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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.

Allergens↗

Injective versus sublingual immunotherapy in Alternaria tenuis allergic patients.

Little knowledge is available on molds either from the environmental or clinical points of view. The latter is mainly due to the lack, until recent times, of purified and standardized extracts. Injective immunotherapy, largely used for patients allergic to mites and pollens, is regarded with some concern for molds. On the other hand, mold-related allergic symptoms (i.e.,from Alternaria tenuis) have in Italy an incidence of about 5 percent, mainly in young patients. We have therefore run a pilot study comparing the efficacy and safety of injective (SIT) and sublingual (SLIT) immunotherapy by administering the same purified and standardized extract of Alternaria tenuis to a total of 23 patients for two years, according to an open experimental plan. Excellent tolerance was shown to SLIT while four (two medium-grade) side effects appeared with SIT. Clinical improvement, subjectively stated comparing symptoms (mainly rhinitis) and drug consumption before and after the therapy, confirmed by an increase in the Specific Nasal Provocation threshold, was obtained with both therapies, but with a statistically significant difference in favour of SLIT. Skin reactivity and blood Alternaria tenuis specific IgE, total IgG and IgG changed in the SIT-treated group, while no statistically significant change was shown in the SLIT-treated group. These results are in good agreement with previous reports on SIT and SLIT with other inhalant allergens (mites, grasses), and suggest the potential use of SLIT for Alternaria allergy, mainly in young patients, when there are concerns about the safety of and compliance with the traditional injective therapy.

Administration, Sublingual↗

[Factors influencing the biosynthesis of beta-galactosidase in Alternaria tenuis].

The effect of pH, carbon sources, and some organic substances on the biosynthesis of beta-galactosidase was studied in Alternaria tenuis on a defined medium that had been optimized by the method of mathematical planning of experiments. Optimal conditions for the production of the enzyme and its liberation into the cultural broth were maintained by adding 2 per cent soya flour to the medium and 0.1 M phosphate-citrate buffer pH 3.0. The production of the enzyme was increased by 3 to 4 times. The biosynthesis of beta-galactosidase by Alternaria tenuis is of an induced nature.

Alternaria↗

Nuclear distribution in Alternaria tenuis.

Nuclear distribution and behaviour during vegetative growth and spore formation in Alternaria tenuis was studied utilising the HC1-Giemsa staining technique. The vegetative mycelium and conidia are predominantly monokaryotic. Anastomoses, followed by nuclear migrations, have been recorded. Intercellular nuclear migrations have only been observed in germinating conidia. Nuclear behaviour during conidial formation indicates that the conidia are homokaryotic. Cytological differences have been found in different monocoidal isolates of A. tenuis.

Alternaria↗

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.

Allergens↗

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.

Allergens↗

Tolerance of immunotherapy with a standardized extract of Alternaria tenuis in patients with rhinitis and bronchial asthma.

We studied the safety of immunotherapy with an Alternaria extract in patients with rhinitis and bronchial asthma. The few studies that have investigated immunotherapy with mold allergens suggest that they cause adverse reactions more frequently than do other extracts. All treatments prescribed by our allergology service with Alternaria immunotherapy between 1988 and 1996 were recorded and analyzed. In all cases a biologically standardized depot extract of Alternaria tenuis containing 5 BU/ml was used according to a conventional immunotherapy schedule. During the study period 129 patients received immunotherapy with Alternaria extract. Of the 3,892 doses given, 1.95% led to adverse reactions, which occurred in 39.5% of the patients. Most of the adverse reactions were systemic and mild, and reproduced the underlying disease. The risk of adverse reactions was significantly higher in children, patients with asthma, and during the initial phase of treatment. Patients who suffered from adverse reactions had a significantly higher level of total and specific IgE. It was concluded that tolerance of Alternaria mold extract was worse than for other allergenic extracts, although most reactions recorded were mild. The risk of adverse reactions was greater in children and patients with asthma, and during the initial phase of immunotherapy.

Adolescent↗

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.

Allergens↗

[Production and properties of a beta-galactosidase preparation from Alternaria tenuis].

Different methods of the preparation of fungal beta-galactosidase from the 72-hour culture of Alternaria tenuis were tested: lyophilization of the culture liquid, precipitation with ethanol, acetone, ammonium sulphate. Optimal results were obtained with precipitation by 1.5 acetone volume. Studies of the properties of fungal beta-galactosidase demonstrated that the preparation retained its activity during 22 month storage at 5 degrees C. The fungal preparation had pH optimum at a more acidic zone (4.2 versus 6.9), was active in a wider pH range 2.8-5.7 and 6.2-7.5), had a much higher temperature optimum (65 degrees and 30 degrees) and better thermostability as compared with the yeast preparation. Data on other properties of the preparation are presented.

Alternaria↗

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.

Alternaria↗

Comparison of mRAST and CAP with skin end point titration for Alternaria tenuis and dermatophagoides pteronyssinus.

There has been a recent explosion of new in vitro tests for the diagnosis of allergies. At present there is no general agreement on which type of in vitro test is best. Recently our hospital switched in vitro testing from the modified radioallergosorbent system (mRAST) to the Pharmacia CAP system (CAP). While changing in vitro testing techniques, 47 patients were tested with both the mRAST and CAP tests. Comparisons were made between the mRAST and CAP results of Alternaria tenuis and Dermatophagoides pteronyssinus allergens. These results were then compared with the results of patients who also underwent intradermal skin testing based on end point titration techniques.

Allergens↗

Comparison of Alternaria tenuis extracts prepared from different raw materials.

Alternaria extracts, prepared from three different sources under the same conditions, were compared by several biochemical/immunochemical methods. Two raw materials (A,G) contained mostly mycelia. The third raw material (C) contained mycelia (35%) and spores (65%). Extracts A and G were different in total allergenic potency, antigen/allergen pattern, and in the Alt 1 content. Extract C was of similar total allergenic potency as extract A but showed a somewhat different antigen/allergen pattern and a different Alt 1 content. Although there were some compositional differences among the extracts, all extracts regardless of source materials (spores and mycelia) demonstrated strong RAST inhibition activity and parallelism of the inhibition curves.

Allergens↗