JP2002119281A - Testing methods for allergic diseases - Google Patents

Abstract

(57) [Problem] To provide a method for detecting an early allergic disease. [MEANS FOR SOLVING PROBLEMS] GM-CSF R β, GM-CSF R from known genes that are thought to be related to allergic diseases, in particular, seven genes that can be used as indicators of early allergic diseases.
α, IL-3Rα, PAF R, bcl-2, bcl-x, and CD44 were found. The present invention provides a method for testing an early allergic disease and a method for screening a compound useful for treatment, using the expression level of these genes in eosinophils as an index.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for testing allergic diseases.

[0002]

2. Description of the Related Art Allergic diseases such as atopic dermatitis are considered as multifactorial diseases. These diseases are caused by the interaction of the expression of many different genes, and the expression of these individual genes is affected by multiple environmental factors. For this reason,
It is very difficult to elucidate the specific gene that causes a specific disease.

[0003] Further, it is considered that allergic diseases involve the expression of genes having mutations or defects, and the overexpression or reduction of the expression level of specific genes. To understand the role gene expression plays in disease,
It is necessary to understand how genes are involved in the pathogenesis and how external stimuli such as drugs alter gene expression.

[0004] In the diagnosis of allergic diseases, questionnaire, family history, and confirmation of the patient's past illness are currently important factors. In addition, in order to diagnose allergy based on more objective information, a test method using blood as a sample and a method for observing a patient's immunological response to an allergen have been implemented. As an example of the former,
Examples include allergen-specific IgE measurement, leukocyte histamine release test, and lymphocyte blastogenesis test. The presence of allergen-specific IgE is evidence of an allergic reaction to the allergen. However, some patients may not always be able to detect allergen-specific IgE. In addition, due to its measurement principle, tests must be performed on all allergens required for diagnosis. The leukocyte histamine release test and lymphocyte blastogenesis test measure the response of the immune system to allergens in vitro.
This is the method of observation. These methods are complicated in operation. On the other hand, a method of using an immune response observed when a patient is actually brought into contact with an allergen for diagnosis of allergy (the latter) is also known. Such tests include brick tests, scratch tests, patch tests, intradermal reactions, or provocation tests. Although these tests can directly diagnose a patient's allergic reaction, they can be said to be highly invasive tests that actually expose the subject to an allergen.

[0005] In addition, test methods have been attempted to prove the involvement of allergic reactions regardless of allergens. For example, if the serum IgE level is high, it can be estimated that the patient has an allergic reaction. The serum IgE value is information corresponding to the total amount of allergen-specific IgE. Although it is easy to determine the total amount of IgE regardless of the type of allergen, IgE may be low in patients with diseases such as non-atopic bronchitis.

[0006] Eosinophil count and ECP value are diagnostic items related to a delayed-type reaction following allergic type I allergy or an allergic inflammatory response. The number of eosinophils is said to reflect the development of allergic symptoms. ECP (eosinophil cationic prot), a protein contained in eosinophil granules,
ein) is also strongly activated with asthma attack. These diagnostic items certainly reflect allergic symptoms. However, in reality, it is a general finding that an increase in eosinophils becomes remarkable as the allergic symptoms progress. Therefore, when there is a clear increase in eosinophils, remarkable allergic symptoms are often accompanied. Therefore, the number of eosinophils cannot be used as an index for an early allergic disease. Therefore, it would be useful to provide a marker for an allergic disease, which has a low risk to the patient and can easily obtain information necessary for early diagnosis. Since such a marker is considered to be deeply involved in the development of allergic diseases, it may be an important target not only in diagnosis but also in control of allergic symptoms.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an index which enables a test for an early allergic disease. Furthermore, an object of the present invention is to provide a method for testing an initial allergic disease and a method for screening a candidate compound for a therapeutic drug for allergic disease based on the index.

[0008]

Means for Solving the Problems It is considered that genes involved in early allergic diseases are located further upstream of allergic symptoms and play a role in inducing the expression of other genes. The present inventors thought that if such a gene could be identified, it would be possible to diagnose early allergic diseases by examining the expression state. The present inventors also thought that such a gene could be an important target also in treating allergic diseases. Effective drugs for early allergic diseases, not only in the early stages of allergy, but also after severe
It may be an effective treatment for the essential causes of the condition. Such therapeutic agents can be expected to have pharmacological actions that lead to the cure of allergies, not merely palliative treatment.

The present inventors have searched for genes having different expression states between patients with early allergic diseases and healthy persons based on such a concept. Eosinophils were selected as targets for comparing gene expression states. Eosinophils are an important indicator of allergic symptoms. Therefore,
In eosinophil cells, genes that cause differences in expression levels are thought to be closely related to allergic symptoms. In addition, by examining gene variation in patients with early allergic disease, it was thought that genes related to early allergic disease could be found. As a result of analyzing the expression state of genes in peripheral blood eosinophils based on such a strategy, the present inventors found that all of the following genes had increased expression in eosinophils of patients with early allergic diseases. I confirmed that. The abbreviation is described in parentheses after the name of each gene. Hereinafter, the name of each gene is described in abbreviated notation. Granulocyte macrophage colony stimulating factor receptor β (GM-CSFRβ), granulocyte macrophage colony stimulating factor receptor α (GM-CSFRα),
Interleukin 3 receptor α (IL3Rα), Bcl-2, Bcl-x,
Platelet activating factor receptor (PAFR), and CD44

[0010] All of these genes have been shown to be associated with eosinophil activation and apoptosis. The fact that fluctuations in the expression levels of these genes were observed in eosinophils of patients with early allergic diseases may be said to indicate a deep link between the genes and allergic symptoms. Based on the above findings, the present inventors have found that the use of these genes and proteins encoded by these genes as indices makes it possible to test for early allergic diseases and completed the present invention. . In addition, the present inventors have found that a therapeutic agent for an early allergic disease can be screened by using the expression levels of these genes or the activities of the proteins encoded by these genes as indicators, and completed the present invention. did. That is, the present invention relates to the following inspection methods and screening methods. [1] An initial method for testing an allergic disease, including the following steps. a) GM-CSFRβ, GM-CSFR in eosinophil cells of a subject
measuring the expression level of any gene selected from the group consisting of α, IL3Rα, Bcl-2, Bcl-x, PAFR, and CD44 b) Expression of the gene in eosinophil cells of a healthy subject [2] the allergic disease is atopic dermatitis,
The inspection method according to [1]. [3] The test method according to [1], wherein the expression level of the gene is measured by cDNA PCR. [4] The test method according to [1], wherein the expression level of the gene is measured by detecting a protein encoded by the gene. [5] GM-CSFRβ, GM-CSFRα, IL3Rα, Bcl-2, Bcl-x,
PAFR, and a polynucleotide comprising any base sequence selected from the group consisting of CD44, or an oligonucleotide having a length of at least 15 bases having a base sequence complementary to its complementary strand, Reagent for testing allergic diseases. [6] GM-CSFRβ, GM-CSFRα, IL3Rα, Bcl-2, Bcl-x,
An initial allergic disease test reagent comprising an antibody that recognizes a peptide containing the amino acid sequence of any protein selected from the group consisting of PAFR and CD44. [7] A method for screening a therapeutic agent for an early allergic disease, comprising the following steps. (1) GM-CSFRβ, GM-CSFRα, IL3Rα, Bcl-2, Bcl-x,
Contacting a candidate compound with cells expressing any gene selected from the group consisting of PAFR and CD44 (2) measuring the expression level of the gene;
(3) Step of selecting a compound that reduces the expression level of the gene as compared to a control [8] The method according to [7], wherein the cells are established leukocytes.

