Bronchoprovocation studies in basidiospore-sensitive allergic subjects with asthma

Bronchoprovocation basidiospore-sensitive with asthma Manuel John

Lopez,

MD, Juliet

E. Salvaggio,

studies in allergic subjects

R. Voigtlander,

MD New Orleans,

BS, Samuel

B. Lehrer,

PhD, and

La.

Bronchoprovocation challenge with basidiospore extracts was performed in eight subjects with asthma and with positive wheal-and-flare skin reactivity and RAST to basidiospore allergens. Five of the eight patients demonstrated a signiJicant decrease in FEV, ranging from 20% to 47% after basidiospore-extract challenge. Both immediate and late-phase reactivity was observed. Six atopic control subjects with asthma and with negative skin reactivity to basidiospore extracts did not exhibit sign$cant bronchospasm after basidiospore challenge. These results demonstrate that basidiospore extracts can induce bronchospasm in subjects with asthma who demonstrate IgE antibodies to basidiospore allergens. (J ALLERGY CLIN IMMUNOL 1989;84:242-6.)

Basidiomycetes, the most morphologically advanced of the fungi, include bracket fungi, rusts, smuts, mushrooms, and puffballs. The class basidiomycetes is estimated to contain between 20,000 and 25,000 species, and these fungi are a very important component of the air-spore load. Gregory and Hirst’ reported high atmospheric concentrations of basidiospores between June and September in the area of Harpendend, England. In Cardiff, Wales, basidiospores constituted 29% of the spore catch, compared to 25% for Cladosporium .2 Davies et al. 3 reported that basidiospores and ascospores comprised 54% of the total spore catch at Liverpool and 29% at London. In the United States, Kramer et aL4 reported that in Kansasbasidiospores comprised 24.3 % of the spore catch, second only to Cladosporium spores. We have noted that asthma “epidemics” of considerable magnitude occurring during the late summer and fall months were associated with high total spore and basidiospore catches.5. 6 Since the atmospheric concentrations of these spores is high in many areas of the world, they are of considerable interest as potentially important allergens. In previous studies we have demonstrated that a significant number of patients in New Orleans with

From the Allergy and Clinical Immunology Section, Department of Medicine, Tulane University School of Medicine, New Orleans, La. Received for publication May 4, 1988. Revised Dec. 20, 1988. Accepted for publication Jan. 27, 1989. Reprint requests: Manuel Lopez, MD, Tulane Medical School, 1430 Tulane Ave., Room 7209, New Orleans, LA 70112. l/1/13023

2

PBS: PD,,:

Phosphate-buffered saline ProvocatIve dose of methachohne

causing a

a history of rhinitis and/or asthma have positive wheal-and-flare skin reactivity’.’ and basidiosporespecific IgE antibodies to extracts from basidiospore allergens. To demonstrate a cause-and-effect relationship between these allergens and asthma, bronchoprovocation challenge studies were performed in selected subjects with asthma who demonstrated skin reactivity and RAST to basidiospore extracts. MATERIAL Subjects

AND METHODS

All study subjects had a clinical history compatible with bronchial asthma and required conventional bronchodilators for asthma control. Reversibility of bronchospasm (15% increase in FEV, over baseline) was demonstrated in all subjects after two inhalations of albuterol. Group I was composed of eight patients selected on the basis of history of asthma, positive prick skin tests (defined as a 2 mm wheal greater than wheal of the negative saline control) to two or more common inhalant allergens, positive methacholine challenge (PD,,, < 10 mg/ml of methacholine concentration) and positive prick skin tests to basidiospore extracts. Group II was composed of six atopic subjects with asthma with negative prick skin tests to basidiospore extracts, positive prick skin tests to two or more common allergens, and positive methacholine challenge. All subjects gave informed consent before inhalation challenge.

