WO2002066979A1

WO2002066979A1 - Method of detecting antigen causative of bronchial asthma

Info

Publication number: WO2002066979A1
Application number: PCT/JP2002/001403
Authority: WO
Inventors: Akio Mori; Kazuo Akiyama; Kazutoh Takesako; Ikunoshin Kato
Original assignee: Takara Bio Inc.
Priority date: 2001-02-22
Filing date: 2002-02-19
Publication date: 2002-08-29
Also published as: JPWO2002066979A1

Abstract

A method of identifying an antigen causative of bronchial asthma wherein the production of IL-5 by T cells due to the stimulation with the antigen is used as an indication; a kit for identifying the causative antigen; and remedies or preventives. Moreover, a method of detecting an antigen causative of bronchial asthma is provided.

Description

Measuring method for bronchial asthma causing antigen

The present invention relates to a method for identifying a causative antigen, a kit for identifying a causative antigen, and a therapeutic or prophylactic agent for bronchial asthma. Further, the present invention relates to a method for detecting an antigen causing bronchial asthma. Background art

Bronchial asthma mainly occurs in childhood, and about 2/3 of the group of atopy-type patients who have IgE antibodies against antigens such as ticks accounts for about 2/3 of the population. Non-atopic (also known as endogenous, infectious) patient groups account for approximately 1 Z 3 of the disease.

Atopic bronchial asthma is recognized as an immediate hypersensitivity caused by IgE antibodies to antigens and mast cells. In atopic bronchial asthma, treatment with anti-inflammatory drugs such as steroid drugs and anti-allergic drugs and bronchodilator drugs and treatment to suppress airway hyperresponsiveness, as well as binding to patient-derived IgE antibodies It is more effective to avoid the antigen identified as the causative factor and to perform desensitization therapy using the antigen by identifying the antigen and further identifying the causative antigen by skin test. On the other hand, in non-atopic bronchial asthma, the method of identifying the causative factor is not yet known, and only treatment for suppressing airway hyperreactivity as symptomatic treatment is performed.

The involvement of antigens such as Candida, mite extract, and house dust in atopic bronchial asthma and dermatitis is well known. Endogenous Candida has been studied as a causative factor of atopic bronchial asthma, and it has been reported that acid protease is the causative antigen. In addition, antifungals are effective against atopic dermatitis It has been reported. As described above, with regard to atopic diseases, various findings regarding the causal factors and the like have been obtained. However, there is no report on the usefulness of these findings for non-atopic bronchial asthma and the findings on the causative factors.

By the way, bronchial asthma is not just a transient immediate hypersensitivity as described above, but it is recognized that persistent eosinophilic inflammation is essential. In other words, airway mucosa of patients with bronchial asthma has cell infiltration and inflammatory findings characterized by accumulation of eosinophils. Unlike normal inflammation, which is characterized by accumulation of monocytes, macrophages, and neutrophils, such as bacterial infections, eosinophils selectively accumulate, so they are called eosinophilic inflammation. Eosinophils in bronchial asthma are seen in patients with bronchial asthma who have significant infiltration of activated helper T cells in the area of airway inflammation and the correlation between the number of eosinophils infiltrating the airway mucosa and the number of τ cells. There have been reports suggesting that sexual inflammation may be associated with CD4-positive helper T cells (Kay AB.``Helper '' (CD4 +) T cells and eosinophils in allergy and asthma) Am. Rev. Respir. Dis. 1992; 145: S22-6]. However, there is no report on the identification and identification of the causative antigen of bronchial asthma from such a viewpoint. Disclosure of the invention

The present invention provides a method for identifying a causative antigen of bronchial asthma, particularly a non-atopic bronchial asthma, and a bronchial bronchi capable of easily and quickly identifying a causative antigen of bronchial asthma. An object of the present invention is to provide a kit for identifying an antigen causing asthma, and a therapeutic or preventive agent for non-atopic bronchial asthma, which comprises an antigen causing bronchial asthma. Another object of the present invention is to provide a method for detecting an antigen causing bronchial asthma, which can detect the presence of an antigen causing bronchial asthma in a test sample.

