Int. Archs Allergy appl. Immun. 55: 78-81 (1977)

IgE-Type Antibodies to Ascaris Antigens in Man Moriyasu Tsuji, Takaharu Hayashi, Shoso Yamamoto, Yasukazu Sakata and Takashi Toshida Departments of Parasitology anti Dermatology, Hiroshima University School of Medicine, and Clinical Department of Oto-rhino-laryngology, Hiroshima Prefectural Hospital, Hiroshima

Many studies have demonstrated that Ascaris antigens produce reaginic antibodies in animals [2, 10] and primates including man [3, 8, 14]. It has been reported that Ascaris suum infections in guinea pigs also induce the production of IgE-type antibod­ ies [2], Although the elevation of IgE globu­ lin levels has been observed in Ascaris-in­ fected patients [7], no in vitro direct evi­ dence for the presence of IgE antibodies to Ascaris antigens in man has been reported so far. Crude Ascaris antigen was therefore pre­ pared from hog A. suum body fluid ob­ tained by puncturing the posterior end of the worms. The pooled fluid was filtered with filter paper, dialyzed against distilled water for 48 h at 4 °C, and lyophilized. Crude Ascaris antigen was fractionated by

Sephadex G-200 column chromatography. Three peaks were obtained and each peak was pooled as indicated in figure 1 (frac­ tions I, II and III). Each fraction was di­ alyzed against distilled water for 48 h at 4 C, lyophilized and stored at -20 C until used. Reaginic serum to Ascaris antigens was obtained from one of the authors (T. H., 40 years of age). He had suffered from Ascaris infection and been treated at 10 years of age. Since then, he had no evidence of reinfection of Ascaris. At 35 years of age, he did skin window experiments [11] to in­ vestigate the eosinophil chemotactic ability of Ascaris antigens, in which approximately 100 //g of crude Ascaris antigen was applied 3 times on his scratched skin at intervals of 1 month. 2 weeks after the last application of

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Abstract. IgE antibodies to Ascaris antigens were detected by radioallergosorbent test (RAST) in the serum obtained from a person experimentally sensitized with Ascaris suum antigens prepared from the body fluid of worms. This serum passively sensitized human skin in vitro and histamine release from the sensitized skin was observed by challenge with antigens. The allergenicity of A. suum antigens was present over a very broad spectrum of molecular size. The present results suggest that specific IgE antibodies to Ascaris antigens might be in /Hcam-infected patients and allergic symptoms which belong to type 1 reaction might be mediated by IgE in these patients.

Fraction II

Fig. I. Fractionation of crude Ascaris antigen by Sephadex G-200 column chromatography. The crude Ascaris antigen was dissolved in 0.05 M NaCl at the concentration of 140 mg/ml and 2 ml of the sample was applied to a 2.6 X 45 cm column of Sephadex G-200 which had been equilibrated with 0.05 M NaCI. The col­ umn was eluted with 0.05 M NaCl at a flow rate of 10 ml/h and 4-ml fractions were collected. Each peak was pooled as indicated in the figure.

A scans antigen with the skin window tech­ nique, skin test was carried out by injecting the same Ascaris antigen (10/tg/ml) into his skin intradermally, and strong positive reac­ tions were observed. 2 months after the skin test, blood was taken and the serum was stored at -20 °C until used. The serum was used within 1 week for the following experi­ ments. To detect IgE-type antibodies to Ascaris antigens in the reaginic serum, radioallergosorbent test (RAST) was carried out essen­ tially by the method of Foucard et al. [4], Ascaris antigens (crude antigen, fractions I, II and III) were coupled to CNBr-activated filter paper discs by the procedure of Ceska et al. [1], As shown in table I, mean ra­ dioactivity of 12SI-labelled anti-IgE bound to the discs coupled with the crude antigen, fractions I, II and III was 1,140, 1,632, 1,069 and 604 cpm, respectively. That of

nonantigen-coupled control discs was 479 cpm. The specificity of the uptake was confirmed by control experiments in which nonatopic normal serum was used in place of the reaginic serum. No significant differ­ ence between radioactivity bound to antigen-coupled discs and that to nonantigencoupled discs was observed. Mean uptake of radioactivity by each disc was less than 437 cpm in the control experiments. These results suggest the existence of IgE-type an­ tibodies to Ascaris antigens in the reaginic serum obtained from /Iscam-antigen-sensitive person. To confirm this, antigen-evoked histam­ ine release from human skin passively sensi­ tized with the reaginic serum was carried out by the method described in detail else­ where [5]. The previous study has indicated that antigen-evoked histamine release from passively sensitized human skin might be mediated by IgE in reaginic serum [5], Hu­ man skin slices were sensitized with the re­ aginic serum (1:5 dilution) and challenged with the crude antigen, fractions I, II and III (100 /ig/ml). As shown in table I (exp. 1), significant histamine release from the sensi­ tized skin slices was obtained by the crude antigen, fractions I and II. In experiment 2, the small, but significant, histamine release by fraction III was also observed. The small release might be due to the use of the skin slices obtained from different donors, be­ cause in experiment 2 the magnitude of his­ tamine release by fraction III was similar with that by the crude antigen. It has been reported that A scans extracts have the property to release histamine via nonimmunologic mechanisms [13). However, no sig­ nificant histamine release from nonsensitized human skin by Ascaris antigens used in the present studies was observed. Thus,

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IgE to Ascaris Antigens

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Tsuji/Hayashi/Yamamoto/Sakata/Toshida

Table I. Uptake o f 1-’’l-labelled anti-lgE by -4«-arà-antigen-couplcd discs and in vino histamine release from passively sensitized human skin by Ascaris antigens Antigen

