE was relatively weak. Control conjugate showed no antibody binding. These results indicated that the N-terminus of Alt a I is an antibody binding site. Moreover, peptide-KLH conjugate and nonconjugated peptide induced both IgG and IgE antibodies in Balb/c mice that recognized both native Alt a I allergen and peptide-BSA conjugate. Since the free peptide was able to induce antibodies in vivo, the peptide may also possess a T cell epitope.

A cDNA clone coding for a novel allergen, Cla h III, of Cladosporium herbarum identified as a ribosomal P2 protein.

Mold allergens represent a major cause of atopic disorders. Progress in the molecular characterization of allergens has been hampered by batch-to-batch variation and poor yields in mold extracts. In the present study, we established a cDNA library in lambda ZAP II by using mRNA isolated from a major allergen-producing mold, Cladosporium herbarum. From this library, a novel allergen has been cloned and sequenced. The clone encodes a full length protein of 111 amino acids with a molecular mass of 11.1 kDa and pI of 3.94. By using sequence homology analysis, the allergen was found to belong to the ribosomal P2 protein family. Approximately 60% peptide sequence homology was found between the cloned protein and other known fungal ribosomal P2 proteins. In addition to conserved C-terminal sequences and serine blocks for phosphorylation in all eukaryotic ribosomal P2 proteins, this protein also contains a potential N-glycosylation site at position 15-17. Northern blots demonstrated that mRNA molecules of this gene are present even at late stages of culture. The gene was expressed in Escherichia coli by using the pMAL-2c system, and murine antisera against the recombinant allergen (rCh2.1) were generated. The antisera revealed that the native allergen corresponding to rCh2.1 was present in extracts prepared by grinding mycelia under liquid nitrogen in the presence of protease inhibitors, but absent in extracts prepared by conventional methods. This result indicates the usefulness of recombinant allergens in characterization and standardization of mold allergenic extracts.

Two-dimensional immunoblot analysis of allergens of Cladosporium herbarum.

In the present study, allergens of Cladosporium herbarum were analysed by SDS-PAGE, two-dimensional gel electrophoresis and immunoblot. The SDS-PAGE under reduced conditions followed by western blotting analysis revealed 10 human IgE-binding components with molecular masses ranging from 15 to 143 kDa. Within the same molecular mass range, five components were detected under non-reduced conditions. Examination of the specificities of 10 individual sera revealed that two patients were sensitive to almost all the allergens, whereas the majority reacted with only some components. Two-dimensional immunoblot was employed to further analyse the allergens in the extract. A silver-stain of the two-dimensional gel revealed that there were more than 30 different protein components visible in the extract. Immunoblot of the proteins separated under denatured conditions disclosed that 17 spots were reactive with human atopic sera.

Purification and characterization of Alt a-29 from Alternaria alternata.

A major protein component reactive with pooled human atopic sera was isolated from a lyophilized broth extract of Alternaria alternata 34-016. By successive chromatography on Whatman DE-52, Sephadex G-100 and Mono Q HR5/5, a low molecular weight antigen was obtained. Comparison with standard proteins on Sephadex G-100 indicated its molecular weight was 31 kD. Non-reduced samples run on SDS-PAGE showed a band at 29.2 kD which reacted strongly with human IgE. After reduction, it produced a doublet pattern on SDS-PAGE with MW 14.5 and 16.0 kD. The doublet pattern was confirmed by Western blotting with pooled human atopic sera. IEF of the protein showed a major component with a PI of 4.15 and two minor components at 4.25 and 4.40. Immunoblots of the IEF bands showed all three were reactive with human IgE. Ion exchange chromatography of the protein on Mono Q HR5/5 resulted in three resolved components, all of which are immunoreactive. Together with the IEF data, this suggests that there are several conformational or structural isoforms of this protein.

Studies on alternaria allergens. VI. Stability of the allergen components of Alternaria tenuis extracts under a variety of storage conditions.

The allergenic potencies of samples of two commercial Alternaria tenuis extracts stored under various conditions were compared at 3 months intervals over a period of 1 year. Aliquots of the extracts at 5.12 mg/ml were maintained in: saline; 50% glycerol-saline mixture; 0.4% phenol-saline; 0.4% phenol-saline containing 0.03% human serum albumin, and samples were also kept in lyophilized form. The samples were stored at -20, 4, 24, 37 and 56 degrees C. Their potency was measured by radioallergosorbent inhibition assay and by mouse IgE passive cutaneous anaphylaxis (PCA) tests. The lyophilized extracts fully maintained their activity at all temperatures for 12 months. Aqueous extracts with or without preservatives also maintained their potency if stored at -20, 4 and 24 degrees C, but showed a significant loss of PCA activity at 56 degrees C, after 3 months. A deleterious effect of phenol was observed in one of the extracts stored at -20 degrees C for 3 months, by PCA tests. The use of preservatives to retain potency over a period of 12 months is therefore unnecessary and may be deleterious. The results indicate that, while storage of A. tenuis in a lyophilized form is best at all temperatures, aqueous extracts are also remarkedly stable when stored at -20, 4 and 24 degrees C, over a 12-month period.

