JP2006174740A - Disease marker of allergic disease and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tool for the sure and easy diagnosis of allergic diseases including asthma and especially provide a means for contributing to the development of medicines effective for controlling the secretion of sputum and remodeling of epidermal cell. <P>SOLUTION: The invention provides a disease marker for allergic diseases containing a polynucleotide having a base sequence containing at least 15 consecutive bases of amphiregulin gene and/or a polynucleotide complementary to the above polynucleotide, a disease marker for allergic diseases containing an antibody recognizing amphiregulin, a method for the diagnosis of allergic diseases using the disease marker, and a method for screening a substance suppressing the expression or function of amphiregulin. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to disease markers for allergic diseases and uses thereof. Specifically, the present invention relates to a marker suitable for diagnosis such as asthma sputum test and its use.

Bronchial asthma is characterized by reversible airflow obstruction and airway hyperreactivity. Goblet cell hyperplasia has been established as a pathological feature of mild, moderate, and severe asthma. Abnormal goblet cell count is accompanied by changes in accumulated and secreted mucins. Mucus hypersecretion is often a prominent feature, especially in the status of asthma attacks. The presence of excessive secretion of mucus is associated with a significant reduction in the forced expiratory volume per second (FEV ₁ : 1 second volume). Currently, topical application of glucocorticoids is widely recognized as a first line treatment for bronchial asthma. Although treatment with steroids has been reported to prevent the development of allergen-induced goblet cell hyperplasia in animal models, once goblet cell hyperplasia has been established, the effect is reduced. Furthermore, in humans, treatment with corticosteroids such as glucocorticoids is largely ineffective in the relationship between tissue remodeling and mucus production, both pathologically and clinically. Excessive secretion of mucus is an important cause of morbidity and death in asthmatic patients and there is currently no effective treatment, so further therapeutic targets and strategies are urgently needed.

  As diseases accompanied by excessive vaginal secretion, asthma, chronic obstructive pulmonary disease (COPD), bronchitis due to infection, and the like are known. Although some patients with COPD have asthma, it is possible to distinguish COPD from asthma based on chest image findings and clinical symptoms. Asthma patients and COPD patients with no complication of infection are sticky, elastic, with white wrinkles. On the other hand, infectious bronchitis often produces yellowish wrinkles. From this point of view, it is currently determined whether or not asthma is associated with infection by qualitative examination based on the characteristics of sputum. Although 30% of asthmatic patients have chronic epilepsy and 70% have been shown to be a critical symptom during epileptic seizures, a test method for epilepsy based on indices characteristic of asthma has been established. Not.

  At the experimental level, animals sensitized and exposed to allergens develop significant goblet cell hyperplasia (Non-Patent Documents 1 and 2). The pathogenesis underlying allergen-induced goblet cell hyperplasia in mice is probably due to upregulation of expression of mucin genes (eg, MUC2 gene, MUC5AC gene, etc.) IL-4, IL-13 and IL- Various mediators including 9 are believed to be involved, such as the epidermal growth factor (EGF) system, the disintegrin and metalloprotease families, and ion channels such as gob-5 (hCLCA1 in humans). The main effector cells appear to be the Th2 subtype of CD4 + lymphocytes. However, IgE high affinity receptor (FcεRI) knockout mice challenged with allergens had less airway inflammation and goblet cell hyperplasia and lower levels of IL-13 in lung homogenates compared to controls. (Non-Patent Document 3). Furthermore, in the human airway epithelial cell culture system, only IL-9 but not IL-4 or IL-13 increased the expression of the mucin gene (Non-patent Document 4).

  Mast cells play a central role in immediate inflammatory allergic reactions that can lead to asthma. Crosslinking FcεRI on mast cells activates signaling pathways that lead to degranulation, synthesis of de novo arachidonic acid metabolites and production of various cytokines / chemokines. The present inventors have reported that human mast cells express epiregulin as one of specific genes mediated by FcεRI (Non-patent Document 5). Epiregulin is a member of the EGF family, and the EGF family consists of EGF, amphiregulin (AREG), heparin-binding EGF, transforming growth factor-α (TGF-α), betacellulin, epiregulin and neuregulin.

