J. Biosci., Vol. 3 Number 1, March 1981, pp. 77-82. © Printed in India. Animal model for testing human Ascaris allergens

KRISHNA MUKERJI*, R. P. SAXENA, S. N. GHATAK and K. C. SAXENADivision of Biochemistry, Central Drug Research Institute, Lucknow 226 001 MS received 26 March 1980; revised 11 October 1980. Abstract. Guinea pigs immunized through the nasal route by an antigenic preparation ofhuman Ascaris lumbricoides produced predominantly homocytotropic antibodies. The sensi-tization of the homologous skin required a latency period. Hypersensitivity reaction was triggered within 30 min. of the antigen challenge. The antibodies were sensitive to heat and β–mercaptoethanol treatments and appeared to be similar to the IgE type of immunoglobulins of man and rabbit. The antigenic preparation elicited an immediate type of skin hypersensitivity reaction also in human subjects harbouring the Ascaris parasite. The guinea pig modelappears suitable for testing the activity of Ascaris allergens. Keywords. Ascaris lumbricoides; homocytotropic antibodies; IgE immunoglobulins; guineapig.

Introduction

Incidence of hypersensitivity to Ascaris antigens has been observed among thelaboratory workers infected by the worm (Sadun, 1972). During the purification of trypsin (Mukerji et al., 1976) and chymotrypsin (Mukerji et al., 1977) inhibitors from Ascaris lubricoides var. hominis in this laboratory, some of the workersshowed immediate hypersensitivity reaction to the trichloracetic acid-soluble preparation of the crude inhibitors. Attempts were made to purify the allergenic factor and an animal model was developed to test the allergen. The details of this model are described in this paper. Materials and methods Collection of worms and extraction of allergens

Ascaris lumbricoides var. hominis were collected from both in-patients and out-patients of King George's Medical College, Lucknow, and were processed accord- ing to the method described earlier (Mukerji et al., 1976). The trichloroacetic acid- soluble material and the first peak, obtained by gel filtration through Sephadex-G-75 (Mukerji et αl., 1976) were used as the source of allergens for standardization ofthe animal model. Trichloroacetic acid was removed by extensive dialysis againstdistilled water (500 ml changes every 4 h) at 4°C. * Present address: Indian Institute of Experimental Medicine, Jadavpur, Calcutta 700 032.

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78 Mukerji et al Immunization schedule Earlier studies (Pathak, 1977) from this laboratory reported that the guinea pig wasa suitable model for testing the activity of the allergens isolated from the pollens of Chenopodium album. The same animal species was used in the present study.Male guinea pigs (250-300 g) were administered 0.1 ml of allergen solution (2 mg/mlin 150 mM NaCl) in each of the nostrils by a dropper. The dose was repeated every 4th day till 6 such doses were given. The animals were bled one week after the administration of the last dose, serum was collected, inactivated to destroy comple- ment and stored at — 10°C. A group of animals was also immunized by the sameschedule using a subcutaneous route.

Passive skin tests

Normal guinea pigs were sensitized by intradermal administration of 0.1 ml of the immunized guinea pig serum on preshaven skin. After 24 h the animals werechallenged with the antigen at the sensitized site and the area of wheal formed wasmarked after 30 min unless otherwise mentioned, and the area measured. Thearea of wheal formed in the unsensitized guinea pig skin (diameter 10 mm) was subtracted from the experimental reading.

Skin tests in Ascaris positive subjects

Skin test was performed on human subjects, who were infected with Ascaris asdetermined by stool examination, by intradermal injection of 0.05 ml allergen solution (10 µg/ml protein) in the forearm and the area of the wheal was measured.

Heat stability of antibodies

Sera of the immunized animals were heated for 2 h at 56°C followed by immediatecooling in crushed ice.

β-Mercaptoethanol treatment

The antibodies were treated with β-mercaptoethanol essentially according to the method of Dobson et al. (1971). Serial dilutions of the immune serum were incubated with equal vol. of 200 mM β-mercaptoethanol at 37° C for 1 h followed by dialysis against 100 vol of 200 mM iodoacetamide for 24 h. The material was further diaiyzed against 3 changes of (100 volumes) phosphate buffer saline (100mM phosphate buffer, pH 7.2; 150 mM NaCl).

Precipitating and agglutinating antibodies

Detection of precipitating and agglutinating antibodies was made by immunodiffu-sion in agar and by the indirect haemagglutination method respectively asdescribed by Kabat and Mayer (1961).