[9] A method for screening a therapeutic drug for an early allergic disease, comprising the following steps. (1) a step of administering a candidate compound to a test animal; (2) GM-CSFRβ, GM-CSFRα, IL in eosinophil cells of the test animal
Measuring the expression intensity of any gene selected from the group consisting of 3Rα, Bcl-2, Bcl-x, PAFR, and CD44; and (3) reducing the expression level of the gene as compared to a control. Step of selecting a compound to be reduced [10] A method for screening a therapeutic agent for an early allergic disease, comprising the following steps. (1) GM-CSFRβ, GM-CSFRα, IL3Rα, Bcl-2, Bcl-x,
Contacting a candidate substance with a cell into which a vector containing a transcription regulatory region of any gene selected from the group consisting of PAFR and CD44 and a reporter gene expressed under the control of this transcription regulatory region has been introduced; (2) a step of measuring the activity of the reporter gene; and (3) a step of selecting a compound that reduces the expression level of the gene as compared to a control. [11] An initial allergic disease comprising the following steps: How to screen for therapeutic drugs. (1) GM-CSFRβ, GM-CSFRα, IL3Rα, Bcl-2, Bcl-x,
Contacting a candidate substance with any protein selected from the group consisting of PAFR and CD44, (2) measuring the activity of the protein, and (3) activity of the protein as compared to a control Selecting a compound that reduces the [12] [7],

A remedy for allergic diseases, comprising as an active ingredient a compound obtainable by the screening method according to any one of [9], [10], and [11]. [13] GM-CSFRβ, GM-CSFRα, IL3Rα, Bcl-2, Bcl-
An agent for treating an early allergic disease, which comprises, as a main component, any gene selected from the group consisting of x, PAFR, and CD44, or a part of the antisense DNA. [14] GM-CSFRβ, GM-CSFRα, IL3Rα, Bcl-2, Bcl-
A transgenic gene having increased expression intensity in eosinophil cells of any gene selected from the group consisting of x, PAFR, and CD44, and / or any gene functionally equivalent to these genes Use of non-human vertebrates as model animals for early allergic disease.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, an allergic disease is a general term for diseases associated with allergic reactions. More specifically, it can be defined that an allergen is identified, a deep link is established between exposure to the allergen and the development of a lesion, and the lesion has an immunological mechanism. Here, the immunological mechanism means that leukocyte cells show an immune response by allergen stimulation. Examples of the allergen include a mite antigen and a pollen antigen. Representative allergic diseases include bronchial asthma, allergic rhinitis, atopic dermatitis, hay fever, and insect allergy. Allergic diathesis is a genetic factor transmitted from a parent with an allergic disease to a child. An allergic disease that develops familially is also called an atopic disease, and a genetically transmitted factor that causes it is atopic predisposition. Atopic dermatitis is a generic term given to atopic diseases, particularly those accompanied by skin inflammatory conditions.

The initial allergic disease in the present invention can be said to be a state in which the immune response to the white blood cells constituting the allergic reaction does not appear as a remarkable numerical change. In particular, numerical fluctuations of eosinophils, which are important indicators of allergic reactions, are closely related to the development of allergic symptoms. Therefore, for example, a state in which the number of eosinophils does not fluctuate but some allergic symptoms appear can be said to be an initial allergic disease. Some allergic symptoms include mild skin symptoms such as itching, rash, and redness in the case of atopic dermatitis. That is, for example, a patient who is generally diagnosed with mild atopic dermatitis can be referred to as an early allergic disease patient.

[0013] The method for testing an early allergic disease of the present invention comprises the steps of: GM-CSFRβ, GM-CSFRα, IL in eosinophils of a subject;
Measuring the expression level of any gene selected from the group consisting of 3Rα, Bcl-2, Bcl-x, PAFR, and CD44, and comparing it with the measurement value of a healthy subject. As a result of the comparison between the two, when the expression is higher than that in a healthy subject, the subject is determined to have an early allergic disease. In the present invention, the above seven genes that can be used as indicators of early allergic diseases are collectively referred to as indicator genes.
Unless otherwise specified, the indicator gene is used as a term indicating one or more arbitrary genes selected from the above seven genes.

In the method for testing an initial allergy according to the present invention, an indicator gene for which the expression level or activity is to be measured is selected from the seven indicator genes. Therefore, the test based on the present invention can be performed by measuring the expression level or activity of any one of the seven genes. Furthermore, in the present invention, test accuracy can be improved by measuring a plurality of types of indicator genes. For example, as a first group,
By measuring a combination of at least one indicator gene selected from the group consisting of GM-CSFRβ, GM-CSFRα, and IL3Rα, and Bcl-2 and / or Bcl-x as a second group, An improvement in inspection accuracy can be expected. Furthermore, it is also significant to measure CD44 as an indicator gene of the third group. In particular, when the following three genes are combined as indicator genes, the pathology of early allergy can be grasped sharply. GM-CSFRβ (first group) Bcl-2 and / or Bcl-x (second group) CD44 (third group) These genes are all activated by eosinophils based on different mechanisms. Is considered to be involved in Therefore,
By measuring the expression levels and activities of these genes in combination, qualitative changes in eosinophils in early allergies can be known from many aspects. In order to capture changes in eosinophils from multiple aspects, it is desirable to measure PAFR as a fourth group of genes. By performing an initial allergy test based on the results of simultaneous measurement of a plurality of indicator genes, the accuracy of diagnosis of an early allergic disease, which is difficult to diagnose, is improved.

In the present invention, the expression level of an indicator gene includes the transcription of these genes into mRNA and the translation into proteins. Therefore, the method for testing an allergic disease according to the present invention is performed based on a comparison between the expression intensity of mRNA corresponding to the gene or the expression level of a protein encoded by the gene.

The measurement of the expression level of the indicator gene in the test for allergic disease in the present invention can be carried out according to a known gene analysis method. Specifically, for example, a hybridization technique using a nucleic acid that hybridizes to the gene as a probe or a gene amplification technique using a DNA that hybridizes to the gene of the present invention as a primer can be used.

The probe or primer used for the test of the present invention can be set based on the nucleotide sequence of the indicator gene. The nucleotide sequence of the indicator gene is known. The GenBank accession numbers of the nucleotide sequences of the indicator genes are shown below. In general, genes of higher animals are frequently associated with polymorphism. Also, there are many molecules that generate isoforms consisting of mutually different amino acid sequences in the process of splicing. Even if the gene has a mutation in the nucleotide sequence due to polymorphism or isoform, any of the genes having the same activity as the indicator gene and involved in early allergy are included in the indicator gene of the present invention. GM-CSFRβ (M59941, M38275), GM-CSFRα (NM_006140), IL
3Rα (M74782), Bcl-2 (M13994, M13995), Bcl-x (U7239
8), PAFR (D10202, D90433), and CD44 (U40373)

As the primer or the probe, a polynucleotide comprising the nucleotide sequence of the indicator gene or a polynucleotide containing at least 15 nucleotides complementary to a complementary strand thereof can be used. Here, the “complementary strand” refers to one strand of a double-stranded DNA consisting of A: T (U in the case of RNA) and G: C base pairs with respect to the other strand.
Further, `` complementary '' is not limited to a completely complementary sequence in at least 15 consecutive nucleotide regions,
It is sufficient that they have at least 70%, preferably at least 80%, more preferably 90%, even more preferably 95% or more homology on the base sequence. The homology of the nucleotide sequences can be determined by an algorithm such as BLAST.