RAST scrum

Histamine release, % radioactivity, cpm total

disc

passive exp. 1 sensitization

exp. 2

7.0 0.6

Crude

R N

20,891 20,347

1,140 388

s n

26.0 4.3

Fraction 1

R N

20,420 20,417

1,632 423

s n

23.1 3.3

Fraction II

R N

20,375 20.794

1,069 358

s n

23.8 2.9

Fraction III

R N

19,866 20,445

604 316

s n

6.8 0

No antigen

No

20,414

479

-

-

-

-

Avcam-antigen-induced histamine release from passively sensitized human skin might be due to an immunologic mechanism. Although the uptake of ,2r*I-labelled an­ ti-lgE by crude antigen, fractions I or 11-coupled discs was not high, it was more than double that in the control experiments. However, the magnitude of allergenicity of all fractions was similar under the present experimental conditions in the histamine re­ lease experiments. It is not clear whether or not the difference of the uptake of radioac­ tivity is due to the amount of specific IgE antibody in each fraction in the reaginic ser­ um. The present results indicate that specific IgE antibodies to Ascaris antigens can be produced in man, as the reaginic serum used was obtained from the person experimental­ ly sensitized with antigens prepared from A.

suum. Previous studies have indicated that most of the components prepared from dif­ ferent A scans species have the same anti­ genicity [12), and, as shown in the results, the allergenic active substances obtained from A. suum were not localized in a limit­ ed part of fraction on Sephadex G-200 frac­ tionation, as previously reported by Hussain et al. [6], These facts raise the possibility that specific IgE antibodies to Ascaris anti­ gens can be present in /4 scam-infected pa­ tients. This problem is under investigation in this laboratory. Allergic symptoms in some patients with Ascaris infection have been observed [9], It is useful to prove the presence of IgE anti­ bodies to Ascaris antigens in man not only in the elucidation of the allergic symptoms but also in the development of diagnosis in /l scam-infected patients.

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R = Reaginic serum obtained from Ascaris-antigen-sensitive person ; N = normal serum; No = no serum in the reaction tube: s = passive sensitization; n = no passive senstization; — = not done. Each value represents the mean of triplicate samples in RAST and that of duplicate samples in histamine release. The dialysate obtained from dialysis of Ascaris body fluid was also tested in RAST and histamine release experiments. Neither significant uptake nor histamine release was observed by the dialysate.

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1 Ceska, M.; Eriksson, R.. and Varga, J.: Ra­ dioimmunosorbent assay of allergens. J. Aller­ gy 49: 1-9 (1972). 2 Dobson, C.; Morseth, D. J„ and Soulsby, E. J. L. : Immunoglobulin E-type antibodies induced by A scaris sum» infections in guinea pigs. J. Immun. 106: 128-133 (1971). 3 Fisherman. E. W.: Induction of immediate cu­ taneous reactivity to an antigen (Ascaris) in cancerous and noncancerous individuals. J. Al­ lergy 33: 12-17 (1962). 4 Foucard, T.; Johansson, S. G. O.; Bennich, H., and Berg, T.: In vitro estimation of allergens by a radioimmune antiglobulin technique using human IgE antibodies. Int. Archs Allergy appl. Immun. 43: 360-370 (1972). 5 Greaves, M. W.; Yamamoto, S., and Fairley, V. M. : IgE-mediated hypersensitivity in human skin studied using a new in vitro method. Im­ munology 23: 239-248 (1972). 6 Hussain, R.; Strejan, G.. and Campell, D. H.: Hypersensitivity to Ascaris antigens. VII. Iso­ lation and partial characterization of an aller­ gen. J. Immun. 109: 638-647 (1972). 7 Johansson. S. G. O.; Mellbin, T., and Vahlquist. Bo.: Immunoglobulin levels in Ethiopian preschool children with special reference to high concentrations of immunoglobulin E (IgND). Lancet i: 1118-1121 (1968). 8 Malley, A.; Amkraut, A. A.; Strejan, G.. and

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Campbell, D. H.: Hypersensitivity to Ascaris antigens. III. Atopic-type hypersensitivity in­ duced in rhesus monkeys. J. Immun. 101: 292-300 (1968). Marsden, D.: Ascariasis; in Beeson and McDermott, Textbook of medicine; 13th ed„ p. 762 (Saunders, Philadelphia 1971). McAninch, J. R. and Patterson, R.: Reaginlike antibody formation in rabbits during a heterogeneous antibody response to Ascaris antigens. Immunology 18: 91-98 (1970). Rebuck. J. W. and Crowley. J. H.: A method of studying leucocytic functions in vivo. Ann. N. Y. Acad. Sci 59: 757-805 (1955). Tsuji, M.: Comparative studies on the antigen­ ic structure of several helminthes by immunoelectrophoresis. Jap. J. Parasitol. 24: 227-236 (1975). Uvnas, B. and Wold, J. K.: Isolation of a mast cell degranulating polypeptide from Ascaris sais. Acta physiol, scand. 70: 269-276 (1967). Weiszer, 1.; Patterson, R., and Pruzansky, J. J.: Ascaris hypersensitivity in the rhesus monkey. J. Allergy 41: 14-22 (1968).

Received: April 8. 1977 Correspondence to: Dr. Moriyasu Tsuji, Department of Parasitology, Hiroshima University School of Medicine, 2-3. Kasumi, 1-chome, Hiroshima (Japan)

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References

IgE-type antibodies to Ascaris antigens in man.

Int. Archs Allergy appl. Immun. 55: 78-81 (1977) IgE-Type Antibodies to Ascaris Antigens in Man Moriyasu Tsuji, Takaharu Hayashi, Shoso Yamamoto, Yas...
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