Fusarium solani: Evidence for common antigenic/allergenic determinants with other Fungi Imperfecti

Aqueous extracts of Fusarium solani and other members of the Fungi Imperfecti were evaluated for the presence of common antigenic/allergenic determinants using skin-prick testing, radio-allergo-sorbent test (RAST) inhibition, and immunoelectrophoretic methods. Prevalence of skin reactivity in forty-four atopic individuals, tested with commercially available fungal extracts, ranged from 27.3% for Alternaria tenuis to 6.8% for Penicillium notatum. No specific patterns of reactivity emerged from statistical analyses of skin test data. In contrast, RAST inhibition demonstrated common allergenic determinants. P. notatum and Aspergillus glaucus inhibited F. solani RAST by 79% and 84%, respectively. This was supported by crossed line immunoelectrophoresis; both P. notatum and A. glaucus had antigenic determinants in common with F. solani. Collectively, these studies suggest that F. solani, P. notatum , and A. glaucus have several common antigenic/allergenic determinants.     

IgE to cross-reactive carbohydrate determinants: analysis of the distribution and appraisal of the in vivo and in vitro reactivity

BACKGROUND: IgE to cross-reacting carbohydrate determinants has already been described by several authors, but their function and distribution are still a matter of debate. In previous studies we showed how the presence of IgE to bromelain could be a useful and simple marker of the presence of IgE to carbohydrate epitopes. METHODS: A survey of 1,831 subjects with a suspected allergic respiratory disease has been carried out by detecting IgE to bromelain. Data were analysed on the basis of demographical and allergological parameters. To find out whether a glycoprotein is capable of triggering an allergic reaction, 1,076 subjects were also skin tested with several purified molecules bearing carbohydrate side chains differing in number, composition and complexity. RESULTS: An overall prevalence of 23% of positive IgE to cross-reacting carbohydrate determinants was recorded. Prevalence varied when subsets of non-allergic (5%), non-pollen-allergic (10%), and pollen-allergic (31%) subjects were considered. Prevalence further increased in subsets with multiple pollen sensitization (71%), and with a previous pollen immunotherapy course (46%), whereas minor differences were found in gender and age distribution. Almost all the allergenic extracts recorded negative in the skin test gave a positive IgE test in vitro. A higher correlation was found mainly with plant-derived allergenic extracts, whereas a lower one was recorded with mites and fungi. Horseradish peroxidase was the only glycoprotein capable of exerting a positive skin test in 21% of the subjects with IgE to cross-reacting carbohydrate determinants, 80% of them having IgE to the HRP molecule. CONCLUSIONS: IgE to cross-reacting carbohydrate determinants are common among the allergic population and the binding to skin test negative allergenic extracts further confirms their poor biological activity. Further studies on horseradish peroxidase should be carried out to define the role of the glycan side chains in its allergenic activity.     

Antigenic activity of nonpollen parts (leaves and stems) of allergenic plants (Parietaria judaica and Dactylis glomerata)

In patients with respiratory allergy to pollen it is common to correlate the onset, duration and intensity of clinical symptoms with the count of atmospheric allergenic pollen grains. Pollen counts, however, may not reflect the total airborne allergen exposure since previous data suggest that pollen allergens may also be carried in microaerosol suspensions. These microdroplets may penetrate deeply into the airways, where pollen grains are too large to penetrate, eventually inducing asthma. The origin of these allergenic aerosols is still uncertain. We investigated whether antigenic activity is present in vegetative parts of allergenic plants. We have used extracts from leaves and stems of Parietaria judaica and Dactylis glomerata to evaluate patients with allergic sensitization to pollen allergens of these plants (19 grass-sensitive patients and 23 Parietaria sensitive). By using skin prick testing and RAST to stem and leaf extracts other than pollen extracts we observed that most patients sensitive to grass or Parietaria pollen had small responses to extracts of stem or leaf. We conclude that allergenic components are present throughout most of Parietaria judaica and Dactylis glomerata plants, most highly concentrated in the pollen but present in the leaves with a trace in the stems.     