Such a polynucleotide can be used as a probe for detecting an indicator gene and as a primer for amplifying the indicator gene. When used as a primer, usually 15 bp to 10
It has a chain length of 0 bp, preferably 15 bp to 35 bp. When used as a probe, it has at least a part or all of the sequence of the indicator gene (or its complementary strand),
DNA with a chain length of at least 15 bp is used. When used as a primer, the 3′-side region needs to be complementary, but a restriction enzyme recognition sequence, a tag, or the like can be added to the 5′-side.

The "polynucleotide" in the present invention can be DNA or RNA. These polynucleotides may be synthetic or natural. Probes used for hybridization
Usually, labeled DNA is used. As the labeling method, for example, the following method can be shown. The term oligonucleotide means a polynucleotide having a relatively low degree of polymerization among polynucleotides. Oligonucleotides are included in polynucleotides. Labeling by nick translation using DNA polymerase I Terminal labeling using polynucleotide kinase Fill-in labeling using Klenow fragment (Be
rger SL, Kimmel AR. (1987) Guide to Molecular Clon
ing Techniques, Method in Enzymology, Academic Pre
ss; Hames BD, Higgins SJ (1985) Genes Probes: A Pr
actical Approach. IRL Press; Sambrook J, Fritsch E
F, Maniatis T. (1989) Molecular Cloning: a Laborat
ory Manual, 2nd Edn. Cold Spring Harbor Laboratory
Press) Labeling by transcription using RNA polymerase (Melton D
A, Krieg, PA, RebagkiatiMR, Maniatis T, Zinn K, Gre
en MR. (1984) Nucleic Acid Res., 12,7035-7056) ・ Incorporation of modified nucleotides without radioisotopes into DNA (KrickaLJ. (1992) Nonisotopic DNA P
robing Techniques. Academic Press)

The test for allergic diseases using the hybridization technique can be performed by using, for example, Northern hybridization, dot blotting, a method using a DNA microarray, or the like. Furthermore, gene amplification techniques such as the RT-PCR method can be used. In the RT-PCR method, the gene amplification process
By using the PCR amplification monitoring method, it is possible to perform more quantitative analysis on the expression of the gene of the present invention.

In the PCR gene amplification monitoring method, a probe labeled on each end with a different fluorescent dye that cancels each other's fluorescence is used to hybridize to a detection target (a reverse transcript of DNA or RNA). PCR reaction proceeds and Taq
5'-3 'exonuclease of polymerase (exonuclea
se) When the same probe is decomposed due to the activity, the two fluorescent dyes are separated and the fluorescence is detected. This fluorescence is detected in real time. Simultaneous measurement of a standard sample with a clear copy number for the detection target enables
Determine the number of copies to be detected in the target sample by the number of linear cycles of PCR amplification (Holland, PM et al., 19
91, Proc. Natl. Acad. Sci. USA 88: 7276-7280; Liva
k, KJ et al., 1995, PCR Methods and Application
s 4 (6): 357-362; Heid, CA et al., Genome Researc
h 6: 986-994; Gibson, EMU et al., 1996, Genome
Research 6: 995-1001). In the PCR amplification monitoring method, for example, ABI PRISM7700 (PE Biosystems)
Can be used.

The method of the present invention for detecting an allergic disease can also be carried out by detecting the protein encoded by the indicator gene. Hereinafter, in the present specification, a protein encoded by the indicator gene is referred to as an indicator protein. As such a test method, for example, a Western blotting method, an immunoprecipitation method, an ELISA method, or the like using an antibody that binds to these indicator proteins can be used.

An antibody that binds to the indicator protein used for this detection can be obtained using a technique well known to those skilled in the art. The antibodies used in the present invention are polyclonal antibodies or monoclonal antibodies (Milstein C, et al., 1983, N
ature 305 (5934): 537-40). For example, a polyclonal antibody against an indicator protein is obtained by extracting blood of a mammal sensitized with an antigen and separating serum from the blood by a known method. As the polyclonal antibody, serum containing the polyclonal antibody can be used. Alternatively, if necessary, a fraction containing a polyclonal antibody can be further isolated from the serum. In order to obtain a monoclonal antibody, immune cells are removed from a mammal sensitized with the above antigen, and are fused with myeloma cells or the like. The hybridoma thus obtained is cloned, and the antibody is recovered from the culture to obtain a monoclonal antibody.

For detection of the indicator protein, these antibodies may be appropriately labeled and used. Further, without labeling the antibody, a substance that specifically binds to the antibody, for example, protein A or protein G can be labeled and detected indirectly. A specific detection method includes, for example, an ELISA method. A protein or a partial peptide thereof used as an antigen can be prepared, for example, by incorporating the gene or a part thereof into an expression vector, introducing this into an appropriate host cell, preparing a transformant, and culturing the transformant to obtain a recombinant. The protein can be obtained by expressing the protein and purifying the expressed recombinant protein from a culture or a culture supernatant. Alternatively, an oligopeptide consisting of an amino acid sequence encoded by these genes or a partial amino acid sequence of the amino acid sequence encoded by full-length cDNA obtained based on SEQ ID NO: 1 is chemically synthesized and used as an immunogen. Can also.

Furthermore, in the present invention, an initial allergic disease can be tested using not only the expression level of the indicator gene but also the activity of the indicator protein in eosinophils as an indicator. The activity of the indicator protein refers to the biological activity of each protein. The activity of the indicator protein can be detected based on a known method.

GM-CSF Rβ and GM-CSF Rα bind granulocyte-macrophage colony stimulating factor (GM-SC
Construct a high affinity receptor using F) as a ligand. GM-C
SF Rβ and GM-CSF Rα are expressed in neutrophils and monocytes, and are also expressed in eosinophils, but not in lymphocytes. Binding to GM-SCF results in cell growth and enhanced function. GM-CSF R β is IL-3 or IL-5
Is a molecule common to the receptor β chain.

IL-3Rα, like GM-CSF Rβ and GM-CSF Rα, builds a high-affinity receptor by binding to a β chain. The β chain is common to GM-CSF R β. IL3R brings about enhanced function on eosinophils by binding to the ligand IL-3. PAF R is a platelet activating factor (PAF; Pla
It is a receptor that uses telet activating factor (1-alkyl-2-acetylglycero-3-phosphocholine) as a ligand. Activates neutrophils, macrophages, and eosinophils in addition to platelets.

[0029] bcl-2 and bcl-x belong to the so-called Bcl-2 gene family, and both are genes having a function of suppressing apoptosis. The splicing isoforms α and β have been reported for Bcl-2. What is actually expressed is mostly α. In the present invention, Bc
Reference to l-2 refers to all isoforms expressed on eosinophils. Similarly, Bcl-x in the present invention refers to all isoforms expressed on eosinophils.

[0030] CD44 is a transmembrane glycoprotein expressed in a wide range of cells in addition to leukocytes. It is known that ligands include hyaluronic acid, collagen types I and VI, and fibronectin. It has been shown that ligands of multiple intracellular proteins undergo phosphorylation via CD44. CD44 is known to have several splicing isoforms. CD44 as an indicator gene in the present invention refers to all isoforms expressed in eosinophils. Therefore, when measuring mRNA, it is desirable to use a base sequence common to isoforms as an index.

For example, GM-CSFRβ, GM-CSFRα, IL3R
Since α and PAFR are both receptor proteins, transduction of a signal into cells by binding of a ligand capable of binding to these receptors is important for the biological activity of each protein. I can say. Signal transduction by ligand binding can be detected using an increase in the calcium concentration of a cell, a change in cell morphology caused as a result of the signal transduction, or the like as an index. Since Bcl-2 and Bcl-x are both apoptosis-suppressing factors, their activities can be compared using apoptosis as an index. Biological activity can also be evaluated using the binding of these factors to molecules that interact with it as an index. CD44 can be evaluated for its biological activity using binding to the ligand, hyaluronic acid, as an index. In particular, fluorescently labeled hyaluronic acid is useful for detecting CD44 activity.