VOLUME NUMBER

Challenge with basidiospores

84 2

TABLE I. Basidiospores inhalation challenge results

Subject

RAST ratio

Il.4

I

6 19 12

2 3

23

Final concentration

4

0.0001 0.0000001

Psrlot~yhe

0.0001 0.000 I 0.001

Coprinus P,siloc~he Ganoderma Psilocvhe

quadri,fidus cuhetw5 lucidum cuhensis

Psilocphe Pleurotus Coprinus Coprinus Psilot:\ te Coprinus

c3henst.s o.weatus quadrijdus quadrifidus cuhatws quadri’dus

1.4

0.001

6

11.0

0.00001

I

1.0 26 19

0.001 0.0001 0.00001

8

Basidiospore extracts Patients were challenged based on skin test results with soluble aqueous basidiospore extracts obtained from Pleurotus

ostreatus,

Coprinus

quadrifidus,

Ganoderma

lucidum,

or Psilocybe cubensis. Extracts were prepared by a method previously described.’ Briefly, basidiomycete caps were placed. gills or pores down, on Whatman No. 1 filter paper (Whatman Inc., Clifton, N.J.) or wax paper and covered with aluminum foil to obtain prints from which spores were scraped with a spatula. Identification or spore preparations, based on color, size, and shape, was confirmed by light microscopy. To prepare extracts, spores were homogenized in a Braun homogenizer (Infors AC, Basel, Switzerland) in 20 ml of ammonium bicarbonate buffer (0.125 moliL, pH 8.1); the homogenate was centrifuged at 48,000 g. The supematant was lyophilized, and the extract was stored at room temperature under desiccation. All extracts were tested for toxicity in female Swiss-Webster mice by the Ames test.

End point titration

cuhen.si.5

Ganoder-mcl lucidum

0.001

5

(mglml)

Extract

0.001

skin test

To determine the initial dose of extract for inhalation challenge, end point skin testing was performed with lyophilized spore extracts suspended in PBS (1 mgiml) material. Tenfold dilutions in PBS of the extracts were freshly prepared on a daily basis. Intradermal skin testing was performed in the upper arms by injecting 0.05 ml of extract dilutions at 15-minute intervals. The end point was defined as the highest extract dilution producing an 8 to 10 mm average diameter wheal and 20 to 29 mm average diameter erythema. PBS was used as a negative control, and histamine. 0.1 mg/ml, as a positive control.

RAST RAST was performed by a method previously described.” Briefly, for coupling to RAST disks, spore extracts were adjusted to 10 mgiml in pH 8.0 borate buffer and coupled to CBNr-activated filter paper disks. For the test, 100 ~1 of patient serum was incubated overnight with a test disk. After

243

-.-__

End point skin test

(mg/ml)

in asthma

FE’.‘, % decrease

i 1). I 3 0.1 i). I 0.1

17 31 24 29

0. I 5

27 0

5

15

5 5 I

9 0 20

24 25

disks were washed, they were incubated overnight with 100 p.1 of “‘I-labeled anti-IgE antiserum, washed three times with saline, and counted in a gamma counter. A RAST binding ~3% of total activity added was considered positive: this value was >3 SD from mean percent binding obtained with atopic control sera from subjects skin test negative to basidiospores.

Bronchial challenge Inhalation challenges were performed according to the method described by Chai et al.“’ Patients were not challenged unless the FEV, was at least 60% of predicted value on the day of the methacholine challenge or 70% on the day of allergen challenge. All short-acting medications were stopped for at least 12 hours and sustained-release medications for at least 24 hours before challenge. Aerosols were generated with a DeVilbiss No. 46 (DeVilbiss Co., Somerset, Pa.) nebulizer attached to a Rosenthal-French (The Johns Hopkins University, Baltimore, Md.) dosimeter. Aerosols were delivered for 0.6 seconds at 20 pounds of pressure during slow inspirations, starting from functional residual capacity. Pulmonary function measurements were performed on a Jones Datamite III spirometer (Jones Medical Instrument Co., Oak Brook, Ill.). and the best of three efforts was recorded.

Methacholine

challenge

On the testing day, a baseline FEV, had to be within 60% of the predicted value. A control FEV, was obtained after five inhalations of PBS. If the postsaline FEV, was within 10% of the baseline value, the patient received five inhalations at IO-minute intervals of increasing methacholine concentrations with the sequence 0.039. 0.156, 0.625, 2.5, and 10 mg / ml. and the PD2, was determined

Basidiospore-extract

challenge

Initial control FEV, was obtained after five inhalations of saline. If the control FEV, was within 10% of presaline baseline FEV, (at least 70% of predicted value), the patient

244

Lopez

et al.