AREG was first purified from the conditioned medium of MCF-7 human breast cancer cells treated with 12-ο-tetradecanoylphorbol-13-acetate (Non-Patent Document 6). The carboxy-terminal side of the AREG molecule exhibits a homology characteristic of EGF and can therefore be classified as a member of the EGF family. AREG binds to the EGF receptor and, like EGF and TGF-α, plays an important role in cell proliferation, cell survival and cell differentiation. AREG is synthesized as a transmembrane precursor with the characteristics of a secreted protein that is finally released by protease cleavage. In vitro, it has been reported that the level of AREG produced in mast cells is higher than the level produced in epithelial cells (Non-patent Document 7).
Am J Respir Crit Care Med 2001; 163: 674-679 Am J Respir Crit Care Med 2000; 161: 627-635 J Immunol 2004; 172: 6398-6406 J Clin Invest 1999; 104: 1375-1382 Blood 2003; 102: 2547-2554 Proc Natl Acad Sci USA 1988; 85: 6528-6532 Am J Respir Cell Mol Biol 2004; 30: 470-478

  An object of the present invention is to provide a reliable and easy diagnostic tool for allergic diseases including asthma, and means for contributing to the development of a drug capable of regulating sputum secretion and epithelial cell remodeling, in particular.

  We hypothesized that human mast cells produce molecules that induce goblet cell hyperplasia after FcεRI aggregation, and that the expression of these molecules is not inhibited by glucocorticoids. The present inventors examined the gene expression profile mediated by FcεRI using a high-density oligonucleotide probe array, and performed clustering analysis depending on the effect of glucocorticoid on the gene expression mediated by FcεRI in human mast cells. The present inventors have found a molecule related to remodeling among cluster genes whose expression was not inhibited by dexamethasone, and gene expression profiles of human blood mononuclear cells, eosinophils and neutrophils Mast cell specific molecules were selected by comparison with. The amphiregulin gene thus selected was included in a subset of genes that are up-regulated by FcεRI aggregation but not down-regulated by dexamethasone pretreatment.

  As a result of examining the effect of amphiregulin on mucin gene expression in human epithelial cells in vitro and in vivo, the present inventors have found a close relationship between the expression of amphiregulin and diseases caused by antigen sensitization, The present invention has been completed. That is, the present invention is as follows.