In the test method of the present invention, eosinophil cells of a subject are usually used as a sample. Eosinophil cells can be prepared from peripheral blood by a known method. That is, for example, heparin-collected blood is fractionated by centrifugation to separate white blood cells. Next, from white blood cells,
Granulocyte cells can be collected by centrifugation with Ficoll or the like, and further, eosinophil cells can be separated by neutrophil depletion using the CD16 antibody. If the separated eosinophils are destroyed to form a lysate, it can be used as a sample for immunological measurement of the protein. Alternatively, if mRNA is extracted from this lysate, it can be used as a sample for measuring mRNA corresponding to the gene. For extraction of eosinophil lysate and mRNA, it is convenient to use a commercially available kit.

Alternatively, the expression level of the gene to be used as an index in the present invention may be measured for whole blood or peripheral blood leukocyte populations without separating eosinophils. In this case, the change in the expression level of the gene in the cell can be obtained by correcting the measured value.
For example, based on a measured value of the expression level of a gene (housekeeping gene) which is specifically expressed in eosinophils and whose expression level does not largely vary irrespective of the state of the cell, the expression of a gene to be used as an index in the present invention Level measurements can be corrected. The indicator gene of the present invention showed an increase in the expression level of eosinophils in the patients in comparison between healthy subjects and patients with early atopic dermatitis. Therefore, an allergic disease test can be performed using the expression level of the indicator gene as an indicator.

The test for an allergic disease in the present invention includes, for example, the following tests. Even if a patient cannot be determined to be an allergic disease by a general test while showing symptoms of suspected allergy, the test according to the present invention can easily determine that the patient is an early allergic disease patient. Alternatively, a test can be performed to determine whether allergic symptoms are improving. The indicator gene of the present invention showed an increased expression level particularly in eosinophils of patients with early atopic dermatitis. Since eosinophils are a representative clinical marker of atopic dermatitis, genes whose expression fluctuates in eosinophils in association with allergic symptoms are useful for judging the therapeutic effect. More specifically, an increase in the expression of the indicator gene in a patient exhibiting the initial symptoms of suspected allergic disease indicates that the patient has an allergic disease.

The present invention also relates to the use of a transgenic non-human animal having an increased expression level of an indicator gene in eosinophil cells as a model animal for early allergic disease. Early allergic disease model animals are useful for elucidating in vivo changes in early allergy. Furthermore, the animal model for early allergic disease is
It is useful for evaluating drugs for allergy.

According to the present invention, it has been revealed that the expression level of the indicator gene in eosinophil cells is increased in eosinophils of patients with early atopic dermatitis. Therefore, animals in which the expression levels of these genes or genes functionally equivalent to these genes in eosinophil cells are artificially enhanced can be used as model animals for early allergic disease. The increase in the expression level in eosinophils includes an increase in the expression level of the target gene in the whole leukocyte population. That is, the expression level of the gene is increased not only by eosinophils but also by the expression level of the gene in the whole leukocyte population. In the present invention, a functionally equivalent gene is a gene that encodes a protein having an activity similar to that of the protein encoded by each indicator gene. A representative example of a functionally equivalent gene is a counterpart of a marker gene in a transgenic animal originally provided in a transgenic animal.

Genes whose expression is increased in early allergic diseases can be said to be genes that regulate the pathology of allergic diseases upstream. In other words, it is considered that allergic pathology is formed by the expression and suppression of various genes located downstream under the influence of genes that initiate activation in early allergic diseases. In other words, genes whose expression increases in early allergic diseases can be said to be genes that play an important role in pathogenesis of allergy. Therefore, drugs that suppress the expression of this gene or inhibit its activity
In the treatment of allergy, it is expected to not only improve the allergic symptoms but also remove the essential cause of allergic pathogenesis.

As described above, genes whose expression increases in early allergic diseases have important significance. Therefore, it is of great significance to use transgenic animals that can be obtained by increasing the expression level of this gene as an animal model for early allergic disease, and to evaluate the role of the gene and drugs that target the gene. is there. In particular, enhancing the expression of any one or all of the genes constituting the above-mentioned three groups, and even the genes constituting the four groups, which are important as indicator genes, is important in the pathogenesis of early allergy. In addition, the initial allergic disease model animal according to the present invention can be used to screen a drug for treating or preventing an early allergic disease, which will be described later, elucidate the mechanism of the initial allergic disease, and furthermore, the safety of the screened compound. Useful for testing. For example, an early allergic disease model animal according to the present invention develops dermatitis,
If a change in a measured value related to any allergic disease is indicated, a screening system for searching for a compound having an action to recover the change can be constructed.

In the present invention, the expression level increase refers to
A state in which the target gene is introduced as a foreign gene and is forcibly expressed, or a state in which the transcription and translation of a gene provided by the host are enhanced, and a state in which the degradation of the translation product protein is suppressed. Mean either. The expression level of the gene can be confirmed, for example, by quantitative PCR as described in Examples. The activity of the protein as a translation product can be confirmed by comparing with the normal state.

A typical transgenic animal is an animal into which a target gene has been introduced and forcibly expressed. Other transgenic animals include, for example, animals in which a mutation has been introduced into the coding region of a gene to enhance its activity or have been modified to an amino acid sequence that is less likely to be degraded. Mutations in the amino acid sequence can indicate substitutions, deletions, insertions, or additions. Others
By mutating the transcriptional regulatory region of the gene, the expression itself of the gene of the present invention can also be regulated.

A method for obtaining a transgenic animal for a specific gene is known. That is, a method in which a gene and an egg are mixed and treated with calcium phosphate, a method in which a gene is directly introduced into a nucleus of a pronuclear stage egg with a micropipette under a phase-contrast microscope (microinjection method, US Pat. No. 4,873,191), A transgenic animal can be obtained by a method using embryonic stem cells (ES cells) or the like. In addition, a method of inserting a gene into a retroviral vector and infecting an egg, a method of introducing a gene into an egg via sperm, and a sperm vector method have also been developed.
The sperm vector method is a genetic recombination method in which a foreign gene is introduced into sperm cells by attaching a foreign gene to sperm or by a method such as electroporation, and then fertilizing the egg to introduce the foreign gene (M. Lavitranoet et Cel
l, 57, 717, 1989).

The transgenic animal used as the animal model for the initial allergic disease of the present invention can be prepared using any vertebrate other than human. Specifically, transgenic animals in which various genes have been introduced or expression levels of which have been modified in vertebrates such as mice, rats, rabbits, minipigs, goats, sheep, and cattle have been created.

Further, the present invention relates to a method for screening a candidate compound for a therapeutic drug for early allergic disease. In the present invention, the expression level of each of the indicator genes is significantly increased in patients having an early allergic disease.
Therefore, by selecting a compound that can reduce the expression level of these genes, a therapeutic drug for early allergic disease can be obtained. In the present invention, the compound that reduces the expression level of a gene is a compound that acts to inhibit any of the steps of gene transcription, translation, and protein activity expression. The method for screening a candidate compound for treating an early allergic disease of the present invention can be performed in vivo or in vitro.
Can also be done. This screening can be performed, for example, according to the following steps. (1) a step of administering a candidate compound to a test animal, (2)
Measuring the expression level of the indicator gene in eosinophils of a test animal, and (3) selecting a compound that reduces the expression level of the indicator gene as compared to a control

As a test animal in the screening method of the present invention, for example, an early allergic disease model animal composed of a transgenic animal in which a human indicator gene is forcibly expressed can be used. When a promoter whose transcription is regulated by a substance such as an appropriate drug is used as the promoter used in the expression vector, the expression level of the exogenous indicator gene in the transgenic animal can be adjusted by administering the substance.