Allergenicity and cross-reactivity of cat and dog allergenic extracts

This study aims to confirm that cat allergen 1 (CAT-1) is a major allergenic determinant in cat-sensitive patients, and to further define the role of other determinants, as well as to identify the determinants responsible for the cross-reactivity between cat and dog extracts. Firstly, the allergenic determinant with an electrophore-tic mobility of 18 kD (corresponding to CAT-1) is indeed a major allergenic determinant being recognized by the majority (75%) of cat-sensitive subjects. Secondly, the cross-reactivity between the two species was confirmed by RAST inhibition. Cat and dog soluble allergens could inhibit, to variable degrees, the binding of serum IgE from cat- and dog-sensitive patients to insolubilized allergens. Binding of serum IgE from subjects sensitive only to cats was inhibited by cat extracts only. These observations suggest the presence of determinants common to the two sources of extracts, and others specific for each species. These data were confirmed by immunoblot analysis. Indeed, an allergenic determinant of 69 kD was found in both cat and dog extracts. Conversely the allergenic determinants with an electrophoretic mobility of 18 and 32 kD were found only in cat extracts, and those at 22 and 24 kD were dog specific. However, surprisingly, serum IgE antibodies from patients sensitive only to cats reacted on immunoblot differently from those of both cat- and dog-allergic subjects. Indeed, the 18 kD determinant was the only one recognized by serum IgE antibodies from subjects sensitive to cats only, as opposed to the patients allergic to both species: then, the 69 kD determinant was strongly recognized and the 18 kD only slightly recognized. If these observations confirmed that CAT-1 is a major allergen in cat-sensitive subjects, they also showed a different profile of reactivity in patients sensitive to cats only and those sensitive to both cats and dogs, which could have diagnostic and therapeutic implications.     

Cupressaceae pollinosis: identification, purification and cloning of relevant allergens

Allergy to Cupressaceae pollen is a worldwide pollinosis caused by several species. Pollen extracts prepared from allergenic species belonging to this family are characterised by low protein and high carbohydrate content. The allergenic components represented in the pollen extracts from different species of the Cupressaceae family show high levels of cross-reactivity when probed with human IgE from allergic subjects and share a number of common epitopes also identified by polyclonal rabbit antisera and monoclonal antibodies. A close relationship has also been described with the Taxodiaceae and Podocarpaceae families. Although both proteic and carbohydrate epitopes appear to be involved in IgE recognition and allergenic cross-reactivity, a large portion of the IgE reactivity of Cupressaceae-allergic patients seems to be associated with sugar moieties present on the relevant allergenic molecules. From this point of view, Cupressaceae/Taxodiaceae allergens constitute a particularly useful model to study IgE cross-reactivity, as they have been shown to display different levels of homology. Moreover, the availability of the purified allergens, together with their recombinant counterparts, may shed light on the actual role played by carbohydrate in allergic sensitisation, IgE recognition and allergenic cross-reactivity.     

Allergenic extracts for specific immunotherapy: to mix or not to mix?

Immunotherapy for allergic diseases give rise to questions about when a decision must be taken to define the number of extracts to be used in a single treatment. This represents a long-lasting matter of debate between American and European allergists, which seems to be without real solution. Through the use of extract-based versus molecule-based diagnostic approaches we suggest a possible solution to this controversial issue. We used four model patients previously tested with allergenic extracts and later on selected on the basis of a panel of available allergenic molecules. Their reactivity patterns in term of extracts and molecules were compared and the decision for allergenic extract immunotherapy was made choosing either the 'classical' approach or the molecular approach. From our study it seems that molecules could offer the solution to the 'mixing' issue of allergenic extracts. This innovative approach seems to provide a solution for both the American and the European approach.     