[1] A disease marker for an allergic disease comprising a polynucleotide having a base sequence of at least 15 consecutive bases of an amphiregulin gene and / or a polynucleotide complementary to the polynucleotide.
[2] The disease marker according to [1], wherein the allergic disease is asthma, atopy or allergic rhinitis, and is used as a probe or primer in the examination of the disease.
[3] The disease marker according to [1] or [2], wherein the allergic disease is asthma, and is used for sputum testing.
[4] A method for diagnosing allergic diseases, comprising the following steps (A) and (B):
(A) A step of measuring the expression level of an amphiregulin gene in a biological sample of a subject, and (B) a step of determining the presence or absence of an allergic disease based on the measurement result of (A).
[5] A method for diagnosing allergic diseases, comprising the following steps (a), (b) and (c):
(A) a step of binding RNA prepared from a biological sample of a subject or a complementary polynucleotide transcribed from the RNA and the disease marker according to any one of [1] to [3] above,
(B) a step of measuring RNA derived from a biological sample bound to the disease marker or a complementary polynucleotide transcribed from the RNA using the disease marker as an index,
(C) A step of determining the presence or absence of an allergic disease based on the measurement result of (b).
[6] The determination of the presence or absence of allergic disease in step (c) is an indicator that the amount of binding to the disease marker is increased by comparing the measurement result obtained for the subject with the measurement result obtained for the normal subject. The diagnostic method according to [5], which is performed as described above.
[7] A disease marker for an allergic disease comprising an antibody that recognizes amphiregulin.
[8] The disease marker according to [7], wherein the allergic disease is asthma, atopy or allergic rhinitis, and is used as a probe in the examination of the disease.
[9] The disease marker according to [7] or [8], wherein the allergic disease is asthma and is used for sputum testing.
[10] A method for diagnosing allergic diseases, comprising the following steps (A) and (B):
(A) A step of measuring the expression level of amphiregulin in a biological sample of a subject, and (B) a step of determining the presence or absence of an allergic disease based on the measurement result of (A).
[11] A method for diagnosing an allergic disease comprising the following steps (a), (b) and (c):
(A) a step of binding a protein prepared from a biological sample of a subject and the disease marker according to any one of [7] to [9] above,
(B) measuring a protein derived from a biological sample bound to the disease marker using the disease marker as an index, and (c) determining the presence or absence of an allergic disease based on the measurement result of (b) .
[12] The determination of the presence or absence of allergic disease in the step (c) is an indicator that the amount of binding to the disease marker is increased by comparing the measurement result obtained for the subject with the measurement result obtained for the normal subject. [11] The method for diagnosing allergic diseases according to [11] above.
[13] A screening method for a substance that suppresses the expression of amphiregulin, comprising the following steps (a), (b), and (c):
(a) contacting the test substance with a cell capable of measuring the expression of amphiregulin,
(b) measuring the expression level of amphiregulin in the cell contacted with the test substance, and comparing the expression level with the expression level of amphiregulin in the control cell not contacted with the test substance;
(c) A step of selecting a test substance that decreases the expression level of amphiregulin based on the comparison result of (b).
[14] A screening method for a substance that suppresses the binding activity of amphiregulin to an EGF receptor, comprising the following steps (a), (b), and (c):
(a) contacting the test substance with amphiregulin and EGF receptor;
(b) measuring the binding activity of the amphiregulin contacted with the test substance to the EGF receptor and comparing the activity with the binding activity of the control amphiregulin not contacted with the test substance to the EGF receptor;
(c) A step of selecting a test substance that suppresses the binding activity of amphiregulin to the EGF receptor based on the comparison result of (b).
[15] The screening method according to [13] or [14] above, which is a method for searching for an active ingredient of a therapeutic agent for allergic diseases.
[16] A screening method for a substance that suppresses vaginal secretion, comprising the following steps (a), (b) and (c):
(a) contacting the test substance and amphiregulin with a cell capable of measuring mucin expression;
(b) measuring the expression level of mucin in the cell contacted with the test substance, and comparing the expression level with the expression level of mucin in the control cell not contacted with the test substance;
(c) A step of selecting a test substance that suppresses sputum secretion based on the comparison result of (b).
[17] The screening method according to [16] above, wherein the vaginal secretion is associated with an allergic disease.
[18] A therapeutic agent for allergic diseases, comprising an antibody that binds to amphiregulin.
[19] A therapeutic agent for allergic diseases, comprising as an active ingredient a substance that suppresses the expression level of amphiregulin or the binding activity to the EGF receptor.
[20] The therapeutic agent according to [19] above, wherein the substance is an antibody against amphiregulin or a dominant negative mutant or an expression vector containing a nucleic acid molecule encoding the antibody or dominant negative mutant.
[21] The therapeutic agent according to any one of the above [18] to [20], which has a sputum secretion inhibitory action or a remodeling inhibitory action.
[22] A therapeutic agent for allergic diseases comprising a substance that suppresses the expression of the amphiregulin gene as an active ingredient.
[23] The therapeutic agent according to [22] above, wherein the substance is an antisense nucleic acid, ribozyme, decoy nucleic acid or siRNA against an amphiregulin gene.
[24] The therapeutic agent according to the above [22] or [23], which has a sputum secretion inhibitory action or a remodeling inhibitory action.

  According to the disease marker of the present invention, it is possible to provide an index of allergic diseases such as asthma, atopy, and allergic rhinitis, and an easy and accurate diagnosis of the disease can be performed. According to the method for diagnosing allergic diseases of the present invention, the relationship with the action of amphiregulin expressed specifically in mast cells is clarified, and diagnosis relating to sputum secretion and epithelial cell remodeling becomes possible. According to the screening method of the present invention, it becomes possible to develop a novel therapeutic agent for allergic diseases based on the mechanism of action of amphiregulin. According to the therapeutic agent of the present invention, the action of amphiregulin can be specifically regulated and allergic diseases with few side effects can be treated.