J. ALLERGY

ALLERGEN: Psrkxybs cubensrs END POINT SKIN TEST I 0.0001 RAST = 14.8

CLIN. IMMUNOL. AUGUST 1989

ALLERGEN:Psrlocybe cubensis END POINT SKIN TEST E 0.0001 mglml RAST I 11.4

mglm,

.‘\i


.OOOl ALLERGEN

FIG. 1. Dose response of baseline postsaline

mcovwcd (mg/ml)

,001 .Ol CONCENTRATION

to basidiospore challenge.

1 ,001 .Ol .I ALLERGEN CONCENTRATION

extract

challenge

in four

ALLERGEN:

ALLERGEN:Ganoderma lucidurn END POINT SKIN TEST E 0.001 mg/ml RAST : 8

,001

.Ol

ALLERGEN

FIG. 2. Dose response of baseline postsaline

.l

CONCENTRATION

to basidiospore challenge.

patients.

FEV, reported

PSi/OCybe

R&XlYe,Xl

.OOl ALLERGEN

challenge

received five inhalations at lo-minute intervals of serially tenfold increasing concentrations of basidiospore extracts. The starting allergen concentration for each patient was calculated at lOO-fold higher than the end point skin test results. Doses were continued until PD,, was observed or a concentration of basidiospore extract of 5 mg/ml was reached. The criteria for positive challenge was established as PD,, or more from the postsaline control value. Patients were observed for 5 hours after challenge for safety precautions.

RESULTS The two groups of subjects with asthma had a similar degree of nonspecific airway hyperreactivity. Mean PD*,, was 0.78 mg/ml for the control group and 1.10 mg/ml for the basidiospore-sensitive group. No control subject with asthma demonstrated significant bronchial response to basidiospore extracts up to a concentration of 5 mg/ml. Results of end point skin tests, RAST, and bronchial challenge in the basidiospore-sensitive subjects with asthma are summarized in Table I. Twelve challenges were performed. Eight challenges were positive and four negative. Five of the eight patients demonstrated

in four

.Ol

CONCENTRATION

patients.

as percent

Cubf?nSiS

END POINT SKIN TEST I 0.001 RAST q 10.7

(mg/ml)

extract

R*Cc.“*rCd (mglml)

mg/ml

.l (mg/ml)

FEV, reported

as percent

a drop in FEV, ranging from 20% to 47% after inhalation challenge. The dose response in four basidiospore-sensitive subjects with asthma is illustrated in Figs. 1 and 2. One subject (Fig. 3) demonstrated a significant late reaction approximately 3 hours after the immediate response. All subjects recovered without prolonged bronchospasm need for steroids or hospitalization. DISCUSSION Studies of basidiospores as aeroallergens are important in view of their high atmospheric concentration during the summer and fall’, 6. ‘. ‘I. I2 and the association of these concentrations with asthma epidemics in the city of New Orleans.6 Our current results demonstrate that basidiospore extracts can induce immediate bronchospasm in subjects with asthma with positive skin tests to basidiospore allergens but not in atopic subjects with asthma with negative skin reactivity to basidiospores. One patient also demonstrated a significant late-phase response after immediate response, but no attempts were made to optimize the conditions for late-phase responsiveness. Further stud-

Challenge

VOLUME534 NUMBER2

ALLERGEN: Psilocybe cubensis DOSE: 0.1 mg/ml POST CHALLENGE FEVl

0

lh

2h

3h

4h

TIME FIG. 3. Inhalation challenge time as percent of baseline postsaline

course in a patient. challenge.

