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ABSTRACT Trichosanthin(TCS) is a potent allergen in mice. It can reproducibly induce specific IgE responses in C57BL /6J mice without the help of adjuvant alum. TCS can bring out the IgE

responses to ovalbumin(OVA), while OVA itself could not induce IgE responses to it. However, TCS only works when OVA immunization is given one day after TCS immunization. Either time lag in

OVA immunization, or immunization of both antigens at the same time, or OVA immunization given first, all has no effect on the induction of IgE responses to OVA. Through analysis of the

antibody specificity of hybridoma clones, it indicated that specific antibodies to TCS or OVA were secreted by independent B cell clones. The IgE antibodies showed no polyreactivity to

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IMMUNOLOGICAL INTEGRITY OF TETANUS TOXOID VACCINE ANTIGEN Article Open access 23 June 2021 INTRODUCTION Trichosanthin (TCS) is an effective protein component of a Chinese herb medicine,

which, has long been used in history for abortion induction1. Recently it has been found that TCS has anti-HIV effects2, which is arousing much interest in it. Its amino acid sequence, DNA

sequence, and 3D structure have all been worked out3, 4. In clinical application it occasionally induce anaphylaxis in certain patients5. Previously we had done a number of studies on the

TCS specific IgE responses in mice immunized with TCS and adjuvant alum6. Since using the protein alone can induce anti-TCS IgE responses in human body, we intend now to study the TCS-IgE

responses without the influence of adjuvant in mice. That would be possibly more closer to the status happened in human body. Furthermore cross-reactivity of IgE antibodies in sera with

different allergens in some of common allergenic determinants on different allergens is suggested to be the reason. However that it may be due to certain structural characteristics in

antigen binding site of IgE molecules was also suggested8. And the possibility of allergens may influence different lymphocyte clones through mediators or cells that involved in the

induction of IgE antibody responses within microenvironment could not be excluded also. In this paper we intend to establish a reproducible TCS-specific IgE antibody responses without the

use of adjuvant to investigate its allergenic potential, and try to study the cross reactivity of allergen at cell clone level. MATERIALS AND METHODS ANIMALS * 1 Mice: C57BL/6J mice, female

and 12 weeks of age * 2 Rats: Wistar rats, female with 200g body weight. REAGENTS * 1 Antigens * 1) TCS: Crystalized TCS in Trichosanthin Injection Solution (TCS-J), produced by Jin-San

Pharmaceutical Factory, 1.2 mg /ml, was used as immunogen. Refined TCS (TCS-W), produced by Wu-Han Biological Products Research Institute, 2mg /ampule was used in the passive cutaneous

anaphylaxis(PCA) test as a challenger and in ELISA as a coating antigen. TCS-J has now been commonly used in clinics for a number of years. Considering TCS-J induced immune responses would

be more closer to clinical status, we used it as the immunogen. However we used TCS-W as a challenger to detect the formed TCS-J-specific IgE reponses in mice. The reason is that the source

of TCS-W is available and abundant in our hands. And we have intentionally run parallel experiments to compare the PCA results following TCS-J or TCS-W challenge. The results showed no

differences (unpublished data). * 2) Ovalbumin (OVA), produced by Shanghai Dong-Feng Factory for Reagents. * 2 Evan's Blue, purchased from Shanghai Chemical Reagent Company. It was

dissolved in phosphate buffered saline (PBS) and was adjusted to 1% concentration ready for use. IMMUNIZATION The TCS-J or OVA were diluted or dissolved in PBS respectively and was each

adjusted to 10 _μ_g/ml and 20 _μ_g/ml. Each mouse was intraperitoneally injected with 0.5 ml antigen solution. The protocol of immunization was referred in below. ASSAYS OF THE ANTIBODY

SPECIFICITY AND TITER * 1 ELISA: Indirect ELISA was used to detect the specific antibody in antiserum or in hybridoma culture supernatant. The procedure were briefly as follow. TCS-W or OVA

were used as the coating antigens. 1:50 diluted antiserum or hybridoma culture supernatants were used as the first antibody. Peroxidase labelled goat anti-mouse Ig (anti-MIg) or goat

anti-mouse IgE (anti-MIgE) were used as the second antibody. Tetramethyl benezidine (TMB) and hydrogen peroxide were used as substrate. * 2 PCA: The assay was used to detect the

antigen-specific IgE antibody in antiserum or hybridoma culture supernatant. According to Ovary9, the assay was carried out briefly as follows. On the shaved back of rats, 0.1 ml antiserum