  In this specification, the abbreviations of amino acids, (poly) peptides, (poly) nucleotides, etc. are defined by IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984). ], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in the field.

  In this specification, “gene” or “DNA” is used to include not only double-stranded DNA but also each single-stranded DNA such as a sense strand and an antisense strand constituting the DNA. The length is not particularly limited. Therefore, in this specification, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing human genomic DNA and a single-stranded DNA containing DNA (positive strand) and a sequence having a sequence complementary to the positive strand. Both double-stranded DNA (complementary strand) and fragments thereof are included. The “gene” or “DNA” includes not only “gene” or “DNA” represented by a specific base sequence (SEQ ID NO: 1) but also a biological function equivalent to the protein encoded by these. “Gene” or “DNA” encoding a protein (eg, homologs (homologs, splice variants, etc.), variants and derivatives) is encompassed. The “gene” or “DNA” encoding the homologue, variant or derivative is specifically any one of the specific base sequences represented by SEQ ID NO: 1 under the stringent conditions described below. Examples thereof include “gene” or “DNA” having a base sequence that hybridizes with the complementary sequence.

For example, as a gene encoding a homologue of a human-derived protein, genes of other species such as mouse and rat corresponding to the human gene encoding the protein can be exemplified, and these genes (homologues) are homologous genes (http: / /www.ncbi.nlm.nih.gov/HomoloGene/). Specifically, a specific human base sequence is matched by BLAST (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993, http://www.ncbi.nlm.nih.gov/BLAST/) ( Get the accession number of the sequence (Score is the highest, E-value is 0 and Identity is 100%). The UniGene Cluster ID (number indicated by Hs.) Obtained by inputting the accession number to UniGene (http://www.ncbi.nlm.nih.gov/UniGene/) is input to the homologene. From the list showing the correlation of gene homologues between the other species gene and the human gene obtained as a result, genes of other species such as mice and rats as homologues corresponding to the human gene indicated by a specific base sequence Can be selected.
The gene or DNA does not ask for distinction of the functional region, and can include, for example, an expression control region, a coding region, an exon, or an intron.

  When the term “amphiregulin gene” or “amphiregulin DNA” is used in the present specification, the human amphiregulin gene (DNA) represented by the specific base sequence (SEQ ID NO: 1) unless otherwise specified by the SEQ ID NO: ) And its homologues, mutants and derivatives, etc. (DNA). Specifically, the human amphiregulin gene (GenBank Accession No. NM_001657) described in SEQ ID NO: 1, its mouse homolog (eg, GenBank Accession No. NM_009704), rat homolog (eg, GenBank Accession No. NM_017123) Etc. are included.

  In the present specification, “protein” or “(poly) peptide” includes not only “protein” or “(poly) peptide” represented by a specific amino acid sequence (SEQ ID NO: 2) but also biological functions thereof. To the same extent, homologues (homologs and splice variants), mutants, derivatives, matures, amino acid modifications, and the like are included. Here, examples of homologs include proteins of other species such as mice and rats corresponding to human proteins, and these were identified by HomoloGene (http://www.ncbi.nlm.nih.gov/HomoloGene/) It can be identified a priori from the base sequence of the gene. Variants also include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences modified by artificial deletions, substitutions, additions and insertions. . Examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and still more preferably 97% homologous to a protein or (poly) peptide without mutation. The amino acid modifications include naturally occurring amino acid modifications and non-naturally occurring amino acid modifications, and specific examples include phosphorylated amino acids.