[0045] A drug candidate compound is administered to the model animal in which the indicator gene has been forcibly expressed in this manner, and the effect of the compound on the expression of the indicator gene in eosinophils of the peripheral blood of the model animal is monitored. The effect of the drug candidate compound on the expression level can be detected. The change in the expression level of the indicator gene in the eosinophils of the test animal can be monitored by the same method as the above-described test method of the present invention. Furthermore, if a drug candidate compound that reduces the expression level of the indicator gene is selected based on the result of this detection, the drug candidate compound can be screened. By such screening,
Drugs involved in various ways in the expression of the indicator gene can be selected. Specifically, for example, drug candidate compounds having the following action points can be found. Activation of signal transduction pathways that lead to expression of indicator genes, increase in transcriptional activity of indicator genes, stabilization or inhibition of degradation of transcripts of indicator genes, etc.

In the in vitro screening, for example, there is a method in which a candidate compound is brought into contact with cells expressing the indicator gene, and a compound that reduces the expression level of these genes is selected. This screening can be performed, for example, according to the following steps. (1) contacting a candidate compound with a cell that expresses the indicator gene; (2) measuring the expression level of the indicator gene; and (3) selecting a compound that reduces the expression level of the indicator gene as compared to a control. Process

In the present invention, cells expressing the indicator gene can be obtained by inserting the indicator gene into an appropriate expression vector and introducing the vector into an appropriate host cell. Usable vectors and host cells may be any as long as they can express the gene of the present invention. Examples of host cells in the host-vector system include Escherichia coli, yeast, insect cells, animal cells, and the like, and any available vector can be appropriately selected.

As a method for introducing a vector into a host, a biological method, a physical method, a chemical method and the like can be mentioned. Examples of the biological method include a method using a viral vector, a method using a specific receptor, a cell fusion method (HVJ (Sendai virus), polyethylene glycol (PEG), an electric cell fusion method, micronucleus fusion). Method (chromosome transfer)). Examples of the physical method include a microinjection method, an electroporation method, and a method using a gene particle gun. Examples of the chemical method include a calcium phosphate precipitation method, a liposome method, a DEAE dextran method, a protoplast method, an erythrocyte ghost method, an erythrocyte membrane ghost method, and a microcapsule method.

In the screening method of the present invention,
As a cell that expresses the indicator gene, an established leukocyte cell can also be used. Eol, YY-
Examples include leukocyte-derived cell lines such as 1, HL-60, TF-1, and AML14.3D10. Among leukocyte cell lines, cell lines derived from eosinophils are suitable for the screening method of the present invention. Cell lines derived from eosinophils are as follows. Eol YY-1 AML14.3D10

Eol (Eol-1: Saito H et al, Establishmen
t and characterization of a newhuman eosinophilic
leukemia cell line.Blood 66, 1233-1240, 1985)
It can be obtained from Hayashibara Research Institute. Similarly, YY-1 (Oga
ta N et al, The activation od the JAK2 / STAT5 pathw
ay is commonly involved in signaling through the h
uman IL-5 receptor. Int.Arch. Allergy Immunol., Su
ppl 1, 24-27, 1997) is provided by Site Signal Laboratory. AML14.3D10 (Baumann MA et al, The AML
14 and AML14.3D10 cell lines: a long-overdue model
for the study of eosinophils and more.Stem Cell
s, 16, 16-24, 1998) is a Research Ser.
vice, VA Commercial Center Available from Paul CC at Dayton.

In addition, the undifferentiated leukocyte cell line HL-60
Clone 15 (ATCC CRL-1964) can be differentiated into eosinophils and used as an eosinophil cell line if cultured for about one week in the presence of butyric acid. Eosinophils can be distinguished morphologically by the presence of eosinophil granules in polymorphonuclear nuclei. Morphological observation is performed by Giemsa staining or Diff Quick staining. In general, human leukocyte cell lines containing eosinophils can be established by cloning immortalized cells from a leukemia patient sample. Therefore, those skilled in the art can also obtain an eosinophil cell line by a known method, if necessary.

In the screening method, first, a candidate compound is added to the established leukocyte cells. Thereafter, the expression level of the indicator gene in the established leukocyte cells is measured, and a compound that reduces the expression level of the gene is selected.

In the screening method of the present invention, the expression levels of the indicator genes are determined not only by the expression levels of the proteins encoded by these genes but also by the corresponding mRs.
Comparison can also be made by detecting NA. In order to compare expression levels by mRNA, the step of preparing an mRNA sample as described above is performed instead of the step of preparing a protein sample. Detection of mRNA and protein can be carried out by a known method as described above.

Further, based on the disclosure of the present invention, a transcription regulatory region of the gene of the present invention can be obtained, and a reporter assay system can be constructed. The reporter assay system refers to an assay system that screens for a transcription regulatory factor acting on the transcription regulatory region, using the expression level of a reporter gene located downstream of the transcription regulatory region as an index. Examples of the transcription control region include a promoter, an enhancer, and a CAAT box, a TATA box, and the like usually found in a promoter region. As the reporter gene, a CAT (chloramphenicol acetyltransferase) gene, a luciferase gene, a growth hormone gene and the like can be used. Many of the indicator genes of the present invention have transcription regulatory regions already clarified. Alternatively, the transcription regulatory region of the indicator gene in the present invention can be obtained as follows. That is, first, based on the nucleotide sequence of the cDNA disclosed in the present invention, a human genomic DNA library such as a BAC library or a YAC library is screened by a method using PCR or hybridization.
Obtain a genomic DNA clone containing the sequence of the cDNA. Based on the sequence of the obtained genomic DNA, the transcription control region of the cDNA disclosed in the present invention is estimated, and the transcription control region is obtained. The obtained transcription regulatory region is cloned so as to be located upstream of the reporter gene to construct a reporter construct. The resulting reporter construct is introduced into a cultured cell line to obtain a transformant for screening. By contacting a candidate compound with this transformant, a compound that controls the expression of a reporter gene can be screened.

The test candidate compounds used in these screenings include, in addition to compound preparations synthesized by existing chemical methods such as steroid derivatives, compound preparations synthesized by combinatorial chemistry, extracts of animal and plant tissues, A mixture containing a plurality of compounds such as a culture of a microorganism, and a sample purified from the mixture, and the like.

The compounds selected by the screening method of the present invention are useful as therapeutics for early allergic diseases. The therapeutic agent for an allergic disease of the present invention contains a compound selected by the screening method as an active ingredient, and a physiologically acceptable carrier, excipient,
Alternatively, it can be produced by mixing with a diluent or the like. The therapeutic agent for allergic diseases of the present invention can be administered orally or parenterally for the purpose of improving allergic symptoms. As oral preparations, granules,
A dosage form such as a powder, a tablet, a capsule, a solvent, an emulsion, or a suspension can be selected. Injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like.

The dosage varies depending on the age, sex, weight and condition of the patient, the therapeutic effect, the administration method, the treatment time, and the type of active ingredient contained in the pharmaceutical composition. 0.1 mg to 500 mg per serving
, Preferably in the range of 0.5 mg to 20 mg. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be required. Hereinafter, the present invention will be described specifically with reference to Examples.
The present invention is not limited to these embodiments.