That is, the present invention C 1] The amount of IL-15 produced from T cells due to the stimulation of the test substance was measured, and an increase in the amount of IL-15 production was observed as compared to T cells not stimulated with the test substance. The test substance is identified as a causative antigen of bronchial asthma, a method for identifying a causative antigen of bronchial asthma,

(2) (a) culturing T cells in the presence of a test substance, and

(b) measuring the amount of IL-5 produced from T cells,

When the difference between the amount of IL-5 produced from T cells and the T cells cultured in the absence of the test substance is 1 Opg / ml or more, the test substance is The identification method according to the above (1), which is identified as a causative antigen of asthma.

(3) the identification method according to the above (1) or (2), wherein the bronchial asthma is non-atopyic bronchial asthma;

[4] a kit for identifying an antigen causing bronchial asthma, comprising an antigen capable of increasing the amount of IL_5 produced from T cells upon stimulation;

(5) the kit according to (4), wherein the bronchial asthma is non-atopic bronchial asthma;

[6] a therapeutic or prophylactic agent for non-atopic bronchial asthma, comprising as an active ingredient all or a part of an antigen causing bronchial asthma;

(7) the therapeutic or prophylactic agent according to (6), wherein the antigen causing bronchial asthma has been identified by the identification method according to any of (1) to (3);

[8] a therapeutic or prophylactic agent for non-atopic bronchial asthma, comprising an antifungal agent as an active ingredient;

[9] Measure the amount of IL-15 produced in T cells that have been brought into contact with the test sample, and find that the amount of IL-5 produced is increased compared to the case of T cells that have not been brought into contact with the test sample. When the test sample is determined to contain a bronchial asthma-causing antigen in the test sample, a method for detecting a bronchial asthma-causing antigen,

[10] (a) a step of culturing T cells in the presence of a test sample, and (b) a step of measuring the amount of IL-15 produced from the T cells, When the difference between IL-15 production from T cells is 1 Opg / ml or more compared to when T cells are cultured in the absence of the test sample, bronchi It is determined that an antigen causing asthma is present, the detection method according to the above (9), and the detection method according to the above (9) or (10), wherein the bronchial asthma is non-atopic bronchial asthma.

About. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the results of induction of IL-15 production from T cells by Candida-derived antigens. The abscissa indicates the antigen derived from each Candida, and the ordinate indicates the difference between the case where the antigen was stimulated and the case of the control as IL-15 production amount (pgZml). The numbers in the graph are the average IL-15 production masculine SE (measured average value of 8 cases).

FIG. 2 is a graph showing the results of T cell proliferation induction by Candida-derived antigens. The horizontal axis describes the Candida-derived antigen, the ³ H- thymidine uptake by T cell proliferation in the vertical axis as a relative value to the control shown by 3 H- TdR uptake (SI). The numbers in the graph are the average 3H-TdR uptake soil SE (mean average value of 8 cases). The amount of 3H-TdR incorporation was determined by the following equation:

3H—TdR uptake (S.I.)

= ^[3 H- thymidine uptake by T cells upon addition antigen (c pm)] / ^[3 H- thymidine uptake by T cells of the control (c pm)] BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors have made it possible to identify the causative antigen of non-atopic bronchial asthma (hereinafter simply referred to as asthma), for which no report has been made on the identification of the causative antigen. Therefore, we presumed that non-atopic asthma was persistent eosinophilic inflammation, and focused on the cytokines involved in eosinophil accumulation.

First, we searched for cytotoxicity induced when T cells from non-atopic asthmatics were stimulated with various antigens. As a result, they found that IL-15 production occurs when stimulated with a Candida antigen or a tick antigen. Also, the antigens that produced those IL-5 caused eosinophilic inflammation in inhalation challenge tests and intradermal reactions on the patient. Furthermore, in patients who produce IL-5 by crude Candida antigen, it was shown that purified acid protease derived from Candida caused eosinophilic inflammation in inhalation challenge tests and intradermal reactions in the patients. That is, it has been surprisingly revealed that an antigen capable of inducing IL-15 production from T cells can induce eosinophilic inflammation. Based on this fact, the present inventors found that T cells from antigen-stimulated

It was found that it was possible to identify the antigen responsible for non-atopic asthma using IL-15 production as an index.