  When the term “amphiregulin protein” or simply “amphiregulin (hereinafter sometimes abbreviated as AREG)” is used in the present specification, a specific amino acid sequence (SEQ ID NO: 2) unless otherwise specified by the SEQ ID NO: It is used to include human amphiregulin and its homologues, mutants, derivatives, matured forms, and amino acid modified forms. Specifically, human amphiregulin having the amino acid sequence described in SEQ ID NO: 2 (GenBank Accession No. NM_001657), a mouse homologue thereof (eg, GenBank Accession No. NM_009704), a rat homologue (eg, GenBank Accession No. NM_017123) etc. are included.

  As used herein, the term “antibody” includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, humanized antibodies or Fab fragments or fragments generated from Fab expression libraries, and the like, which have antigen binding properties. Some of the antibodies are included.

  In the present specification, the term “disease marker” refers to a candidate substance useful for diagnosing the presence or absence of allergic disease, the degree of morbidity, the presence or absence of improvement, and the degree of improvement, and for the prevention and treatment of allergic diseases. It is used directly or indirectly for screening. This can specifically recognize and bind genes or proteins whose expression varies in vivo, particularly in bronchial mucosal epithelial cells or mast cells in connection with the development of allergic diseases (poly) ( Oligo) nucleotides or antibodies are included. Based on the above properties, these (poly) (oligo) nucleotides and antibodies are used as probes for detecting the genes and proteins expressed in vivo, tissues and cells, and (oligo) nucleotides are used in vivo. It can be effectively used as a primer for amplifying the gene expressed in 1.

  In the present specification, the “biological tissue” to be diagnosed refers to a tissue or cell in which the expression of the amphiregulin gene of the present invention increases with an allergic disease caused by antigen stimulation. Specific examples include bronchial mucosal epithelial cells, mast cells, eosinophils and the like.

  As used herein, “allergic disease” refers to a disease that develops upon antigen stimulation, and includes, for example, asthma, atopic dermatitis, allergic rhinitis, food allergy, and the like. Preferred are asthma, atopic dermatitis, and allergic rhinitis involving excessive secretion of mucus and increased remodeling due to increased expression of the amphiregulin gene, more preferably steroid-resistant asthma, atopic dermatitis, allergy Rhinitis.

  The disease marker of the present invention is characterized by containing a polynucleotide having at least 15 consecutive bases and / or a polynucleotide complementary thereto in the base sequence of the amphiregulin gene.

  Specifically, the disease marker of the present invention comprises a polynucleotide having at least 15 consecutive bases and / or a polynucleotide complementary thereto in the base sequence of the amphiregulin gene described in SEQ ID NO: 1. Can be mentioned.

  Here, a complementary polynucleotide (complementary strand, reverse strand) is a full-length sequence of a polynucleotide comprising the base sequence of the amphiregulin gene, or a base sequence having a base sequence of at least 15 consecutive bases in the base sequence. A polynucleotide that is basically complementary to a partial sequence (for convenience, these are also referred to as “positive strands”) based on a base pair relationship such as A: T and G: C. It is. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target base sequence of the positive strand, but has a complementary relationship such that it can hybridize with the target positive strand under stringent conditions. You may have. It should be noted that stringent conditions here bind the complex or probe as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be mentioned. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although there is no particular limitation, the more stringent hybridization conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more severe hybridization conditions are “0.1 × SSC, 0.1% SDS, 65 ° C.”. Can do. Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the target base strand, and at least 90%, preferably 95% homology with the strand. An example is a chain composed of a base sequence having the same.

  Here, the polynucleotide on the positive strand side includes not only those having the nucleotide sequence of the amphiregulin gene or a partial sequence thereof, but also nucleotide sequences that are more complementary to the nucleotide sequence of the complementary strand. A chain consisting of can be included.

  Furthermore, the above-mentioned normal and complementary (reverse) polynucleotides may each be used as a disease marker in a single-stranded form, or may be used as a disease marker in a double-stranded form.

  Specifically, the disease marker for allergic diseases of the present invention may be a polynucleotide comprising the base sequence (full-length sequence) of the amphiregulin gene, or a polynucleotide comprising the complementary sequence thereof. Good. Moreover, as long as it selectively recognizes (specifically) these amphiregulin genes or polynucleotides derived from these genes, it may be a polynucleotide comprising the full-length sequence or a partial sequence of its complementary sequence. In this case, examples of the partial sequence include a polynucleotide having at least 15 consecutive base lengths arbitrarily selected from the base sequence of the full-length sequence or its complementary sequence.