Studies on Alternaria allergens. V. Comparative biochemical and immunological studies of three isolates of Alternaria tenuis cultured on synthetic media.

Six extracts were prepared from Alternaria tenuis isolated ATCC 6663, ATCC 16086 and DAOM (Agriculture Canada) 183905 grown separately on two synthetic media, revised tobacco and Czapek's, and their biochemical and immunological properties were examined. High-performance liquid chromatography revealed considerable variations in UV absorbance and carbohydrate profiles among extracts from the different isolates. These differences were less marked among samples of the same isolate cultured on different media. Enzyme screening showed that all extracts contained large amounts of phosphatases and glucosidases and moderate quantities of esterases. Only the alpha-galactosidase activity showed any correlation with allergenic activity. No significant variation was observed in isoelectric focusing patterns. Extensive antigenic cross-reactivity even between the different isolates was found in precipitin studies. In mouse IgE passive cutaneous anaphylaxis tests, all extracts gave reactions of similar intensity. In direct RAST and RAST inhibition assays, ATCC 16086 grown on revised tobacco medium was found to be the most potent and approached the activity of an extract from a commercial material (B-I). DAOM 183905 grown on either medium was next in potency while ATCC 6663 samples were the least potent. The results indicate that it is possible to obtain extracts of high allergenic potency for standardization purposes from growth of selected A. tenuis isolates on a chemically defined medium.

Adjuvant and immunogenic properties of gonococcal R-type lipopolysaccharide in reaginic antibody formation in mice.

The ability of gonococcal R-type lipopolysaccharide (LPS) to function as an adjuvant and as an antigen in the formation of IgE and IgG1 antibody responses in mice was investigated. LPS failed to induce LPS-specific IgE or IgG1 under a variety of experimental conditions. LPS was capable of enhancing IgE and IgG1 antibody responses to gonococcal protein (ZAB), but its adjuvant effect was weaker than that of A1(OH)3. The LPS-induced anti-ZAB IgE antibody titers showed a cycling phenomenon with time, but the IgG1 response was delayed and peaked only once.

Studies on alternaria allergens. IV. Comparative biochemical and immunological studies of commercial Alternaria tenuis Batches.

The biochemical and immunological characteristics of a series of commercial samples of Alternaria tenuis were examined, including samples from two different companies. There was a considerable variation in the biochemical parameters such as the gel filtration profile on Sephadex G-100, isoelectric focussing pattern and immunodiffusion precipitin band pattern. The content and proportion of two high molecular weight polysaccharides, one a galactomannan and the other a glucan, also varied greatly. In contrast, the allergenic properties of the samples were more consistent. In rat IgE passive cutaneous anaphylaxis tests, extensive cross-reactivity was observed among batches from the same company, but this cross-reactivity was weaker when batches from the two companies were compared. In direct radioallergosorbent tests (RAST) the samples showed similar reactivities, but RAST inhibition tests showed a 40-fold range in activity between samples from one company and 55-fold between batches from the two sources. It was found that the proportions of the major A. tenuis allergen and a related hypoallergenic antigen in the samples were relatively constant, hence RAST inhibition can be used as a measure of allergenic potency despite the presence of this cross-reacting antigen. The lack of correlation between allergenic and biochemical properties of the various samples suggest that a simple biochemical test is unlikely to be a satisfactory replacement for the RAST inhibition assay in standardizing A. tenuis extracts.

Studies on Alternaria allergens. III. Effect of Alternaria tenuis on the humoral response to three T cell-dependent antigens in rats.

The effects of an extract of the saprophytic mold, Alternaria tenuis (AT-CE) on the humoral response to a ragweed allergen extract (DWSR), ovalbumin and sheep red blood cells (SRBC) was investigated in female Wistar rats. Animals pretreated with 100 micrograms or 2 mg AT-CE showed enhancement (p less than 0.05) in the reaginic response (IgE antibody) to DWSR at 25 and 18 days postimmunization. On the other hand, animals posttreated with AT-CE showed substantial reduction in anti-DWSR IgE antibody response. Contrasting results were obtained when ovalbumin was used as an immunizing antigen. There was a remarkable enhancement in the reaginic response to ovalbumin in rats pre- or posttreated with 10 micrograms of AT-CE. Pretreatment with AT-CE did not affect the hemagglutination titers to ovalbumin, while posttreatment with 100 micrograms or 1 mg AT-CE increased the hemagglutination titers of IgM antibody. There was a significant reduction in hemagglutinin, and hemolysin titers to SRBC in animals pretreated with all concentrations of AT-CE; at day 21, suppression was noted in animals pre- or posttreated with all concentrations of AT-CE. On the other hand, greatly increased hemagglutination titers were found in animals posttreated with 100 micrograms or 1 mg AT-CE. Hence, enhancement and suppression can both occur depending on the dose and time of administration of AT-CE together with the nature of the immunizing antigen.