On the other hand, atypical asthma is distinguished from non-atopic asthma in that IgE antibodies are involved (furthermore, an immediate asthmatic response (IAR) involving IgE antibodies occurs). At present, it is generally understood that the pathogenesis of the disease is completely different, but in atopic asthma, as in non-atopic asthma, an acid is understood as a late asthmatic response (LAR). Spheroid inflammation is observed. In addition, the antigen responsible for atopic asthma is IgE antibody production.

It is known to cause IL-15 production together. In other words, it can be said that an antigen that causes IL-5 production in an atopic asthmatic patient is a causative antigen of acute topic asthma. Therefore, it is possible to identify the antigen responsible for atopic asthma using the production of IL-15 from T cells by antigen stimulation as an indicator.

Based on the above findings, the present inventors can identify asthma-based antigens without distinguishing between atopic and non-atopic types by using IL-15 production from T cells by antigen stimulation as an index. And found that the present invention was completed. Therefore, according to the method for identifying asthma-causing antigens of the present invention, asthma-causing antigens that have been regarded as completely different diseases and that have been classified into two disease types, Athobi type 1 and non-Athby type, have been identified. Without such a distinction, identification can be performed simply and reliably using IL-5 production from T cells as an index. In particular, by examining IL-5 production from T cells from individuals (eg, patients) diagnosed with non-atopic asthma without production of IgE antibodies, non-atopic Its technical significance is extremely great because it allows identification of the antigen responsible for atopic asthma.

Specifically, the method for identifying an asthma-causing antigen of the present invention comprises measuring the amount of IL-15 produced from T cells by stimulation of a test substance, and comparing the case of T cells not stimulated with the test substance. By comparison, when an increase in the amount of IL_5 production is observed, the test substance is identified as an antigen causing asthma.

As used herein, the term “test substance” refers to a target substance that is evaluated for its IL-5 production effect on T cells, and includes any substance presumed to be involved in the development of asthma. It is not limited. Examples thereof include mites, house dust, fungi, pollens, pet epidermis, and components contained therein. The test substance also includes a substance which has been known as a causative antigen of atopic asthma, but according to the identification method of the present invention, a causative antigen of non-atopic asthma can be identified. When such a substance is used as a test substance, it is possible to further obtain information on whether or not the substance is a causative antigen of non-atopic asthma. Also, “τ cells” refers to an individual-derived biological tissue specimen ( For example, Τ cells derived from peripheral blood, spleen, etc.), and preferably CD4 positive helper Τ cells derived from peripheral blood, which have been reported to be involved in the development of eosinophilic inflammation. In addition, from the viewpoint of the accuracy of the identification method of the present invention, the T cells are preferably isolated from a biological tissue specimen, but need not necessarily be isolated. Alternatively, peripheral blood mononuclear cells can be directly used in the identification method of the present invention.

An “individual” is preferably a mammal (eg, a dog, a cat, a pen, a dog, a human), and more preferably a human.

The stimulation of T cells by a test substance is performed, for example, by collecting peripheral blood mononuclear cells containing T cells from an individual according to a known method, adding a test substance thereto, and in the presence of the test substance, preferably 35 to It can be performed by incubating at 38 ° C for about 1 hour to 7 days. On the other hand, the peripheral blood mononuclear cells are cultured in the same manner without adding the test substance and without stimulating with the test substance, to serve as a control.

Next, the amount of IL-5 produced from the T cells is measured. Such a measurement can be carried out by the ELISA method for measuring IL-5 protein mass, RT_PCR for quantifying the amount of mRNA, Northern hybridization, or the like. The present invention is not particularly limited, but is preferably determined by the ELISA method. As a result of the measurement, when an increase in the production amount of IL-15 (pg / ml) from T cells stimulated with the test substance was observed as a substantial difference compared to the control, The substance is identified as the causative antigen of asthma. When the difference in the amount of IL-15 produced is expressed by specific numerical values, it is preferably 1 Opg / m1 or more, more preferably 20 pg / m1 or more, and still more preferably 5 Opg / m1 or more. If the difference in the amount of IL-15 production is less than 10 pg / ml, it does not necessarily indicate a substantial difference due to individual differences or the measurement limit of the amount of IL-5 production, etc. In any case, if the difference is not more than 10 pg / m1, the antigen causing asthma can be clearly identified.