  Here, “selectively (specifically)” means that, for example, in Northern blotting, an amphiregulin gene or a polynucleotide derived therefrom can be specifically detected, and in RT-PCR method. Means that an amphiregulin gene or a polynucleotide derived therefrom is specifically produced, but the present invention is not limited thereto, and those skilled in the art can obtain the above-mentioned detection product or product from these genes. Anything that can be determined to be.

  The disease marker of the present invention is, for example, based on the base sequence of the human amphiregulin gene described in SEQ ID NO: 1, for example, primer 3 (HYPERLINK http://www.genome.wi.mit.edu/cgi-bin/ It can be designed using primer / primer3.cgi) or Vector NTI (Infomax). Specifically, a primer or probe candidate sequence obtained by applying the nucleotide sequence of the gene of the present invention to primer 3 or vector NTI software, or at least a sequence partially containing the sequence may be used as a primer or probe. it can.

  The disease marker of the present invention is not limited as long as it has a length of at least 15 consecutive bases as described above. Specifically, the length can be appropriately selected and set according to the use of the marker. .

  When the disease marker of the present invention is used as a primer in the detection of allergic diseases, those having a base length of usually 15 bp to 100 bp, preferably 15 bp to 50 bp, more preferably 15 bp to 35 bp can be exemplified. When used as a detection probe, those having a base length of usually 15 bp to the entire sequence, preferably 15 bp to 1 kb, more preferably 100 bp to 1 kb can be exemplified.

  The disease marker of the present invention is used as a primer or probe according to a conventional method in a known method for specifically detecting a specific gene such as Northern blot method, RT-PCR method, DNA chip analysis method, in situ hybridization method, etc. can do. By the use, the presence or absence or expression level (expression level) of the amphiregulin gene in allergic diseases can be evaluated.

The allergic disease diagnosis method of the present invention comprises the following steps (A) and (B):
(A) A step of measuring the expression level of an amphiregulin gene in a biological sample of a subject, and (B) a step of determining the presence or absence of an allergic disease based on the measurement result of (A).

For example, the allergic disease diagnosis method of the present invention includes the following steps (a), (b) and (c):
(A) a step of binding RNA prepared from a biological sample of a subject or a complementary polynucleotide transcribed from the RNA and the disease marker of the present invention,
(B) a step of measuring RNA derived from a biological sample bound to the disease marker or a complementary polynucleotide transcribed from the RNA using the disease marker as an index,
(C) A step of determining the presence or absence of an allergic disease based on the measurement result of (b).

  When RNA is used as a measurement object, specifically, the diagnostic method of the present invention is performed by using the disease marker of the present invention as a primer or probe, Northern blot method, RT-PCR method, DNA chip analysis method, in It can be carried out by using as an indicator that the amount of RNA or its transcript bound to the disease marker is increased by in situ hybridization analysis or the like.

  As a biological sample to be measured, a part of the biological tissue of the subject (bronchial mucosa, nasal mucosa, gastric mucosa, etc.) is collected by biopsy or the like according to the type of detection means used or body fluid Total RNA prepared according to a conventional method from a sample obtained by collecting cells present in blood (blood, sputum, nasal discharge, gastric juice, etc.) may be used, and various RNAs prepared based on the RNA. These polynucleotides may be used.