[0058]

[Example 1] Expression levels of seven genes in peripheral blood eosinophils and atopic dermatitis. Allergic conditions in blood cell cells isolated from peripheral blood of patients with atopic dermatitis and healthy subjects. The expression fluctuations of the genes considered to be related were analyzed. (1) Subject Table 1 shows the profiles of 10 atopic dermatitis patients who collected blood and 10 healthy subjects. Allergen non-specific (Total IgE), mite and cedar-specific IgE
Was measured by the EIA method. That is, the test serum was reacted with the cap to which the anti-human IgE antibody was bound, and the allergen non-specific IgE antibody or the mite or cedar-specific IgE antibody in the serum was bound. Next, a β-D-galactosidase-labeled anti-human IgE antibody and a substrate solution (4-methylumbelliferyl-β-D
-Galactopyranoside) and reacted to produce a fluorescent material. The reaction was stopped by adding a reaction stop solution, and the antibody concentration was determined from the fluorescence intensity of the standard IgE measured at the same time. LDH
In the measurement of, the reduction rate of NADH due to the reaction between pyruvate and NADH was calculated from the decrease in absorbance by the UV method (Wroblewski-La Due method). For measuring LDH value, use L-type Wako LDH
(Wako Pure Chemical Industries) and 7170 automatic analyzer (Hitachi) were used.
The number of eosinophils can be determined by microscopy and SE-9000 (RF / DC impedance method,
Sysmex). In the table, “○” of the disease state indicates a remission period. Specific IgE (S IgE) is anti-mite Ig
In E, Class 0 to 1 were "-" and Class 2 to 6 were "+". Total IgE (T IgE) is 1000 IU / ml or less as "Low (L)",
The case where it was more than 1000 IU / ml was regarded as “High (H)”. For eosinophils, "A" was used for less than 3%, "B" for 3-7%, and "C" for more than 7%.

[0059]

[Table 1]

(2) Expression analysis and analysis A 3% dextran solution was added to about 10 mL of peripheral blood of patients with mild atopic dermatitis and healthy persons (Table 1), and left at room temperature for 30 minutes to sediment erythrocytes. Collecting the upper leukocyte fraction,
The solution was placed on a Ficoll solution (Ficoll-Paque PLUS; Amersham Pharmacia Biotech) and centrifuged at 1500 rpm for 30 minutes at room temperature. The granulocyte fraction collected in the lower layer was reacted with CD16 antibody magnetic beads at 4 ° C. for 30 minutes, and cells eluted without being trapped by separation using MACS were used as eosinophils in the experiment. The eosinophil prepared as described above was dissolved in Isogen (Nippon Gene; Wako Pure Chemical), and RNA was isolated from this solution according to the protocol attached to Isogen. Chloroform was added, and the mixture was centrifuged with stirring to collect an aqueous layer. Next, isopropanol was added, and the mixture was centrifuged with stirring to collect the total RNA in the precipitate. The recovered total RNA was added with DNase (Nippon Gene; Wako Pure Chemical Industries, Ltd.), reacted at 37 ° C. for 15 minutes, extracted with phenol-chloroform, and recovered by ethanol precipitation. Dn RNA
ase treatment, cDNA synthesis, and expression of various known genes in eosinophils were quantified by real time RT-PCR (ABI7700) using primers and TaqMan probe. Primers and TaqMan probes used for measurement by ABI 7700 were designed by Primer Express (PE Biosystems) based on the sequence information of each gene. The 5 'end of the TaqMan probe is FAM
(6-carboxy-fluorescein) and the 3 'end is TAMRA (6-car
boxy-N, N, N ', N'-tetramethylrhodamine). The nucleotide sequences of the primer and the TaqMan probe used in the experiment are as shown in SEQ ID NOs shown in Table 2, respectively.

[0061]

[Table 2]

As a template, a cDNA reverse transcribed from a total RNA using poly T (12 to 18 mer) as a primer was used. For a standard curve for calculating the copy number, a plasmid clone containing a nucleotide sequence region amplified by both primers was prepared for each gene, and the reaction was performed using the serial dilution as a template.
The composition of the reaction solution for monitoring the PCR amplification is shown in Table 3.

[0063]

[Table 3]

Further, in order to correct the difference in cDNA concentration in the sample, the same quantitative analysis was performed on the β-actin gene as an internal standard for correction, and correction was performed based on the copy number of those genes. And the copy number of the target gene was calculated. For the quantification of β-actin gene, human cDN
A was used as a template. Primers and probes for β-actin measurement were obtained from TaqMan β-actin Control Reagents (PE
Biosystems) was used. The base sequence is as follows. The expression levels (copy / ng RNA) of each gene corrected by β-actin are shown in Tables 4 to 10 and FIGS. 1 to 7. In the table, Blood IDs 1 to 8 are measured values of healthy subjects, and 9 and later are measured values of patients with atopic dermatitis. β actin forward primer (SEQ ID NO: 22) TCA CCC ACA CTG TGC CCA TCT ACG A β actin reverse primer (SEQ ID NO: 23) CAG CGG AAC CGC TCA TTG CCA ATG G β actin TaqMan probe (SEQ ID NO: 24) 5 '-(FAM) ATGCCC-T (TAMRA) -CCCCCATGCCATCCTGCGTp-3' FAM: 6-carboxy-fluorescein TAMRA: 6-carboxy-N, N, N ', N'-tetramethylrhodamine

[0065]

[Table 4]

[0066]

[Table 5]

[0067]

[Table 6]

[0068]

[Table 7]

[0069]

[Table 8]

[0070]

[Table 9]

[0071]

[Table 10]

(3) Statistical Analysis Using the above data, a parametric multiple comparison test and a non-parametric multiple comparison test were performed.
Statistical analysis, The SAS SYSTEM SAS preclinical package Ver
sion 4.0 (SAS Institute Inc.). Table 11 shows the results.

[0073]

[Table 11]

From the group of eosinophil-specific genes and genes considered to be related to the pathology of allergy, GM-CSF R
β, GM-CSF R α, IL-3R α, PAF R, bcl-2, bcl-x, and seven genes of CD44 were significantly different from healthy subjects in peripheral blood eosinophils in patients with mild atopic dermatitis. It was found to be rising (Table 11).

Although CD44 is known as a connective tissue receptor such as fibronectin and hyaluronic acid, it is an eosinophil activation marker in vitro and its expression is enhanced by IL-5. In addition, apoptosis-related protein, bcl-2,
Itro has been reported that expression is enhanced by IL-5. Increased expression of GM-CSF Rα, GM-CSF Rβ, and IL-3Rα increases signal input to eosinophils via cytokine receptors such as IL-5 in early atopic dermatitis , Eosinophils are activated and proliferate and anti-apoptotic
It is shown that it is in a state. The movements of these genes correspond well with the enhanced expression of CD44, bcl-2 and bcl-x. Generally, IL-5 is considered to be a cytokine that acts to activate eosinophils and progress allergic symptoms.

Here, the anti-apoptotic state refers to the state itself in which the apoptosis-suppressing gene bcl-2 is expressed. After culturing the isolated peripheral blood eosinophils for several days,
There have been many reports of experiments examining functions. Eosinophils usually die in 3 to several days when cultured in serum-containing medium alone. However, when peripheral blood eosinophils are cultured with IL-5,
It was reported that bcl-2 was expressed in the cells at this time, and the cells at this time were reported (Clin.Exp.Immunol., 107, 1).
98-204, 1997). However, in reality, eosinophil activation and ant
There are various variations of the i-apoptotic state. For example, the cell lifespan of eosinophils stimulated with interferon gamma is prolonged as with IL-5 stimulation. However, eosinophils at this time do not express bcl-2 (Clin.Ex.
p. Immunol., 107, 198-204, 1997). In other words, in vitro
In fact, the fact that Bcl-2 expression is induced in E. coli cannot predict changes occurring in eosinophils in patients with actual allergic diseases.