Therefore, the method for identifying an antigen causing asthma of the present invention

(a) culturing T cells in the presence of the test substance, and

(b) measuring the amount of IL-5 produced from T cells,

If the difference is greater than 1 OpgZml compared to the T cell culture in the absence of the test substance, the test substance is a cause of asthma Methods that can be identified as antigens can be mentioned. In such a method, the amount of IL-15 produced from T cells in the control (S 1) and the amount of IL-5 produced from T cells cultured in the presence of the test substance (S 2) were each determined. Measure, subtract 32 from 31, and if the subtracted value is 1 O pg / ml or more, the test substance is identified as a causative antigen of asthma. The temperature and period suitable for culturing T cells are the same as described above, and more specific culture conditions and the like may be in accordance with known ones.

In a more preferred embodiment of the method for identifying a causative antigen of asthma of the present invention, a causative antigen specific to non-atopic asthma can be identified. In this embodiment, T cells derived from an individual diagnosed with non-atopic asthma may be used in accordance with the conventional determination of the type of asthma.

By the above identification method, for example, the cause antigens of asthma patients, especially non-atopic asthma patients, which have not been known before, can be clarified, so that antigen evasion and desensitization therapy are effective for all asthma patients. Will be able to be implemented in a practical manner. In particular, an antigen specific to non-atopic asthma is very effective for the treatment or prevention of the asthma. In another aspect of the present invention, the identification of the asthma-causing antigen is performed easily and quickly. And a kit for identifying an asthma-causing antigen. Such a kit includes an antigen capable of increasing the amount of IL-15 produced from T cells upon stimulation. The antigen is not particularly limited as long as it can increase the amount of IL-15 produced from T cells by the stimulation, but it is preferable to use the method for identifying an asthma-causing antigen of the present invention. The identified antigen. Examples of the antigen include, for example, at least one selected from the group consisting of Candida-derived antigen, Candida-derived acidic protease, mite extract, and Derf 1, Derf II, and house dust, which are described in Examples below. Can be mentioned. It is preferable that the antigen is, for example, lyophilized and packaged from the viewpoint of preservation. The antigen must be suitable before use. It can be used after reconstitution in a carrier.

If the antigen contained in the above kit can be added to the medium used for culturing T cells at a concentration in the range of 0.1 to 100 / g / m1, the form, content, There is no particular limitation on the concentration. For example, a lyophilized antigen or a high-concentration antigen solution that can be contained in the kit of the present invention may be dissolved and diluted to an appropriate concentration, and then added to the medium so that the concentration is within the above range. .

The kit may further optionally contain a reagent used for measuring the amount of IL-5 produced from T cells. Further, for example, when it is intended to measure the amount of IL-5 produced from T cells by a colorimetric method, it may contain various concentrations of IL-15 used for calibration.

Identification of the asthma-causing antigen using the kit can be performed according to the method of the present invention for identifying an asthma-causing antigen. That is, when the causative antigen is identified using the kit, the antigen contained in the kit is treated in the same manner as the test substance, and the causative antigen of asthma is identified in the same manner as in the above-described identification method. Since the kit contains various antigens capable of increasing the production of IL-15 from T cells in advance, for example, by identifying the causative antigen using the kit for asthma patients, Patient-specific causative antigens are easily identified. In particular, when the antigen contained in the kit is specific to non-atopic asthma, the asthmatic patient is a non-atopic asthmatic patient, and a causative antigen specific to the asthma is simultaneously identified. This means that more efficient asthma prevention or treatment measures can be taken. In such an embodiment, the kit of the present invention can also serve as a diagnostic kit for non-atopic asthma.

In another aspect of the present invention, there is provided a therapeutic or preventive agent for non-atopic asthma, which contains all or a part of an asthma-causing antigen as an active ingredient. As such a causative antigen, those identified by the method for identifying an asthmatic causative antigen are preferable, and among them, a causative antigen specific to non-atopic asthma is preferable. Therapeutic agent of the present invention Alternatively, specific examples of the causative antigen that can be suitably used in the prophylactic agent include the antigens exemplified in the kit.

In the following, the term “causal antigen” means all or a part of the causative antigen. As used herein, "part of the causal antigen" refers to at least a part of the causative antigen including a structure that determines the specificity and immunogenicity of the antigen-antibody reaction of the antigen, and is preferably an epitope or an antigenic determinant. Means part of the causative antigen. The causative antigen may be a hapten bound to an appropriate carrier.