  When using the Northern blot method, the presence or absence of the expression of the amphiregulin gene in RNA and the expression level thereof can be detected and measured by using the disease marker of the present invention as a probe. Specifically, the disease marker (complementary strand) of the present invention is labeled with a radioisotope (RI) or a fluorescent substance, and hybridized with RNA derived from the biological tissue of a subject transferred to a nylon membrane or the like according to a conventional method. After making soybean, the formed double marker of disease marker (DNA) and RNA, and signal derived from the marker (RI or fluorescent substance) of the disease marker is detected with a radiation detector (BAS-1800II, manufactured by Fuji Film Co., Ltd.) ) Or a method of detecting and measuring with a fluorescence detector. In addition, using Alk Phos Direct Labeling and Detection System (Amersham Pharamcia Biotech), a disease marker (probe DNA) is labeled according to the protocol, hybridized with RNA derived from the biological tissue of the subject, and then labeled with the disease marker. It is also possible to use a method in which a signal derived from a product is detected and measured with a multi-bioimager STORM860 (manufactured by Amersham Pharmacia Biotech).

  When the RT-PCR method is used, the presence or absence and expression level of the amphiregulin gene in RNA can be detected and measured by using the disease marker of the present invention as a primer. Specifically, a pair of cDNA prepared from the disease marker of the present invention is prepared so that a region of the target amphiregulin gene can be amplified using a template prepared from RNA derived from biological tissue of a subject according to a conventional method. A method in which a primer (a normal strand that binds to the cDNA (-strand), a reverse strand that binds to a + strand) is hybridized with the primer, and a PCR method is performed according to a conventional method to detect the resulting amplified double-stranded DNA Can be illustrated. In addition, the detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method can be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and a labeled disease marker is used as a probe and hybridized with the marker to detect it. The produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). Also, prepare an RT-PCR reaction solution according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems) and react with ABI PRISM 7700 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also.

  When DNA chip analysis is used, a DNA chip on which the above-described disease marker of the present invention is attached as a DNA probe (single strand or double strand) is prepared. Examples include a method in which a double strand of DNA and cRNA formed by hybridization with a cRNA prepared by a method and labeled with biotin is detected with fluorescently labeled avidin.

  When using the in situ hybridization method, the biological tissue of the aforementioned subject is collected by biopsy and a section is prepared. A specific antisense probe or sense probe of the disease marker gene of the present invention is prepared. The probe is labeled with an RI label or a non-RI label (eg, DIG label). The section is deparaffinized (in the case of a paraffin section) and pretreated, and then fixed with ethanol or the like. After pre-hybridizing the fixed section and hybridizing with the probe, washing and RNase treatment are performed, and a detection method according to the label (for example, development for RI labeling, immunological detection for non-RI labeling) The presence or absence of the expression of the amphiregulin gene in the living tissue and its expression level can be detected and measured.

  In the diagnosis method of the present invention, the determination of the presence or absence of allergic disease in the step (c) increases the amount of binding to the disease marker by comparing the measurement result obtained for the subject with the measurement result obtained for the normal subject. It is preferable that the measurement is performed using the above as an index.

  When the allergic disease is asthma, a preferable diagnosis is to distinguish asthma by sputum test from other bronchitis. In this case, compared with other bronchitis-derived sputum, the asthma patient-derived sputum has an increased amount of binding to the disease marker of the present invention as an indicator, and the possibility of asthma is high when the amount is high It can be determined that the possibility of bronchitis is high when the binding amount is low.

  In another embodiment, the disease marker of the present invention is characterized by containing an antibody that recognizes amphiregulin.

  The antibody is a tool (disease marker) that can measure whether or not the subject suffers from an allergic disease by detecting the presence or absence of the amphiregulin protein in the biological sample of the subject. ) Is useful.

  The antibody is also useful as a tool (disease marker) for detecting changes in the expression of amphiregulin protein in the prevention and prediction of symptoms of allergic diseases described below.

  The form of the antibody is not particularly limited, and may be a polyclonal antibody using an amphiregulin protein as an immunizing antigen or a monoclonal antibody thereof. Furthermore, it may be a chimeric antibody, a single chain antibody, a humanized antibody, or a fragment generated by a Fab fragment or Fab expression library prepared based on the gene encoding the monoclonal antibody.

