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Paulo (ICB-USP), S~o Paulo, SP, Brazil; 2Faculdade de CiOncias FarmacOuticas, Universidade de S~o Paulo,. S~o Paulo, SP, Brazil; 3Faculdade de Medicina, ...
Mycopathologia 131: 75-81, 1995. © 1995 KluwerAcademicPublishers. Printedin the Netherlands.

Biochemical,

75

antigenic and allergenic characterization

of crude extracts of

Drechslera (Helminthosporium) monoceras Everardo A. Menezes 1'*, Walderez Gambale 1, Mahasti S. Macedo 1, Dulcineia S. R Abdalla 2, Claudete R. Paula 1 & Julio Croce 3 1Sef~o de Micologia, Departamento de Microbiologia, Instituto de Ci~ncias Biomddicas, Universidade de S~o Paulo (ICB-USP), S~o Paulo, SP, Brazil; 2Faculdade de CiOncias FarmacOuticas, Universidade de S~o Paulo, S~o Paulo, SP, Brazil; 3Faculdade de Medicina, Universidade de S~o Paulo, S~o Paulo, SP, Brazil Received 18 October 1994; accepted in revised form 19 April 1995

Abstract In a previous study with airborne mould extracts we verified that Drechslera (Helminthosporium) monoceras presented stronger reactions than those presented by 42 other moulds isolated in S~o Paulo city. In the present study, we evaluated the biochemical composition and the antigenicity of crude extracts obtained from vegetative and conidial stage ofD. monoceras using Czapeck broth (CB) modified and tris-HC1 for extraction. The maximum values of total proteins and lipids were verified in the crude extract obtained in the 28th day of growth, and maximum values of carbohydrates were observed in the extracts of the 16th, 22nd and 26th days. The fractionated proteins by SDS-PAGE presented bands with molecular weights between 14.4 to 67 Kd; the 28th day extract showed a larger number of bands. The carbohydrates and amino acids were characterized by thin-layer chromatography. The antigenicity of the crude extracts was verified by immunodiffusion reaction in agar against rabbit hyperimmune sera. Precipitation lines were observed in all studied extracts and common antigenic molecular populations. Based on the above results, the 28th day extract was selected to verify the induction of IgE antibody responses in immunizations of Balb/c and cAF-1 mice, and titer by passive cutaneous anaphylaxis test using Wistar rats. The maximum titers obtained were 160 in cAF-1 mice and 1.280 in Balb/c mice. The results suggest that the 28th day extract contains allergenic fractions and should be chosen for future studies related to fractionation, characterization and standardization in diagnostic methods and immunotherapy. Key words: Allergens, AntigenicityDrechslera (Helminthosporium)monoceras, Fungi, Induction of IgE antibody, SDS-PAGE Introduction Airborne moulds are a significant cause of respiratory allergies. In the city of Sao Paulo, Brazil, there are 45 genera of airborne moulds, with the most frequent isolates being Cladosporium, Epicoccurn, Rhodotorula, Penicillium, Aspergillus, Aureobasidiurn, Phorna, Alternaria, Candida, Fusariurn, Trichoderma, Cephalosporiurn and Drechslera [1 ]. In a previous study with airborne mould extracts we verified that D. rnonoceras presented stronger reactions * Part of the thesis of E.A. Menezes to get degree of Doctor "Fractionation and characterization of allergenic extract of Drechslera (Helminthosporium)monoceras'.

than those presented by 42 others moulds isolated in Silo Paulo city, as measured in skin tests of asthamatic patients [2], an observation not reported in other countries. Drechslera is a genus of the subdivision Deuteromycotina, class Hyphomycetes, family Dernatiaceae. It is a soil saprophyte and is associated with pathogenicity for plants such as maize, oats and sugar cane. Dispersion is by air and the fungus is frequently isolated from sputum and skin, although it is rarely responsible for infectious diseases in man [3]. The correct diagnostic of allergy to fungi as well as immunotherapy, fundamentally depend on well characterized extracts. Listed in the literature are about

76 340 fungi associated with respiratory allergies, but just four fungal extracts are characterized and standardized: Alternaria, Cladosporium, Aspergillus and Penicillium [4, 5]. In the present study, as a main stage for future fractionation, purification, characterization, and standardization of allergenic extract, the biochemical composition, antigenicity and allergenicity of crude extracts obtained from the vegetative and conidial stages of D. monoceras were evaluated by using tris-HC1 for extraction.

and lipid [9] content. The proteins were fractioned by SDS-PAGE [ 10]; carbohydrates and amino acids were characterized by thin-layer chromatography [11 ]. Antigenic analysis. The antigenicity of extracts obtained from several growth stages were studied by gel-double diffusion [ 12]. The antiserum was produced in rabbits (Rex white) by repeated subcutaneous injections of extract of D. monoceras-28th day emulsified with Freund's incomplete adjuvant (1:1). Initially, 1000 pl of suspension were administered and repeated after 14, 28 and 42 days. On the 50th day the rabbits were bled and the serum was separated.