Although it is possible to use the causative antigen itself as the therapeutic or prophylactic agent of the present invention, it is preferable to provide them as a pharmaceutical composition. For example, such compositions include a causative antigen, one or more pharmaceutically acceptable carriers (eg, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, etc.), and optionally other And those containing a therapeutic or prophylactic component.

The compositions include those suitable for oral (preferably inhalation), nasal, and parenteral (eg, subcutaneous, transdermal, intradermal, intramuscular, intravenous, and rectal administration). It can be adjusted as appropriate according to the symptoms of the individual who needs to administer the composition. The compositions may, if desired, be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

Compositions suitable for oral and / or nasal administration include, for example, capsules, tablets, powders or granules each containing a predetermined amount of the causative antigen as an active ingredient, as a solution or suspension in aqueous or non-aqueous liquid Alternatively, it can be provided as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. Compositions for inhalation may be provided in any manner known to be effective (eg, metered dose inhalers). Further, the composition may contain a known adjuvant (for example, cholera toxin, cholera toxin B subunit, and variants thereof). ' Compositions for parenteral administration include, for example, water-soluble and water-insoluble sterile injectable solutions which can contain antioxidants, buffers, bacteriostats, solutes which are isotonic with the blood of the individual to be dosed. And aqueous and non-aqueous sterile suspensions which may contain suspending and bulking agents. The compositions may be presented in single or multidose containers, for example, sealed ampoules and vials, and added to a sterile liquid carrier immediately before use, for example, water for injection. It may be provided in lyophilized form, which only needs to be done. In addition, as a composition for rectal administration, for example, it can be provided as a suppository in combination with a usual carrier such as polyethylene glycol.

The content of the asthma-causing antigen in the composition is not particularly limited, and can be appropriately adjusted as desired. For example, the preferred causal antigen content in the composition is preferably between 0.01 and 50 g / ml.

The dose of the therapeutic or prophylactic agent of the present invention is not particularly limited as long as the desired effects of the present invention can be obtained. Generally, for example, in the case of humans, the dose is preferably administered orally or parenterally within the effective dosage range of the causative antigen, preferably from 0.2 ng to 0.1 mg / kg body weight per adult. However, since the type and degree of asthma and individual differences (age, sex, etc.) in each individual, the dose of the causative antigen as an active ingredient is not limited to this range. In addition, the administration method, the number of administrations, the administration period, and the like are not particularly limited.

According to the therapeutic or prophylactic agent of the present invention, by producing an antibody against the causative antigen to be administered, neutralization of an antigen involved in the development of asthma in an individual, in particular, an antigen specific to non-atopic asthma Therefore, it is possible to effectively treat or prevent non-atopic asthma, which could not be effectively performed until now. For example, by administering the therapeutic or prophylactic agent of the present invention, IgA and IgG are produced in the nasal discharge and oral cavity, and can effectively work for removing antigens around the bronchi. Therefore, the therapeutic or prophylactic agent of the present invention is effective for mucosal immunotherapy. Also, When the causative antigen contained as an active ingredient in the therapeutic or prophylactic agent of the present invention is specific to non-atopic asthma, the therapeutic or prophylactic agent also functions as a diagnostic composition for non-atopic asthma I can do it.

Furthermore, by referring to the results of the method for identifying an asthma-causing antigen of the present invention, a more effective therapeutic or preventive agent for non-atopic asthma can be provided. For example, if the causative antigen is identified as a Candida-derived antigen, an effective antifungal agent (eg, fluconazole, itraconazole, amphotericin B, etc.) that is effective in killing Candida is used as the active ingredient. A therapeutic or preventive agent for non-atopic asthma can be provided. According to the therapeutic or prophylactic agent, the root cause of asthma can be directly eliminated, so that a very excellent therapeutic or preventive effect can be obtained. The therapeutic or prophylactic agent is also included in the present invention. The therapeutic or prophylactic agent may contain only an antifungal agent as an active ingredient.However, from the viewpoint of expecting a therapeutic or preventive effect over asthma in general, as an active ingredient, an antigen causing asthma, Among them, those containing both the antigen causing non-atopic asthma are preferable. The content of the antifungal agent in the therapeutic agent or the prophylactic agent is not particularly limited, and varies depending on each antifungal agent, and can be appropriately selected. However, a desired amount depends on the dosage form and the route of administration. What is necessary is just a content which can acquire an effect. The dosage of the antifungal agent is generally, for example, preferably 1 g to 5 mg / kg body weight per day for an adult when intravenously injected into a human. In addition, the production method and administration method of the therapeutic or prophylactic agent are the same as described above.