The present inventors have shown that eosinophils expressed bcl-2 in patients with early atopic dermatitis, as in the case of IL-5 stimulation in vitro. In other words, eosinophils in patients with early atopic dermatitis
It was revealed to be in an ic state. Unlike moderate or severe cases, the number of eosinophils in the blood is not significantly increased in patients with mild atopic dermatitis. Therefore, the fact that qualitative changes in eosinophils are induced in patients with early atopic dermatitis gives very important information in the diagnosis of allergic diseases. By examining bcl-2 expression in peripheral blood eosinophils, eosinophil pathogenesis and signs of allergy may be diagnosed earlier. Bcl-2
Similarly, it has recently been reported that CD44 also enhances expression of eosinophils by inducing IL-5 stimulation in vitro (Am.
J. Res. Cell Mol. Biol., 18, 860-866, 1998). However, similarly to Bcl-2, CD44
The fact that D44 expression is induced cannot predict changes occurring in eosinophils in patients with actual allergic diseases.

The above findings indicate that eosinophils show an IL-5-dependent activation state and an anti-apoptotic state in mild atopic dermatitis in a healthy state, Measuring the expression of these seven genes has shown diagnostic value in atopic dermatitis.

By the way, in patients with eosinophilia (HES) or the like, it is known that the morphology of peripheral blood eosinophils changes in a condition where eosinophils increase in the blood. Eosinophils whose form has been changed are called low-density eosinophils because their specific gravity decreases with the change in form. Low-density eosinophils often show an activated state, such as enhanced expression of Fc receptor or increased production of active oxygen and leukotriene. In the case of atopic dermatitis, it was also found that such eosinophils appeared in correlation with clinical symptoms. It can be said that the indicator gene in the present invention indicates a precursor of eosinophil morphological change.

[0080]

Industrial Applicability According to the present invention, in a patient with early stage atopic dermatitis, the expression of genes involved in eosinophil activation and pathological changes is enhanced at a stage where the number of peripheral blood eosinophils is not significantly changed. It was found to be. Genes whose expression is increased prior to eosinophilia can be used as a very sensitive indicator of allergic symptoms. It is usually difficult to diagnose allergic symptoms at the stage where there is no increase in eosinophils. However, the index provided by the present invention enables early diagnosis, which is difficult with conventional diagnostic indices. Since early diagnosis has been made possible, an accurate treatment method can be selected even for an early allergic disease.

Eosinophil recruitment is an important step in allergic reactions. Therefore, prior to the eosinophils increase,
Genes with increased expression in eosinophils are thought to play an important role in allergic diseases, especially in the early stages. Moreover, the genes used as indices in the present invention are all genes that are considered to be related to allergic symptoms in some form. There are many genes that may be related to allergic symptoms, and among them, the present invention, which has clarified the relationship between specific genes and early allergic diseases, is particularly significant. As described above, all of the genes that serve as indices in the present invention are associated with their pathological state, and their suppression is a target of a therapeutic strategy for allergic diseases, and suppressing them is important.
It is expected to be useful as a new clinical diagnostic index for monitoring in such new treatment methods.

The indicator gene provided by the present invention comprises:
Regardless of the type of allergen, its expression level can be easily known. Therefore, the pathology of the allergic reaction can be comprehensively grasped. In addition, the method of testing for allergy according to the present invention can analyze the expression level of peripheral blood eosinophils as a sample, and therefore has low invasiveness to patients. Moreover, regarding gene expression analysis, unlike the measurement of proteins such as ECP, highly sensitive measurement using a small amount of sample is possible. Gene analysis technology has been increasing in throughput and lowering its price year by year. Therefore, the method of testing for allergy according to the present invention is expected to be an important diagnostic method at the bedside in the near future. In this sense, the diagnostic value of these disease-related genes is high.

[0083]

[Sequence List] SEQUENCE LISTING <110> Genox Research, Inc. The President of National Children's Hospital <120> Method for examination for allergosis <130> G1-A0005 <140> <141> <160> 24 <170> PatentIn Ver. 2.1 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 1 tggagtggcc tctggttatg 20 <210> 2 <211> 22 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 2 gggaactagg gagacagacg ag 22 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: an artificially synthesized TaqMan probe sequence <220> <221> misc_binding <222> (1) <223> Label FAM (6-carboxy-fluorescein) <220> <221> misc_binding <222 > (29) <223> Label TAMRA (6-carboxy-N, N, N ', N'-tetramethylrhodamine) <400> 3 ctgcagacct ggtattcacc ccaaactca 29 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> De scription of Artificial Sequence: an artificially 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<212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized TaqMan probe sequence <220> <221> misc_binding <222> (1 ) <223> Label FAM (6-carboxy-fluorescein) <220> <221> misc_binding <222> (27) <223> Label TAMRA (6-carboxy-N, N, N ', N'-tetramethylrhodamine) <400 > 9 atgaaagcaa aggctcagca gttgacc 27 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 10 tgtgggagct gcatcctact tc 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 11 ctcaaagcag cgagtgacgt t 21 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized TaqMan probe sequence <220> <221> misc_binding <222> (1) <223> Label FAM (6-carboxy-fluorescein) <220> <221> misc_binding <22 2> (26) <223> Label TAMRA (6-carboxy-N, N, N ', N'-tetramethylrhodamine) <400> 12 tcatcctgga ctccaccaac acagtg 26 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 13 acatgacccc accgaactca 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 14 tggaggagct cttcagggac 20 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized TaqMan probe sequence < 220> <221> misc_binding <222> (1) <223> Label FAM (6-carboxy-fluorescein) <220> <221> misc_binding <222> (24) <223> Label TAMRA (6-carboxy-N, N , N ', N'-tetramethylrhodamine) <400> 15 aggccacaat cctcccccag ttca 24 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 16 gcgtagacaa ggaga tgcag gt 22 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 17 ggtcattcag gtaagtggcc at 22 <210> 18 <211 > 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially stynthesized TaqMan probe sequence <220> <221> misc_binding <222> (1) <223> Label FAM (6-carboxy -fluorescein) <220> <221> misc_binding <222> (23) <223> Label TAMRA (6-carboxy-N, N, N ', N'-tetramethylrhodamine) <400> 18 ttggtgagtc ggatcgcagc ttg 23 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 19 gacctctgca aggctttcaa 20 <210> 20 <211> 21 <212> DNA <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 20 tccgatgctc agagctttct c 21 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Ar tificial Sequence: an artificially synthesized TaqMan probe sequence <220> <221> misc_binding <222> (1) <223> Label FAM (6-carboxy-fluorescein) <220> <221> misc_binding <222> (23) <223> Label TAMRA (6-carboxy-N, N, N ', N'-tetramethylrhodamine) <400> 21 accttgccca caatggccca gat 23 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 22 tcacccacac tgtgcccatc tacga 25 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 23 cagcggaacc gctcattgcc aatgg 25 <210> 24 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized TaqMan probe sequence <220> <221> misc_binding <222> (1) <223> Label FAM (6-carboxy-fluorescein) <220> <221> misc_binding <222> (7) <223> Label TAMRA (6-carboxy-N, N, N ', N' -tetramethylrhodamine) <400> 24 atgccctccc ccatgccatc ctgcgt 26

[Brief description of the drawings]

FIG. 1 is a diagram showing the expression level (copy / ng RNA) of GM-CSF R β gene in eosinophils of healthy subjects (open columns) and patients with early atopic dermatitis (hatched columns).