Diagnosis and Immunotherapy of Mould Allergy

In order to screen for mould allergy, extracts of five common atmospheric moulds (Cladosporium, Alternaria, Penicillium, Aspergillus and Mucor) from various manufacturers were investigated in 130 patients (5-60 years old) with clinical symptoms indicating possible mould allergy. The patients were screened by skin prick test (SPT) and radioallergosorbent test (RAST). SPT seemed to be more sensitive than RAST as a diagnostic screening procedure (80% positive reactions to one or more species compared to 50%). With a partially purified, standardized preparation of Cladosporium herbarum more positive reactions were obtained than with crude extracts without evidence of any unspecific reactions. The difference between commercial and standardized extracts is most probably a result of a variation of both the biological potency of allergenic determinants and the allergenic composition. A considerable number of negative RAST reactions with standard discs were found in patients with positive skin reactions to partially purified Cladosporium, but RAST seemed to be more sensitive than SPT with the other commercial mould extracts. Based on the screening, a very convincing tendency to IgE-reactivity to other moulds was found in patients reacting to Cladosporium, the most common cause of mould allergy. The results confirm the inadequacy of most mould extracts used in diagnostic procedures and strengthen the value of using standardized extracts.     

Cross-reactivity between antigens of fungal extracts studied by RAST inhibition and immunoblot technique

We have used the RAST-inhibition technique in homologous and heterologous inhibitions of the Aspergillus fumigatus, Alternaria tenuis, and Cladosporium herbarum RAST assays followed by the immunoblot technique to assess the degree of shared allergenic determinants in these extracts. In the Aspergillus RAST, there was little or no inhibition with Cladosporium and Alternaria, but considerable cross-reactivity was found between Alternaria and Cladosporium. Inhibition by Alternaria of the Cladosporium RAST was found in a dose-dependent fashion in one serum, whereas all four sera in the Alternaria RAST were inhibited by Cladosporium in this fashion. Logit transformation of the Alternaria RAST-inhibition curves produced common slopes with Alternaria and Cladosporium in three of the four sera, indicating their immunologic identity. The immunoblot technique confirmed the degree of cross-reactivity found by RAST inhibition among the molds. This evidence of common allergenic determinants in these fungi could help to explain the observation that mold-allergic patients often have skin test reactions to several fungi.     

Mapping of Bet v I epitopes by using murine monoclonal antibodies.

The different determinants of birch pollen extracts, as shown by SDS-PAGE analysis, range from 10 to 94 kDa. These determinants were then electrotransferred on nitrocellulose strips and allowed to react with human IgE Ab from sensitive patients in order to identify the allergenic determinants. Several minor (43, 35, 28 and 21 kDa) and the major (17 kDa) allergenic determinants were identified. Murine monoclonal antibodies (mAb) were then produced against the major allergenic determinant (Bet v I) and their specificity confirmed by immunoblot. One of them, mAb 3F10, was used to affinity-purify the Bet v I. The purity of this material was confirmed by SDS-PAGE analysis and its reactivity on immunoblot against human IgE ensured its biological activity. These mAb were then gathered on four families based on their pattern of reactivity with Bet v I. Indeed four different epitopes on the molecule were identified. Binding inhibition studies using two of them (mAb 5F9 and 8F12) suggested that the epitopes of Bet v I recognized by these mAb are not overlapping. On another hand, the binding of 8H7 and 3F10 was partially inhibited by 5F9 and the binding of 3F10, by 8F12. These data suggest that those two latter epitopes are somewhat overlapping. Finally, the mAb 5F9 could inhibit the binding of human IgE on the affinity-purified Bet v I up to 40% and then shares a common idiotope with human specific IgE Ab of allergic patients.     

Specific IgE response before and after rush immunotherapy with a standardized allergen or allergoid in grass pollen allergy

Allergen extracts contain a wide variety of allergenic determinants and the sensitization of allergic subjects is extremely heterogeneous. Specific immunotherapy is usually performed with multiple component extracts and it is therefore important to determine whether this treatment may elicit the onset of newly developed IgE sensitivities. By means of nitrocellulose immunoblotting technique, the IgE sensitivities of 20 patients were characterized before and after specific immunotherapy. Ten patients had a rush immunotherapy with a standardized orchard grass pollen extract and ten others underwent a rush immunotherapy with a 6-grass pollen allergoid. The allergenic profiles of the patients before and after immunotherapy were qualitatively similar. In some patients an increase of orchard grass pollen-specific IgE was observed and the allergenic profile was quantitatively different. These results suggest that rush immunotherapy with either an aqueous non-modified extract or an allergoid does not elicit the onset of new IgE sensitivities. Multicomponent allergenic extracts may therefore be used in the treatment of patients.     