Further, as one aspect of the present invention, there is provided a method for detecting an asthma-causing antigen in a test sample. Specifically, the detection method is to measure the amount of IL-15 produced in T cells that have been brought into contact with the test sample, and to compare this with the T cells that have not been brought into contact with the test sample (control). A method for detecting an asthma-causing antigen, wherein it is determined that an asthma-causing antigen is present in the test sample when an increase in the IL-15 production amount is observed. Such a detection method can be performed according to the method for identifying an antigen causing asthma of the present invention. Wear. That is, the contact between the test sample and the T cells may be performed by using the test sample instead of the test substance in the step of stimulating the cells with the test substance in the identification method. Can be performed. The test sample is not particularly limited as long as the presence of the test substance can be expected. For example, food, drinking water, domestic water, house dust, indoor and outdoor airborne substances, and the like can be mentioned. Next, the amount of IL-5 produced from the Τ cells is measured in the same manner. As a result of the measurement, when an increase in the amount of IL_5 produced from the cells in contact with the test sample was observed as a substantial difference compared to the control, the antigen causing asthma in the test sample was included in the test sample. Is determined to exist. The specific numerical value of the difference in the amount of IL-5 production is the same as in the case of the identification method. Therefore, in a preferred embodiment of the method for detecting an antigen causing asthma of the present invention,

(a) culturing T cells in the presence of a test sample, and (b) measuring the amount of IL-15 produced from T cells,

If the difference is greater than or equal to 1 O pg / ml compared to the case where the T cells were cultured in the absence of the test sample (control), A method for detecting an asthma-causing antigen, which is determined to include an asthma-causing antigen in a sample.

In addition, the use of T cells derived from individuals diagnosed as non-atopic asthma according to the conventional determination of asthma type can detect the presence of a causative antigen specific to non-atopic asthma in test samples. can do.

According to these detection methods, an excellent effect of detecting asthma-causing antigens present in food and drink or the environment with high specificity is exerted, and asthma can be prevented from being caused by contact with the antigens. In particular, an excellent effect is exhibited. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples. Example 1 Induction of IL-5 Production by Peripheral Blood Mononuclear Cells in Patients with Non-Atopic Asthma by an Antigen and Inhalation Induction by Each Antigen

Venous blood was collected from 30 asthmatic patients, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque specific gravity centrifugation. The cells were suspended in AIM-V medium (Gibco BRL) at 2 × 10 ^δ cells / μl and cultured in a 24 μl plate (manufactured by Koning). Here, a physiological saline solution of the antigen [Candida extract (manufactured by Torii Pharmaceutical Co., Ltd.)] was added and stimulated so that the final concentration of the antigen was 10 gZml. In the control, physiological saline alone was added instead of the physiological saline solution of the antigen. After culturing for 6 days, the culture supernatant was collected and the amount of IL-5 was measured by ELISA, and the difference between the case of stimulation with the antigen and the case of the control was examined.

As a result, the increase in IL-5 production induced by Candida extract was less than 20 pg / m1 in 254 patients (Group A), while it was 20 pg / m1 or more in 55 patients (Group B). Showed an increase in IL-5 production. The average age of patients in group A was 47.0 ± 1.1 years, the average increase in IL-5 was 9.7 ± 0.6 pgZml, and the average age of patients in group B was 46.2 ± 2. The average increase in IL-15 at age 2 was 142.3 ± 34.9 pg / ml.

Among the patients in group B, venous blood was collected again from six patients with normal values (RA STfiO. 35 PRU / ml or less) based on the measurement of antigen-specific IgE antibodies in serum. PBMC was separated by specific gravity centrifugation. The cells were suspended in AIM-V medium at 2 × 10 ⁶ cells / well and cultured in a 24-well plate. Various antigens (Candida extract, tick extract (Torii Pharmaceutical Co., Ltd.), Candida albicans-derived secreted acidic protease (Candida acid protease, Takara Shuzo)) were used as saline solution, and the final concentration of each antigen was 10 zgZml. It was added and stimulated. In the control, physiological saline was used instead of the saline solution of the antigen. Brine only was added. After culturing for 6 days, the culture supernatant was collected and the amount of IL-5 was measured by the ELISA method, and the difference between the case of stimulation with the antigen and the case of the control was examined. Table 1 shows the results. In Table 1, do not list the difference simply as IL-5 (pg / 1).