FEV,

ies are needed to evaluate the incidence of late-phase asthmatic reactions to basidiospores. Although it has been demonstrated that an atmospheric concentration of basidiospores is high in many areas of the world, the role of these fungi in respiratory allergic diseases has been difficult to study. In contrast to fungi from the imperfecti class, basidiomycetes grown in vitro on synthetic media lack tertiary structure and cannot sporulate. Thus, a major difficulty in studying basidiomycete allergy has been the lack of sufficient quantities of basidiospores to prepare extracts. To overcome these problems we have developed methods for collection of spores from basidiomycetes growing in our environment. Spores from a number of basidiomycetes growing in the Gulf South regions have been identified and collected. With allergens derived from these species, we have demonstrated that 32% of a group of atopic subjects with respiratory allergic disease tested demonstrated positive skin reactivity by prick test to one or more basidiospore species. The prevalence of skin reactivity exceeded that to extracts from species of the fungi imperfecti class, suggesting that those were among the fungal species eliciting more allergic reactivity.” Similar results were obtained with the same basidiospore extracts in Seattle, Wash.13 Studies by Santilli et al. I4 in Connecticut and Giannini et al. I5 in Arizona have also demonstrated significant skin reactivity to spore extracts from basidiomycetes. In addition, up to 22% of atopic patients skin tested with extracts from basidiospores collected in the wild in New Zea-

with

basidiospores

in asthma

245

land had positive prick tests,” and Cutter et al.” reported incidence of 28% positive skin test to the basidiospore Gunoderma in 115 subjects with asthma. A major question is the relationship between basidiospore sensitivity demonstrated by skin test and RAST and respiratory symptoms. In contrast to pollen allergens, Bruce” reported skin sensitivity could not serve as a predictor of bronchial responsiveness in a group of subjects with asthma sensitive to fungal spores. Perhaps the differences in those allergens can be attributed to differences in allergenic extracts used and degree of sensitivity of the study populations. Studies by Herxheimer et al.‘” demonstrated that in 14 patients with summer asthma exacerbations, basidiospores likely played an important precipitating role. In the United States, it is known that basidiospores are important components of the air-spore load and that a significant percentage of allergic subjects with asthma have IgE antibodies against basidiospore allergens. Little is known, however, regarding the bronchial response to basidiospores in basidiosporesensitive subjects with asthma. To our knowlege, no previous inhalation challenge studies with basidiospore extracts have been reported in this country, and our current data now suggest that basidiospores are clinically important aeroallergens in production of IgE-mediated bronchial asthma. Since the study was performed in a limited number of subjects with asthma, our data do not provide information regarding the prevalence of airway responsiveness in basidiospore-sensitive subjects with asthma. Further investigations with larger numbers of patients are necessary to provide definitive information on the prevalence of basidiospore-induced respiratory allergy. REFERENCES I.

7li.

3.

4.

5.

6.

7

Gregory PH, Hirst JM. The summer axspore at Rothamsted in 1952. J Gen Microbial 1957;17:135. Hyde HA, Adams KF. Airborne allergens at Cardiff 1942-59. Acta Allergy 1960;15(suppl 7):159. Davies RR, Denny MJ, Newton LM. A comparison between the summer and autumn air-spore at London and Liverpool. Acta Allergol 1963;18:131. Kramer CL, Pady CM, Rogerson CT, et al. Kansas aeromycology. II. Materials, methods, and general results. Tran Kansas Aca Sci 1959:62:184. Salvaggio JE. Seabury J. New Orleans asthma. IV. Semiquantitative airborne spore sampling. 1967 and 1968. J ALLERGY CLIN IMMUNOL 1971;48:82. Salvaggio JE, Seabury J, Schoenhardt E. New Orleans asthma. V. Relationship between Charity Hospital asthma admission rates, semiquantitative pollen, fungal spore counts, and total particulate aerometric sampling data. J ALLERGY CLIN IMMUNOL. 1971:48:96. Lopez M. Salvaggio J, Butcher B. Allergenicity and immunogenicity of basidiomycetes. J ALLERGY CLIN IMMUNOI. 1976:57:480.

Lopez et al.