diluted to 1:10 with PBS or 0.1 ml of hybridoma culture supernatant were intradermally injected for priming. On the next day 2 mg TCS-W or 3 mg OVA dissolved in 1% Evan's blue were

intravenously injected into the tail vein of the rat. After 30 minutes blue patches appeared at the primed site were accounted as positive reaction, i.e. the presence of antigen-specific IgE

in the test samples. CONSTRUCTION OF HYBRIDOMAS The primed lymphocytes were from mesenteric lymph node of mice immunized according to immunization protocol for Group B (see below). Five

days before fusion the mice from Group B were boosted again. The tumor fusion partner was either SP2 /0 or NS-1 myeloma ceils. The fusion was carried out according to the routine method,

including selection by HAT medium and screening. RESULTS DOES MINUTE AMOUNT OF TCS ALONE WITHOUT ADJUVANT COULD INDUCE ANTI-TCS IGE RESPONSES? The ELISA results of TCS specific serum titer

from mice immunized ip with 5 _μ_g TCS-J at 2-week interval are shown in Tab 1. It shows that all mice after two immunizations produced anti-TCS antibodies. And the antisera also contained

anti-TCS IgE antibodies since PCA results indicated positive reaction. DOES TCS EFFECT THE INDUCTION OF OVA-SPECIFIC IGE RESPONSES? Through immunization of different combination of TCS-J and

OVA, the influence of TCS on the induction of OVA-specific IgE responses in C57BL /6J mice could be observed. The protocol for the combined immunization of TCS-J and OVA is shown in Tab 2.

Tab 3 shows the experimental results. Results from Group F show that OVA alone could not induce IgE responses in mice. However, under the influence of TCS, it could induce OVA-specific IgE

responses (Group B). But TCS effects only when it was used one day before OVA immunization. In case of TCS used five days before, it showed no promotion effects on the induction of

OVA-specific IgE responses(Group A). On the other hand, either TCS and OVA given at the same time (Group C) or OVA given first and then immunization with TCS (Group D), none could induce

OVA-specific IgE responses, although the anti-TCS IgE responses were all induced in these groups. These results have been repeatedly reproduced in separate experiments. ANTI-OVA IGE AND

ANTI-TCS IGE ARE FROM SEPARATE NONES OR FROM ONE CLONE? The hybridomas constructed by the fusion of mesenteric lymph node cells from Group B mice and myeloma cells were tested for their

antibody specificities against TCS and OVA. Tab 4 shows the results of specific antibody titer of antiserum from lymphocyte donor. It can be seen that both antibodies against TCS and

antibodies against OVA are present, and both contains TCS- and OVA-specific IgE, since the PCA tests are both positive. The specificities of the antibodies secreted by hybridomas in separate

wells tested in two experiments are listed in Tab 5. In Experiment 1, although there are some wells that are both TCS and OVA positive in Ig-ELISA, not a single well shows both positive

reaction to TCS and OVA in PCA test. It means that there is not presence of both anti-TCS and anti-OVA IgE activities in a same well. In fact only five OVA-specific IgE wells were detected.

Since the fusion rate is quite low, 18.4 %, it can assume that each well contains a single clone, and the anti-TCS or anti-OVA IgE antibodies are secreted by individual clones. There is no

indication of the cross reactivity or polyreactivity at the antibody molecule level. Experiment 2 further provides evidence for the assumption. This time there only appears two wells that

are both TCS and OVA positive in the IgE-ELISA. And the majority of the wells are either TCS-IgE positive (72 wells) or OVA-IgE positive (75 wells). After further cloning of the cells in

double positive wells, antibody secretion in one well was lost. All the wells growing out cells were negative. The other one turned to single positive. The cloning rate is 19%. All the wells

only showed positive reaction to TCS in IgE-ELISA without simultaneous positive reaction to OVA. DISCUSSION It has well been known that IgE mediates immediate hypersensitivity, an allergic

response. When allergens cross-link the allergen-specific IgE receptors on cell surface of mast cells or basophils, it will activate these cells, leading to the release of allergic

mediators, thus bringing about the allergic responses. Therefore induction of IgE antibody responses is the characteristics of allergen. In the present study, it indicates that minute amount

of TCS can reproducibly induce specific IgE responses in mice. TCS is a potent allergen. As a potent allergen, what about the allergic potential of TCS? For this, we studied the effects of