The inhalation induction test was performed in accordance with the Japanese Society of Allergology standard method. The subject's respiratory function before loading was measured using Peakman 8 (manufactured by Chiis Giken) under maximum expiratory effort. Inhalation was performed by using a nebulizer (Devilbiss) and injecting 5 LZmin of compressed air for 2 minutes. After inhaling physiological saline alone as a control and confirming that there was no decrease in respiratory function, 1 g / m1 for Candida acid protease, and a 10,000-fold diluted aqueous solution for tick extract and power plant extract were used. After inhalation for 2 minutes, respiratory function was measured 10 minutes later. When no more than 15% reduction in respiratory function was observed, an antigen solution 10 times more concentrated was inhaled and respiratory function was measured in the same manner. Inhalation loading was carried out to a 100-fold diluted solution of Candida acid protease and a 100-fold diluted solution of Candida extract mite extract 100 Oz g / ml.

The respiratory function was measured 10 minutes after the inhalation load of the antigen solution, and thereafter, the respiratory function was measured every hour for delayed asthmatic reactions. The results are shown in Table 1. In both cases of IAR and LAR, if it is 5%, it is determined that the reaction is not recognized.

As is evident in Table 1, in asthmatic patients without IgE antibody, when an increase in IL_5 production from T cells was observed as a substantial difference in antigen reactivity as compared to control, Clear LAR was observed following inhalation of the antigen. The antigen also produced a delayed reaction in a separate skin test. These results indicate that it is possible to identify the antigen causing asthma using the production of IL-15 from T cells as an index. table 1

Antigen I L-5 (pg / ml)

I AR LA

1 force 239.3 <5 20.0

2.286.6 Ku 5 19.5

Acid protease 152.4 2.4 5 51.3 Mite extract 10 10 <5 5 5

3 591.0 5 13.0

Tick extract 271.0 <5 20.2

4 211.4 C 5 9.1

Acid Proteases 10 <5 <5 5 Candida Extract 79.6 Ku 5 19.1

Tick extract 191.9 5 20.0

6 Candida extract 55.6 Ku 5 15.8

Tick extract 10 10 5 10

rate (%)

L

Example 2 Induction of IL-15 production and lymphocyte (T cell) proliferation by Candida-derived antigens against PBMC in patients with non-atopic asthma

Does not have the I gE antibodies against mite antigen, non than patients atopic asthma to be diagnosed, the venous blood was collected, PB MC a 2 X 1 0 ⁶ cells harvested by Fuikoru one pack density centrifugation method / as a Uweru suspended in AIM-V medium at 24 © E Le plates at 37, and cultured in a 5% C_〇 ₂ incubator within one. The final concentration of each antigen is 10 / g / ml in a saline solution of each Candida-derived antigen [Candida extract, Candida acid protease, Candida mannan A (Candida, albicans-derived cell wall typeA mannan, manufactured by Takara Shuzo)]. As well as stimulated. In the control, physiological saline alone was added instead of the physiological saline solution of the antigen. After culturing for 6 days, the culture supernatant was collected, and the amount of IL-5 was measured by ELISA, and the difference between the case of stimulation with the antigen and the case of the control was examined. Fig. 1 shows the results for eight cases (5 cases for Candida mannan A) that reacted against Candida-derived antigens. In FIG. 1, the difference is referred to as IL-15 production (pgZm1). Put on.

In addition, AIM- the PB MC suspended in V medium was placed in a 1 X 1 0'll be ⁵ cells / Uweru sea urchin 9 6 © El plate. The antigen was added to 10 βg / ml, and after culturing for 6 days, ³ H-thymidine (0.5 CiZ perl) was added. After 18 hours of culture, the cells were collected, and the amount of ³ H-thymidine incorporation into the cells was measured as an indicator of T cell proliferation. In the control, physiological saline alone was added instead of the physiological saline solution of the antigen. Fig. 2 shows the results for eight cases (5 cases for Candida mannan A) that react with Candida-derived antigens.