Allergenic and antigenic relationship between three species of storage mite and the house dust mite, Dermatophagoides pteronyssinus

We have explored the antigenic and allergenic relationship between the house dust mite Dermatophagoides pteronyssinus and three species of storage mite, Glycyphagus destructor, Acarus siro, and Tyrophagus longior. Crossed immunoelectrophoresis demonstrated that all the mite extracts contained multiple antigens but that there was only limited cross-reactivity between the different species. Six sera were obtained from workers exposed to storage mites and with occupationally related lower respiratory tract symptoms. All workers had specific IgE to D. pteronyssinus and to one or more of the storage mites. The pattern of reactivity varied between the different sera, two responded primarily to D. pteronyssinus and A. siro and four sera to D. pteronyssinus and G. destructor. Only weak responses were observed to T. longior. RAST-inhibition and affinity-absorption experiments demonstrated that D. pteronyssinus had at least three groups of distinct allergenic determinants, determinants specific to D. pteronyssinus, determinants shared with A. siro, and determinants shared with G. destructor. Similarly, both A. siro and G. destructor have specific allergenic determinants and determinants shared with D. pteronyssinus. The findings demonstrate the complexity of the immunologic responses to the different mite species.     

Allergenic cross-reactivity among pollens of Urticaceae

Common antigenic determinants have been observed between Parietaria and Urtica dioica pollen. The four Parietaria pollens selected (P. judaica, P. officinalis, P. lusitanica and P. mauritanica) are shown to possess a high allergenic homology. IgE-binding structures, homologous to the P. judaica main allergenic polypeptide (Pj10), were found in the other species by immunodetection. Monoclonal antibodies specific to the Pj10 polypeptide recognized proteins from the four Parietaria pollens. Skin prick test and RAST inhibition yielded results that also indicated a high allergenic cross-reactivity among these pollens, with homologous peptides bearing common antigenic and allergenic determinants. On the other hand, U. dioica pollen showed only a slight allergenic similarity to Parietaria. The potential allergenic activity of these pollens is discussed.     

Cross antigenic and allergenic properties of the house dust mite Dermatophagoides farinae and the storage mite Tyrophagus putrescentiae

The crossed antigenicity and allergenicity of the storage mite Tyrophagus putrescentiae (TP) and the house dust mite Dermatophagoides farinae (DF) were characterized by means of crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis. DF extracts exhibited 32 antigens and as many as eight were demonstrated to be allergens. DF feces exhibited 20 antigens and six of these were allergens. Twenty antigens and two allergens were demonstrated for TP. Two antigenic and allergenic determinants were shared by DF and TP, and two determinants were also shared by DF feces and TP feces. TP feces and DF shared two antigenic and allergenic determinants. Our results demonstrated that the two mites and their feces extracts contain multiple antigens and allergens.     

Allergenic cross-reactivity of cytochromes c of Kentucky bluegrass and perennial ryegrass pollens