Material and methods

Culture. The strain ICBUSP K-l-16; CBS 15426 of D. monoceras used in this study was maintained on potato agar with mineral oil; this strain has been used for production of allergenic extracts since 1973. Subcultures for the experiments were made on Czapeck broth (CB) as modified by Yunginger et al. [6] (NaNO3 2.0 g, K2HPO4 1.0 g, MgSO4 0.5 g, KC10.5 g, FeSO4 0.01 g maltose 15 g, dextrose 15 g, tryptone 10 g, distilled water 1000 ml, pH 6.8-7.0). Growth curve and morphological study. The inoculum was obtained from a 5-day-old colony on CB and the absorbance adjusted to 0.105-0.132 in a spectrophotometer at 550 nm. The growth curve was measured by dry weight, samples were taken every two days during the 30 days of the culture. The morphological study was done under the same conditions using macro and microscopic analysis [3] Extract preparation. The in0culum obtained from the growth curve study was added to five Roux flasks with 200 ml of CB. The fungal growth was interrupted by addition of thimerosal 1 : 10,000 on the 4th, 10th, 16th, 22nd and 28th day. The fungal mass was filtered and washed threefold with acetone, air dried for 48 hours at 37 °C and powdered. The extraction was made by addition of tris-HC1 0.05 M at a concentration of 10 mg dry weight per 100 ml of tris-HC1. The solution was maintained at p H 8, 4 °C, with agitation overnight and then centrifuged at 1,500 rpm for 10 minutes. The supernatant was dialysed against NH4HCO3 0.1 M pH 8.4 for 24 hours with three changes of the liquid [6]. Biochemical analysis. The extracts obtained from the 4th, 10th, 16th, 22nd and 28th day of the fungal growth were evaluated for total carbohydrate [7], protein [8],

Induction of IgE antibody responses. To study the induction of IgE antibodies by D. monoceras components, eight BALB/c and eight cAF-1 mice were immunized with the extract obtained from the 28th day of growth [13]. Current IgE sensitization was achieved by intraperitoneal injections of the antigen at day 0, 30 and 50. For the first injection, 0.5 ml of extract with 50 #g or 200 pg of protein were mixed with 7.5 mg of AI(OH)3. For second and third injections (30th and 50th day), the Balb/c mice received the same doses and the cAF-1 mice were injected with 1 #g or 5 Izg,respectively, of soluble extract without adjuvant. Blood was removed after 10, 20, 30, 40, 50 and 60 days. IgE titers in mouse sera were determined by passive cutaneous anaphylaxis (PCA) performed on Wistar rats [14]. The tests were done in triplicates; the rats were challenged with 700 #g of antigen in 0.25% of Evans blue. The reciwocal of the highest dilution giving a positive reaction (5 mm of diameter) was recorded as the PCA titer.

Results

The phases of D. monoceras growth are illustrated in Fig. 1. Until the 12th day of incubation only the presence of vegetative mycelia was verified. The conidial phase began on the 14th day of growth. The maximum total protein and lipid contents was observed at day 28th of growth and maximum total carbohydrates was obtained after day 16th (Table 1). The percentage of these components are listed in Fig. 2. The SDS-PAGE pattern for the extracts is illustrated in Fig. 3. The extracts obtained from 4th and 10th day of D. monoceras growth presented two bands of molecular weight approximately of 60 and 67 kD. On the extracts from 16th and 22nd day, five bands were

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identified - 18.5, 30, 36, 60 and 67 kD and on the extract from the 28th day seven bands were identified, the five mentioned and two bands of 14.4 and 20.1 kD. By chromatography of this extract, the amino acids glutamic acid, alanine, asparagine, cystein, phenylalanine, glycine, histidine, iso-leucine, leucine, methionine, proline, tyrosine, tryptophan and valine; and the carbohydrates glucose, mannose and galactose were

detected. No reactions were observed with the extracts obtained from the young cultures (4th and 10th days) against the hyperimmune serum. The other extracts (16th, 22nd and 28th days) presented four precipitation bands indicating different antigenic population and total identity (Fig. 4). To study the induction of IgE antibodies by D. r n o n o c e r a s components, cAF-1 mice immunized with

78

Fig. 3. SDS-PAGEof extracts obtained with tris-HCl from the 4th, 10th, 16th, 22nd and 28th days of Drechslera monoceras growth on Czapeck broth.

Fig. 4. Doublediffusion-in-gelof extracts obtainedfrom 4th, 10th, 16th, 22nd and 28th days of Drechslera monoceras growth againstrabbit hyperimmuneserum [8].