As it is clear from FIGS. 1 and 2, Candida extract and obvious production induction of IL- 5 from T cells upon stimulation with Candida acid protease, and T cell proliferation by ³ H- thymidine Uptake was observed. On the other hand, stimulation with Candida mannan A did not provide enough data for induction of IL-5 production and T cell proliferation to be a substantial difference compared to controls. In any case, these results identify Candida acid protease as at least a causative antigen for these asthmatics. Example 3 Production of kit for identifying asthma-causing antigen

The following components are individually packaged to produce a kit for identifying the antigen causing asthma.

[Antigen] Candida albicans-derived acid protease (lyophilized product): lmg [Carrier] Physiological saline: 1 ml When using this kit, the whole amount of the above antigen is dissolved in 1 ml of physiological saline to obtain The antigen solution thus obtained is added to the medium in 1/1000 volume, and the medium is used for culturing T cells. Example 4 Production of a therapeutic agent for non-atopyic asthma

The respective components are mixed as follows, and a therapeutic agent for non-atopic asthma (antisensitizing therapeutic antigen) is produced according to a known method.

That is, 1 mg of Candida albicans-derived acid protease (freeze-dried product) is dissolved in physiological saline to which 0.5% phenol is added, and this is used as a stock solution of an antigen for the treatment of desensitization. In treatment, the above stock solution is diluted appropriately before use. Industrial applicability

According to the present invention, there is provided a method for identifying an antigen causing asthma, which is capable of identifying an antigen causing asthma, particularly non-atopic asthma. Also provided is a kit for identifying an asthma-causing antigen, which can easily and quickly identify an asthma-causing antigen. In addition, there is provided a therapeutic or preventive agent for non-atopic asthma, which comprises an asthma-causing antigen which can be identified by the identification method and the like. Further, the present invention provides a method for detecting an asthma-causing antigen capable of detecting the presence of an asthma-causing antigen in a test sample. Therefore, the present invention provides a very useful technique in the field of industry related to the treatment and prevention of asthma, especially non-atopic asthma.

Claims

The scope of the claims

1. Measure the amount of IL-15 produced from T cells due to the stimulation of the test substance, and the increase in the amount of IL-15 production is observed as compared to the case of cells not stimulated with the test substance. The method for identifying a causative antigen of bronchial asthma, wherein the test substance is identified as a causative antigen of bronchial asthma.

2. (a) a step of culturing the T cells in the presence of the test substance, and (b) a step of measuring the amount of IL-15 produced from the T cells,

When the difference between the amount of IL-5 produced from T cells and the T cells cultured in the absence of the test substance is 1 Opg / ml or more, the test substance is 2. The identification method according to claim 1, which is identified as a causative antigen of asthma.

3. The identification method according to claim 1 or 2, wherein the bronchial asthma is non-atopyic bronchial asthma.

4. A kit for identifying an antigen causing bronchial asthma, comprising an antigen capable of increasing the amount of IL-5 produced from T cells upon stimulation.

5. The kit according to claim 4, wherein the bronchial asthma is non-atopyic bronchial asthma.

6. A therapeutic or preventive agent for non-atopic bronchial asthma, comprising all or a part of the antigen causing bronchial asthma as an active ingredient.

7. The therapeutic or prophylactic agent according to claim 6, wherein the antigen causing bronchial asthma has been identified by the identification method according to any one of claims 1 to 3.

8. A therapeutic or preventive agent for non-atopyic bronchial asthma comprising an antifungal agent as an active ingredient.

9. The amount of IL-15 produced in the T cells contacted with the test sample was measured, and an increase in the amount of IL-5 production was observed as compared to the case of T cells not contacted with the test sample. A method for detecting an antigen causing bronchial asthma, wherein the test sample is determined to contain an antigen causing bronchial asthma.

10. (A) a step of culturing T cells in the presence of a test sample, and (b) a step of measuring the amount of IL-15 produced from the T cells,

When the difference between IL-15 production from T cells is 1 O pg / ml or more compared to when T cells are cultured in the absence of the test sample, 10. The detection method according to claim 9, wherein it is determined that an antigen causing bronchial asthma is present.

11. The detection method according to claim 9 or 10, wherein the bronchial asthma is non-atopyic bronchial asthma.