TCS on the induction of IgE responses to other antigen, which by itself is not an allergen, but when it is used to immunize under the influence of TCS, the consequence is different. The

present study indicates TCS could bring out IgE responses to OVA, which normally is not an allergen. In our experiments also showed that immunization with OVA alone could bring about

OVA-specific antibody responses (unpublished data) but not IgE responses. However when OVA was used in combination with TCS, OVA-specific IgE responses were aroused. Thus it indicates that

TCS enables other unrelated antigen-specific B lymphocyte clones to secrete specific IgE antibodies, while they normally do not switch to secrete IgE antibody. The mechanism seems not to be

due to the presence of common antigenic determinants on TCS and OVA, since they are structurally quite different. And it seems not to be due to polyreactivity or multispecificity of the

antigen binding sites on the IgE antibody molecules, since through the detection of the antibody specificity at clonal level (hybridoma clones), the secreted antibodies of hybridoma cells in

each well were all antigen specific, i.e. they only reacted with one antigen, either TCS or OVA. Double positive reaction of antibodies secreted by hybridoma clones have not been found. In

the process of immune responses, some aspects are antigen-specific, but some are non-specific. However the non-specific ones also play very important roles in immune responses. As we all

know that interleukin-4 plays a very important role in IgE switch10, 11. Some other cytokines and cell surface molecules are shown to be also involved in the regulation of IgE responses12.

After the allergic state created by TCS, when another antigen circulates to the “allergic” micro-environment, the antigen-specific lymphocyte cell clones not only received signals from

specific antigen but may possibly also receive some signals already present in the micro-environment, which just critical and appropriate for the induction of IgE switch in B cells at the

particular stage. In this case OVA-specific clones may receive the appropriate nonspecific signals, IL-4 etc presumably, resulted from TCS stimulation. They may drive the OVA-specific B cell

clones to switch to IgE secretion. The present research lend some support to this possibility, since the condition leading to IgE switch in B cells is very critical. Only when mice were

immunized with OVA one day after TCS was given, the OVA-specific IgE responses can be induced. In this sense it may represent a bystander effect. Furthermore it has been reported that ricin

can enhance the IgE responses to an allergen, bee venom phosphilipase A2 (PLA2)13. They showed it was due to suppression of CD8 T cells by ricin. Ricin has not been reported to be an

allergen and to induce IgE response. It is a toxic protein, while TCS is also a toxic protein, and is reported to be structurally related to ricin A chain14. Whether TCS also through the

suppression of CD8 T cells to bring out the induction of IgE responses is interesting to study. However, there are also reports that showed TCS could activate CD8 T cells to mediate TCS

immune suppression15. Anyway, whatever the true mechanism to be, the present paper demonstrates that a potent allergen can bring out the IgE responses to another unrelated antigen, even the

antigen originally is not an allergen itself. This may afford an explanation for the cross-reactivity of IgE antibodies in sera with different allergens, which are frequently reported in

allergic patients. The “cross-immunization” of allergens may lead to the persistent allergic status. Further study of the cellular and molecular mechanism of the “cross-immunization”

potential oi' allergens may provide clues to the new strategy for the prevention and treatment of allergic diseases, which almost affect 10-15% of the people. REFERENCES * 2nd lab. of

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references ACKNOWLEDGEMENTS The authors thank Ms. Lin Guomei and Mr. Xie Zhigang for their technical assistance. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Shanghai Institute of Cell

Biology, Chinese Academy of Sciences, Shanghai, 200031, China Yongyong Ji, Cuihong Yang & Ming Yeh Authors * Yongyong Ji View author publications You can also search for this author

inPubMed Google Scholar * Cuihong Yang View author publications You can also search for this author inPubMed Google Scholar * Ming Yeh View author publications You can also search for this

author inPubMed Google Scholar ADDITIONAL INFORMATION *The work is specifically dedicated to Prof. Zhen YAO for his 80-yeaxs birthday RIGHTS AND PERMISSIONS Reprints and permissions ABOUT

THIS ARTICLE CITE THIS ARTICLE Ji, Y., Yang, C. & Yeh, M. The influence of Trichosanthin on the induction of IgE responses to ovalbumin under adjuvant-free condition. _Cell Res_ 5, 69–74

(1995). https://doi.org/10.1038/cr.1995.7 Download citation * Received: 06 March 1995 * Revised: 05 May 1995 * Accepted: 13 May 1995 * Issue Date: 01 June 1995 * DOI:

https://doi.org/10.1038/cr.1995.7 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently

available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * Trichosanthin * ovalbumin * allergen * IgE response