The allergenic cross-reactivity of cytochromes c isolated from Kentucky bluegrass (KBG) and ryegrass (RG) pollens was demonstrated by the finding that both cytochromes elicited PCA reactions of comparable titers in rats sensitized with murine reaginic antiserum to either KBG or RG cytochrome c. However, allergenic differences were revealed at limiting doses of the cytochromes c; under these conditions RG cytochrome c elicited PCA reactions only with the homologous reaginic serum, whereas KBG cytochrome c elicited PCA reactions with both murine reaginic sera. Moreover, in experiments involving inhibition of PCA. RG cytochrome c failed to neutralize some of the IgE antibodies of the antiserum to KBG cytochrome c, whereas KBG cytochrome c neutralized all IgE antibodies of both murine reaginic antisera. From these results it may be concluded that: (1) the two cytochromes c share common allergenic determinants, and (2) the KBG cytochrome c possesses additional allergenic determinant(s) not present on RG cytochrome c. The radioallergosorbent test, utilizing KBG cytochrome c discs and a pool of sera of individuals allergic to KBG, was inhibited to the extent of 87 or 41% by the addition of 10μg of KBG or RG cytochrome c, respectively. By contrast both cytochromes c at 10, μg were equally effective in the inhibition (92%) of the binding of the IgE antibodies in this serum pool to RG cytochrome c allergosorbent discs. These experiments confirmed that the two cytochromes share common allergenic determinants.     

Identification and characterization of allergens in two species of tuna fish.

BACKGROUND: In countries where fish consumption is high, allergenic reactions to fish are common among patients diagnosed with food hypersensitivity. For tuna fish, allergenic proteins are not known. In addition, it is not known how the tuna fish extracts should be processed to obtain optimal in vitro diagnostic performance and to preserve labile antigens. OBJECTIVE: The aim of this study was to characterize IgE-binding components of Yellowfin and Albacore tuna fish. METHODS: Various tuna fish extract preparations were fractionated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose and analyzed by using tuna positive patients with different Western blot profiles. The functional activity of the extracts was evaluated by basophil histamine release. RESULTS: Immunoblot analysis showed the majority of patients responding to Yellowfin tuna extract. Inhibition studies using immunoblot analysis and histamine release testing showed a specific protein with a molecular weight of approximately 46 kD that is present in Yellowfin tuna, but absent in Albacore. Only defatted, lyophilized tuna fish extracts were able to induce histamine release from sensitized basophils although IgE-binding components were detected in fresh raw, fresh cooked, and canned tuna fish preparations. CONCLUSION: These studies indicate that patient sera may contain different tuna fish species IgE specific antibodies directed against unique species specific allergens present in Yellowfin and Albacore tuna fish. Possibly, extracts should contain specific allergenic components from both Albacore and Yellowfin to cover the epitope heterogeneity observed in sera from patients developing IgE antibodies against tuna fish.     

High throughput screening: a rapid way to recombinant allergens

Complex allergenic sources such as moulds, foods and mites contain large panels of IgE-binding molecules which need to be cloned, produced and characterized in order to mimic the entire allergenicity of whole extracts reconstituted by mixing single standardized recombinant allergens. Phage display of cDNA libraries allows selective enrichment of allergen-expressing clones using IgE from allergic patients. For the characterization of all different clones present in enriched cDNA libraries in a fast and cost-effective way, however, high-throughput screening technology is required. We have used a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries in order to characterize whole allergenic repertoires from complex allergenic sources. The strategy, based on a combination of phage display and high-density arrays, allowed fast discovery of panels of related structures from different allergenic sources. They cover secreted, cytoplasmic and structural proteins with or without enzymatic activity and offer a rational explanation for the IgE-mediated cross-reactivity frequently encountered in clinical practice.     

Immunological analysis of allergenic cross-reactivity between peanut and tree nuts

BACKGROUND: Peanut and tree nut allergy is characterized by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Peanut allergy is more common than tree nut allergy, but many subjects develop hypersensitivity to both peanuts and tree nuts. Whether this is due to the presence of cross-reactive allergens remains unknown. OBJECTIVE: The aim of this study was to investigate the presence of allergenic cross-reactivity between peanut and tree nuts. METHODS: Western blotting and ELISA were performed using sera from subjects with or without peanut and tree nut allergy to assess immunoglobulin E (IgE) reactivity to peanut and tree nut extracts. Inhibition ELISA studies were conducted to assess the presence of allergenic cross-reactivity between peanut and tree nuts. RESULTS: Western blot and ELISA results showed IgE reactivity to peanut, almond, Brazil nut, hazelnut and cashew nut for peanut- and tree nut-allergic subject sera. Raw and roasted peanut and tree nut extracts showed similar IgE reactivities. Inhibition ELISA showed that pre-incubation of sera with almond, Brazil nut or hazelnut extracts resulted in a decrease in IgE binding to peanut extract, indicating allergenic cross-reactivity. Pre-incubation of sera with cashew nut extract did not cause any inhibition. CONCLUSION: These results show that multiple peanut and tree nut sensitivities observed in allergic subjects may be due to cross-reactive B cell epitopes present in different peanut and tree nut allergens. The plant taxonomic classification of peanut and tree nuts does not appear to predict allergenic cross-reactivity.     