Results

Antibody response in guinea pigs immunized by the nasal and subcutaneous routes

Guinea pigs immunized by the nasal route exhibited a strong skin reaction within30 min of the antigen challenge. However, precipitating and agglutinating anti-bodies were not detected in the sera of these animals. Animals immunized by thesubcutaneous route did not display significant hypersensitivity reactions following

Model for Ascaris allergens 79 the antigen challenge. The presence of precipitating antibodies also could not be seen in the sera of these animals. However, by indirect haemagglutination a weak titre of antibodies (½) could be seen. Characterization of the cytotropic antibodies

Homocytotropic nature

Table 1 shows the data on skin test in guinea pigs passively sensitized with theserum from immunized animals. Strong reaction was observed upto 1/16 dilution of serum. The response was reduced at subsequent dilution but was stilldetectable at 1/128 dilution.

Table 1. Antibody titre of guinea pig serum immunized by the nasal route

a Antisera from 3 guinea pigs were pooled. Each value is the mean of skin tests in two different

animals. The area of skin raised due to injection of 0.15 Μ NaCl has been substracted from the experimental readings.

Time course of optimal binding of antibodies

Guinea pigs sensitized with a constant volume of the antiserum were challenged at different periods after the passive transfer. Significant skin reaction was not visible before 6 h of the antibody transfer (table 2). The response was better after 24 h and was maintained till the end of the observation period (72 h).

Table 2. Sensitization of homologous skin as a function of time

Each value is the mean of two skin tests in two different animals.

80 Mukerji et al Onset and duration of hypersensitivity reaction

Table 3 presents the data on skin reactions measured at different periods after theallergen challenge in the passively sensitized animals. Skin reaction of a fairlylarge magnitude appeared within 30 min of the challenge with a slight increase in the value in the subsequent period up to 3 h. A gradual decrease in the wheal area was noticed by 12 h and the reaction subsided by 24-48 h.

Table 3. Onset and duration of hypersensitivity reaction

a Each value is the mean of two skin tests in two different animals.

Sensitivity of the antibodies to heat and β-mercaptoethanol

Significant reduction in the skin reaction was observed after heating the antiserumat 56°C for 2 h or its treatment with β-mercaptoethanol (table 4). Later treatmentresulted in complete inactivation at 1/64 dilution.

Table 4. Sensitivity of antibodies to neat and β-mercaptoethanol treatments

Skin response in Ascaris positive subjects

With a view to ascertain if the antigenic fraction of human Ascaris that elicited immediate hypersensitivity response in the sensitized guinea pigs also produced comparable response in human subjects, the skin test was performed in individuals who were positive to Ascaris by stool examination. All the cases examined gave a skin reaction of strong or medium grade upon challenge with 1 µg antigen protein (table 5). Ascaris negative patients, however did not show any response.

Model for Ascaris allergens 81

Table 5. Skin test with human Ascaris antigen(s) in Ascaris positive and Ascaris negativesubject

a Each value is mean of two determinations on the same patient.

Discussion

The immunization schedule followed in the present study succeeded in eliciting afailry high titre of homocytotropic antibodies in guinea pigs immunized by thenasal route. The precipitating antibodies of IgG class which might suppress the synthesis of reagenic antibodies and interfere with the hypersensitivity reaction by interacting with the allergen, thereby preventing it from reacting with the sensitized mast cells, were not formed by this schedule.

The observations on the requirement of a latency period for sensitization of homologous skin, persistence of sensitization for a few days, appearance of the skin reaction within 30 min of the antigen challenge and a substantial recovery by 12 h provide confirmatory evidence in favour of the homocytotropic nature of the antibodies that give rise to the immediate type of hypersensitivity reaction (Dobson et al., 1971; Hussain et αl., 1973). Guinea pig antibodies similar to the IgE type of human antibodies are characterized by their sensitivity to heat and β-mercapto- ethanol (Dobson et αl., 1971). The data of the present study on heat and β- mercaptoethanol treatment suggest a fairly good concentration of IgE type of anti- bodies in the sera of immunized guinea pigs. A small amount of homo-cytotropic antibodies belonging to 7Sr2 type (Hussain et αl., 1973) were also present as the reaction continued to increase till 3 h.

The data on skin reaction in Ascaris-positive human subjects show that the antigen fraction which elicits a hypersensitivity reaction in guinea pigs also triggers the skin response in man. Guinea pigs sensitized with the human Ascaris antigen, therefore, are a satisfactory model for testing human Ascaris allergens.

Experimental hypersensitivity to A. suum antigens has been produced in the guinea pig, by injecting embryonated eggs (Strejan and Campbell., 1967 a, b) or repeated administration of Ascaris extract by intramuscular or subcutaneous routes (Strejan and Campbell, 1970, 1971). Immunization under these conditionsrequires longer time and a larger quantity of the antigen. Moreover, in addition to the skin binding antibodies the sera of the immunized animals also contain an appreciable titre of precipitating antibodies which may interfere with the skin reaction.

82 Mukerji et al

Human Ascaris allergen appears to be a glycoprotein. Studies on its characteri-zation will be reported elsewhere.

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