FIG. 2 is a graph showing GM-CSF R α gene expression levels (copy / ng RNA) in eosinophils of healthy subjects (open columns) and patients with early atopic dermatitis (hatched columns).

FIG. 3 shows IL-3Rα gene expression levels (copy / ng RNA) in eosinophils of healthy subjects (open columns) and patients with early atopic dermatitis (hatched columns).

FIG. 4 shows PAFR gene expression levels (copy / ng RNA) in eosinophils of healthy subjects (open columns) and patients with early atopic dermatitis (hatched columns).

FIG. 5 is a diagram showing the expression level (copy / ng RNA) of bcl-2 gene in eosinophils of healthy subjects (open columns) and patients with early atopic dermatitis (hatched columns).

FIG. 6 is a diagram showing bcl-x gene expression levels (copy / ng RNA) in eosinophils of healthy subjects (open columns) and patients with early atopic dermatitis (hatched columns).

FIG. 7 is a view showing CD44 gene expression levels (copy / ng RNA) in eosinophils of healthy subjects (open columns) and patients with early atopic dermatitis (hatched columns).

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61P 37/08 C12Q 1/02 4C086 C12N 5/10 1/68 A C12Q 1/02 G01N 33/15 Z 1 / 68 33/50 Z G01N 33/15 C12R 1:91) 33/50) // (C12N 15/09 ZNA (C12Q 1/02 C12R 1:91) C12R 1:91) (C12N 5/10 C12N 15 / 00 ZNAA C12R 1:91) 5/00 B (C12Q 1/02 C12R 1:91) C12R 1:91) (72) Inventor Ryoichi Hashida 3-35-31 Taishido, Setagaya-ku, Tokyo National Children's Medical Research Within the Center Within Genox Research Institute (72) Inventor Kaoru Ogawa 3-35-31 Taishido, Setagaya-ku, Tokyo National Children's Medical Research Center (72) Inventor Tomoko Fujishima 6-26-1, Jingumae, Shibuya-ku, Tokyo Kirin Brewery Pharmaceutical Business Division Licensing Department (72) Inventor Gozo Tsujimoto 3-35-31 Taishido, Setagaya-ku, Tokyo National Children's Hospital Children's Medical Research Center F-term (Reference) 2G045 AA02 AA29 AA34 AA35 BB01 BB10 BB20 CA15 CB17 DA13 FA16 FB02 FB03 4B024 AA11 BA80 CA04 CA09 CA11 DA03 HA13 4B063 QA01 QA19 QQ08 QQ52 QR80 QR80 QS25 QS36 QX02 4B065 AA93X AB01 BA02 CA44 CA46 4C084 AA17 DA45 DA46 NA14 ZB131 ZB132 4C086 AA01 AA04 EA16 NA14 ZB13

Claims (14)

[Claims] 1. A method for testing an early allergic disease, comprising the following steps: a) Granulocyte macrophage colony stimulating factor receptor β, granulocyte macrophage colony stimulating factor receptor α, interleukin 3 receptor α, Bcl-2, Bcl-x, platelet activation in eosinophil cells of a subject Measuring the expression level of any gene selected from the group consisting of factor receptor and CD44 b) comparing the expression level of the gene in eosinophil cells of a healthy subject 2. The test method according to claim 1, wherein the allergic disease is atopic dermatitis. 3. The method according to claim 1, wherein the expression level of the gene is measured by cDNA PCR. 4. The method according to claim 1, wherein the expression level of the gene is measured by detecting a protein encoded by the gene.
Inspection method described in 1. 5. Granulocyte macrophage colony stimulating factor receptor β, granulocyte macrophage colony stimulating factor receptor α, interleukin 3 receptor α, Bcl-2, Bcl
-X, a polynucleotide comprising any base sequence selected from the group consisting of a platelet activator receptor and CD44, or at least 15 bases having a base sequence complementary to a complementary strand thereof. An initial allergic disease test reagent comprising an oligonucleotide having 6. Granulocyte macrophage colony stimulating factor receptor β, granulocyte macrophage colony stimulating factor receptor α, interleukin 3 receptor α, Bcl-2, Bcl
-An initial allergic disease test reagent consisting of an antibody recognizing a peptide containing the amino acid sequence of any protein selected from the group consisting of x, a platelet activator receptor, and CD44. 7. A method for screening a therapeutic agent for an early allergic disease, comprising the following steps: (1) granulocyte macrophage colony stimulating factor receptor β, granulocyte macrophage colony stimulating factor receptor α,
Interleukin 3 receptor α, Bcl-2, Bcl-
x, a step of contacting a candidate compound with a cell expressing any gene selected from the group consisting of a platelet activating factor receptor and CD44 (2) a step of measuring the expression level of the gene, B) selecting a compound that reduces the level of expression of the gene as compared to a control 8. The method according to claim 7, wherein the cells are established leukocytes. 9. A method for screening a therapeutic agent for an early allergic disease, comprising the following steps: (1) Step of administering a candidate compound to a test animal; (2) Granulocyte macrophage colony stimulating factor receptor β, granulocyte macrophage colony stimulating factor receptor α, interleukin 3 receptor α in eosinophil cells of the test animal , Bc
measuring the expression intensity of any gene selected from the group consisting of 1-2, Bcl-x, platelet activating factor receptor, and CD44, and (3) comparing the expression strength of said gene with respect to a control. Step of selecting a compound that reduces the expression level 10. A method for screening a therapeutic agent for an early allergic disease, comprising the following steps: (1) granulocyte macrophage colony stimulating factor receptor β, granulocyte macrophage colony stimulating factor receptor α,
Interleukin 3 receptor α, Bcl-2, Bcl-
x, a cell into which a vector containing a transcription regulatory region of any gene selected from the group consisting of a platelet activating factor receptor and CD44 and a reporter gene expressed under the control of this transcription regulatory region is introduced. (2) measuring the activity of the reporter gene, and (3) selecting a compound that reduces the expression level of the gene compared to a control. 11. A method for screening a therapeutic drug for an early allergic disease, comprising the following steps: (1) granulocyte macrophage colony stimulating factor receptor β, granulocyte macrophage colony stimulating factor receptor α,
Interleukin 3 receptor α, Bcl-2, Bcl-
x, a protein selected from the group consisting of a platelet activating factor receptor and CD44, and a candidate substance; (2) a step of measuring the activity of the protein; and (3) a control and Selecting a compound that reduces the activity of the protein by comparison 12. A remedy for allergic diseases, comprising as an active ingredient a compound obtainable by the screening method according to any one of claims 7, 9, 10, and 11. 13. Granulocyte macrophage colony stimulating factor receptor β, granulocyte macrophage colony stimulating factor receptor α, interleukin 3 receptor α, Bcl-2, Bc
A therapeutic agent for an early allergic disease, comprising, as a main component, any gene selected from the group consisting of lx, a platelet activating factor receptor, and CD44, or a part of the antisense DNA. 14. Granulocyte macrophage colony stimulating factor receptor β, granulocyte macrophage colony stimulating factor receptor α, interleukin 3 receptor α, Bcl-2, Bc
lx, a platelet activating factor receptor, and any gene selected from the group consisting of CD44, and / or
Alternatively, use of a transgenic non-human vertebrate as an animal model for early allergic disease in which the expression level of any gene functionally equivalent to these genes in eosinophil cells is increased.