Studies on alternaria allergens. II. Presence of two related antigens with contrasting allergenic properties in Alternaria tenius extracts.

Earlier, it was reported that most of the allergenic activity of Alternaria tenuis seemed to reside in the last Sephadex G-100 fraction (G3, molecular weight approximately 20,000). These conclusions were based on results obtained by the in vitro techniques of radioallergosorbent tests (RAST) and RAST inhibition. In the present studies, the allergenic activity of A. tenuis fractions was measured in vivo. It was found that fraction G3 was neither capable of inducing reaginic (IgE) antibodies in rats nor eliciting a skin reaction in rats sensitized with reagins against A. tenuis non-dialyzable fraction (MW greater than 12,000). On the other hand, subfractions G2D3 and G2D4 obtained from the middle fraction of G-100 (G2, MW approximately 30,000-40,000) followed by ion-exchange chromatography on DEAE-cellulose, showed the greatest allergenic activity in vivo, giving high titered reaginic antibodies in rats. When compared by RAST inhibition, the activities of fractions G2D3 and G2D4 were slightly lower than that of G3. Fraction G3 was immunogenic in rabbits. In immunodiffusion tests, G2D3, G2D4 and G3 appeared to be identical and they were cross-inhibiting in RAST inhibition tests. It was concluded that the sizes and multivalent properties of G2 and G3 antigens exerted a strong influence on their allergenic activities in rats (IgE antibody production) but this factor was of little importance for their antigenic activity in rabbits (precipitin antibody production) and their behaviour in the in vitro tests.

Influence of pregnancy on the immune response to ragweed antigen in rats.

The reactivity of spleen lymphocytes in culture and the reaginic antibody (IgE) response to nondialyzable water-soluble ragweed (DWSR) extract were assessed at different intervals of pregnancy in rats. On day 6 of pregnancy, spleen cells showed a significant reduction in their mitogenic response to phytohemagglutinin (PHA) and slightly lowered response to concanavalin A (Con A) when compared to that of control nonpregnant rats. On day 11 of pregnancy, values obtained with PHA were slightly lower and those obtained with Con A similar to those of control rats. However, at day 18 of gestation, PHA values also returned to normal levels. In contrast, IgE antibody response to DWSR from day 4 to 9 of pregnancy was either slightly enhanced or similar to that of control rats and significantly suppressed from day 11 to 18 of gestation. The decline in humoral response to DWSR during late pregnancy is discussed in the text.

Induction of reaginic (IgE) gonococcal antibodies in the rat by a common antigen of Neisseria gonorrhoeae.

An antigen (ZAB) common to Neisseria gonorrhoeae was prepared by stepwise elution of a crude gonococcal antigen (ZA) from columns of diethylaminoethyl cellulose employing 0.02 M phosphate buffers, pH 7.6, containing increasing concentrations of sodium chloride. Rats immunized with ZAB produced reaginic (IgE) antibody which cross-reacted with ZA prepared from eight gonococcal strains by the passive cutaneous anaphylaxis (PCA) test. Heating of the sera at 56 degrees C for 4 h destroyed the PCA activity. The PCA activity of the anti-ZAB rat serum was removed after absorption with ZAB antigen or with rabbit anti-rat IgE but not after absorption with gonococcal lipopolysaccharide or with heat-killed or formalinized gonococci. Treatment of ZAB with trypsin or heating at 100 degrees C for 30 min destroyed or reduced the antigenic activity respectively. Further purification of ZAB by filtration through Sephadex G-100 gave a preparation (ZAB2) which contained the common antigen as shown by the cross-reactivity of anti-ZAB2 rat serum with seven stains of N. gonorrhoeae. Fraction ZAB2 contained material which had a molecular weight less than 13,700 and was associated with the presence of material absorbing at 260 nm. The results of this study indicate that a low molecular weight antigen, which appears to be protein in nature and associated with nuclei acid, is common to the gonococcus and is the main antigenic component inducing reaginic (IgE) antibody in the rat.

Studies on Alternaria allergens. I. Isolation of allergens from Alternaria tenuis and Alternaria solani.

Extracts of Alternaria tenuis and Alternaria solani were separated into dialyzable (molecular weight less than 10,000) and non-dialyzable forms. The latter was further fractionated by gel filtration through Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The dialyzable material was fractionated by gel filtration through Sephadex G-50. The allergenic activities of the fractions obtained from the A. tenuis extract was measured in vitro by the radioallergosorbent test assay and the allergenic potency was measured by radioallergosorbent test inhibition assay. Allergenic activity was detected in most of the non-dialyzable fractions, the majority of the activity being in the last G-100 fraction (MW approximately 20,000) which was predominantly protein in nature. The same component may be responsible for the activity found in the dialyzate and its first G-50 fraction since the immunodiffusion studies indicated that the last G-100 fraction has antigenic components in common with those of the first G-50 fraction. In addition, cross-reactions between A. tenuis and A. solani extracts show that the two species share common antigenic determinants.