50 or 200/~g of extract proteins + adjuvant showed a low primary IgE response. This group of mice received a second and third dose with the soluble antigen and the response increased fourfold. In the group injected with the larger dose, the secondary response was

higher than the group injected with the smaller dose (Fig. 5A). In the Balb/c mice no primary response of IgE was observed, but the second and third doses of antigen + adjuvant increased the secondary (32 times)

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Kinetics of IgE antibody production. (a) In cAF- l mice immunized intraperitoneally with 50/~g or 200/~g of Drechslera monoceras extract + 7.5 mg of AI(OH)3; .L: Booster of 1 #zg (50 ~g (200/~g). (b) in Balb/c mice immunized with 50 fzg or 200 tzg of Drechslera monoceras extract + 7.5 mg of AI(OH)3; .L: Booster with the same dose of immunization plus adjuvant.

and the tertiary responses (10-32 times) of IgE (Fig. 5B).

Discussion There are no studies in the literature on the antigens and allergens o f D r e c h s l e r a m o n o c e r a s . Previous work with skin testing has demonstrated sensitivity to D.

80 Table 1. Total protein, carbohydrateand lipid contents of Drechsleramonoceras extracts obtainedfrom extraction with tris-HC1 accordingto growth time on Czapek broth

Growth time

Proteins Carbohydrates Lipids mg/ml mg/ml mg/ml

4th day 10th day 16th day 22nd day 26th day

0.12 0.66 1.65 1.16 2.20

0.10 0.56 0.70 0.70 0.66

0.27 0.27 0.27 0.13 0.54

monoceras in asthamatic patients in Sao Paulo city [21. The characterization of allergenic fungal extracts has been difficult because of variations in culture media, growth time, and extractor, among other factors [15]. The purpose of this work was to establish basic conditions for preparation of D. monoceras extract and future fractionation, characterization, and standardization for utilization in the diagnosis and irnmunotherapy of allergies caused by this fungus. The culture medium used was based on studies with Alternaria alternata [61 and the extraction with trisHC1 has been utilized with good results in the preparation of allergenic extract s from other fungi [ 13, 16-19]. From the growth curve data and morphological study of D. monoceras, five periods were chosen. For the preparation of extracts: 4th day (vegetative and exponential phase); 16th day (vegetatiye and steady phase); 10th and 22nd day (conidial and steady phase) and 28th day (conidial and decrease phase). Total carbohydrates and proteins increased with growth time, specifically from the conidial and steady phase. The results reported by other authors in relation to growth patterns of fungi are contradictory because results depend on several factors. In 5-week-old Cladosporium herbarum it was verified the relation of 45 to 73% of carbohydrates and 55 to 27% of proteins [20]. In Paracoccidioides brasiliensis, the carbohydrate contents diminished and the protein contents increased in relation to the time of growth [21]. The higher protein content in the present study was obtained on the 26th day extract with a relation of 15% carbohydrates and 75% proteins (Fig. 2). A larger number of protein bands on SDS-PAGE, including the bands with low molecular weight also were observed with this 28th day extract.

A rabbit hyperimmune serum prepared against this extract reacted with four different antigens present in detectable concentrations as measured by immunodiffusion. This was also verified with the extracts obtained on the 16th and 22nd day, but not with the extracts of the 4th and 10th days. This fact was reported in other studies in which it was demonstrated that extracts from cultures 4 or 5 weeks old are more reactive in an immunodiffusion test than from young cultures 4 or 5 weeks old are more reactive in an immunodiffusion test than from young cultures [22-24]. The chromatography outline of carbohydrates varied with the culture time. On the 16th and 22nd days it was found that the sugars glucose, galactose and mannose were similar to the results observed with Cladosporium herbarum extracts from cultures 21 days old [20]. On the 10th and 28th days the presence of mannose was not verified. This variation can only be clarified with further investigations. Based on the high protein content, we chose the extract from the 28th day for the induction of IgE antibody responses in mice. To compare the results with other fungi [13, 25] we chose the Balb/c strain and included a cAF-1 hybrid. The primary response of these hybrid mice was better than that observed with Balb/c, but after the booster with adjuvant, the response of the latter increased more than the former. Bouziane et al. [13], in a similar investigation with the fungus Cladosporium cladosporioides, obtained a titer of 160 in Balb/c mice. Using the same protein dose, we achieved a difference of eightfold in the IgE antibody response on day 60 (1280), which suggests that the Drechslera rnonoceras strain used in this study is a stronger inductor of IgE than C. cladosporioides under the same conditions. Many authors have reported that allergenic fractions of fungi extracts are polypeptides of low molecular weight [6, 19, 26, 27]. As we demonstrated by SDS-PAGE, the 28th extract contained at least four low molecular weight components. The data obtained under the established conditions concerning the culture medium, growth time, and extractor suggest the utilization of this extract for future studies, aiming at the fractionation, characterization and standardization of diagnostic mad immunotherapy methodologies.

References

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Address for correspondence: W. Gambale, ICR/USE Departamento de Microbiologia, Av. Prof. L Ineu Prestes 1374, 05508-900 S~o Paulo, Brazil.