J. ALLERGY

8. Lehrer SB, Lopez M, Butcher BT, et al. Basidiomycete mycelia and spore-allergen extracts: skin test reactivity in adults with symptoms of respiratory allergy. J ALLERGY CLIN IMMUNOL 1986;78:478. 9. Butcher BT, O’Neil CE, Reed M, et al. Basidiomycete allergy: measurement of spore-specific IgE antibodies. J ALLERGY CLAN IMMUNOL 1987;80:803. 10. Chai H, Farr RS, Sheffer AL, Spector SL, Townley RG: Standardization of bronchial inhalation-challenge procedures. J ALLERGY CLIN IMMUNOL 1975;56:323. 11. Gregory P, Hirst J. Possible role of basidiospores as airborne allergens. Nature 1952;170:414. 12. Adams K, Hyde H, Williams D. Woodlands as a source of allergens with specific reference to basidiospores. Acta Allergo1 1968;23:265. 13. Sprenger JD, Altman LC, O’Neil CE, et al. Skin test reactivity to basidiospores (BS) in adults in Seattle with respiratory allergies [Abstract]. J ALLERGY CLIN IMMUNOL 1986;77:200.

CLIN. IMMUNOL. AUGUST 1989

14. Santilli J, Rockwell WJ, Collins RP. The significance of the spores of the basidiomycetes (mushrooms and their allies) in bronchial asthma of and allergic rhinitis. Ann Allergy 1985; 55:469. 15. Giannini EH, Northey WT. Leather CR. The allergenic significance of certain fungi rarely reported as allergens. Ann Allergy 1975;35:372. 16. Hashain SM, Wilson JD, Newhook FJ, Segedin BP. Allergy to basidiospores: immunologic studies. NZ Med J Sci 198.5; 98:393. 17. Cutter AEC, Hasnain SM, Segedin BP, et al. The basidiomycete ganoderma and asthma: collection, quantitation, and immunogenicity of the spores. NZ Med J 1988;101:361. 18. Bruce RA. Bronchial and skin sensitivity in asthma. Int Arch Allergy 1963;22:294. 19. Herxheimer M, Hyde HA, Williams DA. Allergic asthma caused by basidiospores. Lancet 1969;2:3 13.

Allergen-specific induction of interleukin-2 (IL-2) responsiveness in lymphocytes from children with asthma 1. Antigen specificity the induction

and initial

events

of

lzumi Yoshizawa, BS, Takeshi Noma, MD,* Yutaka Kawano, MD,* and Junichi Yata, MD** Saitama and Tokyo, Japan Interleukin-2 (IL-2) responsiveness of Demratophagoides farinae (Df)-stimulated lymphocytes from children with bronchial asthma was studied. Six-day culture of lymphocytes from allergic patients increased after an additional 3 days of incubation with recombinant IL-2. This phenomenon was not observed when the lymphocytes of patients allergic to Df were stimulated with ovalbumin (OVA). Normal lymphocytes stimulated with Df expressed Tat antigen (low-a&%&y IL-2 receptor) but, in contrast to the patients’ lymphocytes, did not absorb nor respond to IL-2. Nonadherent responder cells cultured with Df-pulsed autologous adherent cells acquired IL-2 responsiveness, but those cultured with OVA-pulsed adherent cells did not. The monoclonal antibody to HLA-DQ f ramework (Leu IO and clonab DQ), but not to HLA-DR framework (OKIaI) and HLA-DP (HLA-DP and clonab DP-DR), blocked the antigen-presenting cells from inducing IL-2 responsiveness. Nonadherent responder cells depleted of OKT4 (CDI)-positive cells failed to acquire IL-2 responsiveness, whereas depletion of OKT8 (CD8) cells had no impact. Taken as a whole, the results indicate that De-bearing adherent cells from allergic donors play a key role in presenting Df antigen to allergen-spectfic responder T cells, which are very likely to be members of the OKT4 positive subset. (J ALLERGY CLIN IMMUNOL 1989;84:246-55.)

From the Central Research Laboratory diatrics, National Defense Medical **Department of Pediatrics, Tokyo sity, Tokyo, Japan. Reprint requests: Takeshi Noma, MD, Medical School, 38 Morohongo, tama 350-04. Jauan. l/1/13405

L

246

and *Departments of PeCollege, Saitama, Japan, and Medical and Dental UniverDept. of Pediatrics, Saitama Moroyama, Iruma-gun, Sai-

The presenceof allergen-sensitizedlymphocytes in allergic patients has been demonstrated by in vitro proliferaiive responses’-6 andIL-2 production7by lymphocytes stimulated with antigen. We previously reported an allergen-specific acquisition of IL-2 responsivenessin patients with hen-egg allergy after