Basidiospore allergens: analysis of Coprinus quadrifidus spore, cap, and stalk extracts

Atopic individuals with symptoms of respiratory allergy have been shown to have IgE-mediated reactions to spores from the basidiomycete fungi. Because our earlier studies suggested that parts of the fungus other than spores may contain allergens, the current study was performed. Extracts of Coprinus quadrifidus spores, caps, and stalks were prepared and fractionated by gel filtration column chromatography on Sephadex G-75. Analysis of column fractions of each separation by ultraviolet absorption demonstrated at least three peaks of absorbency in spore, cap, and stalk extracts. Pooled column fractions were analysed by direct radio-allergosorbent test (RAST) using pooled sera from C. quadrifidus skin-test positive subjects. Enhanced allergenic activity was present in the same portion of the column eluate for cap, spore, and stalk fractionations, corresponding to a molecular weight of approximately 10.5–25 kD. Pools with allergenic activity were used to test volunteers by skin prick and RAST. Skin test and RAST activities were similar for each of the three Coprinus extracts, with stalk being the most potent. Evidence of common allergenic epitopes was demonstrated by inhibition of spore RAST by spore, cap, and stalk extracts. These results suggest that C. quadrifidus cap and stalk extracts contain allergens similar to those in spores extract and may provide useful sources of allergen for further study.     

Comparison of the antigenic and allergenic properties of three types of bovine epithelial material.

The antigenic and allergenic characteristics of three bovine epithelial extracts, dander, skin scrapings and whole skin, were compared using IgE-ELISA inhibition and SDS-PAGE with immunoblotting. Cow dander extract was shown to contain more allergenic activity than skin scrapings or whole skin extracts which were needed in about three times higher amounts than cow dander extract to induce the same degree of inhibition in ELISA. Skin scrapings and whole skin extracts contained more high-molecular weight components than dander extract. These components were at least partly serum-derived and reacted often with the IgG but not with the IgE of both the cow-asthmatics and their control subjects. The antigenic characteristics of the low-molecular weight components as well as the allergenic qualities of these three epithelial preparations were generally similar. Using the sera of 49 cow-asthmatic farmers, two major allergens were detected at 20 and 22 kD in all three extracts. Our results show that the highest amount of allergenic material and all the essential allergens are present in cow dander extract. In addition, the normally non-allergenic high molecular weight components are detected in low concentrations in dander extract. Therefore it is concluded that cow dander extract is the best alternative for allergen purification and allergen extract preparation.     

Cockroach allergenic activity: Analysis of commercial cockroach and dust extracts

Previous investigations demonstrated that cockroach whole bodies and feces are important sources of allergens in the induction/exacerbation of bronchial asthma. The current study investigated different cockroach source materials, commercial extracts, and house dust extracts for cockroach allergenic activity. In general, extracts from four different sources of either American or German cockroaches contained similar amounts of allergenic activity by RAST inhibition. Three commercial American cockroach extracts compared by RAST inhibition had similar allergenic activity on an equal protein basis. Skin test results correlated house dust reactivity to both commercial and inhouse cockroach wholebody extracts and to fecal extracts. Six different samples of house dust obtained from vacuum cleaners in the New Orleans area and three commercially obtained house dust extracts contained varying quantities of cockroach allergenic activity by RAST inhibition. These studies demonstrate that commercial cockroach extracts vary in allergenic activity and that all house dust extracts tested contain cockroach allergens.