  Methods for producing these antibodies are known, and the antibodies can also be produced according to conventional methods (Current Protocol in Molecular Biology, Chapter 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, an amphiregulin protein expressed and purified in Escherichia coli or the like according to a conventional method, or an oligopeptide having a partial amino acid sequence of these proteins according to a conventional method is used. It is possible to synthesize and immunize a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, a non-human animal such as a mouse is immunized with an oligopeptide having a partial amino acid sequence of an amphiregulin protein expressed and purified in Escherichia coli according to a conventional method, and the resulting spleen cells and myeloma are obtained. It can be obtained from hybridoma cells prepared by cell fusion with cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).

  Amphiregulin proteins used as immunizing antigens for antibody production are DNA cloning, construction of each plasmid, transfection into a host based on the sequence information (SEQ ID NO: 1 etc.) of the gene provided by the present invention and the like. It can be obtained by culturing the transformant and recovering the protein from the culture. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.

  Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding amphiregulin protein in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured. Then, a protein as an immunizing antigen for producing the antibody of the present invention can be obtained by recovering the target protein from the obtained culture. Moreover, the partial peptides of these amphiregulin proteins can also be produced by a general chemical synthesis method (peptide synthesis) according to the amino acid sequence information (SEQ ID NO: 2 etc.) provided by the present invention and the like.

The allergic disease diagnosis method of the present invention comprises the following steps (A) and (B):
(A) A step of measuring the expression level of amphiregulin in a biological sample of a subject, and (B) a step of determining the presence or absence of an allergic disease based on the measurement result of (A).

  In one embodiment, the allergic disease diagnosis method of the present invention is characterized by using the disease marker of the present invention. Since the disease marker has the property of specifically binding to amphiregulin protein, the amphiregulin protein expressed in animal tissues can be specifically detected.

For example, the diagnostic method for allergic diseases of the present invention can be carried out by a method comprising the following steps (a), (b) and (c):
(a) a step of binding a protein prepared from a biological sample of a subject and the disease marker (antibody) of the present invention,
(b) measuring a protein derived from a biological sample bound to the disease marker using the disease marker as an index,
(c) A step of determining the morbidity of the allergic disease based on the measurement result of (b).

  When a protein is used as a measurement object, specifically, the diagnostic method of the present invention uses the disease marker (antibody) of the present invention as a probe, a Western blotting method, a radioisotope immunoassay method (RIA method). It can be carried out by using as an indicator that the amount of protein binding to the disease marker is increased by a detection means such as ELISA, fluorescent antibody method, immunohistochemical staining.

  Depending on the type of detection means used, a sample of the subject's tissue (bronchial mucosa, nasal mucosa, gastric mucosa, etc.) can be collected with a biopsy, etc., or in a body fluid (blood, sputum, nasal discharge) , Protein prepared in accordance with a conventional method from a sample obtained by recovering cells or the like present in gastric juice or the like, or a protein dissolved in a body fluid can be used.

  More specifically, in the method for diagnosing allergic disease of the present invention, the amount of amphiregulin protein bound to the marker is increased by Western blotting or the like using the disease marker (antibody) of the present invention. This can be implemented by using this as an index.

When using Western blotting, the disease marker of the present invention is used as a primary antibody, followed by a secondary antibody labeled with a radioisotope such as ¹²⁵ I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP), or the like. Using a secondary antibody (an antibody that binds to the primary antibody), the signal derived from the radiolabeled isotope or fluorescent substance of the obtained labeled compound can be detected with a radiation measuring instrument (BAS-1800II, manufactured by Fuji Film Co., Ltd.), a fluorescence detector, etc. This can be done by detecting and measuring. In addition, after using the disease marker of the present invention as a primary antibody, it was detected using the ECL Plus Western Blotting Detection System (Amersham Pharmacia Biotech) according to the protocol and measured with a multi-bioimager STORM860 (Amersham Pharmacia Biotech) You can also

  When using the immunohistochemical staining method, for example, amphiregulin-positive cells can be measured using an enzyme-labeled antibody and its chromogenic substrate in accordance with the method described in Example 4 described later.

  For example, in the case of sputum tests, allergic diseases are diagnosed by the expression level of the amphiregulin gene in the sputum of the subject, or the amount, function or activity of the amphiregulin protein (hereinafter referred to as “protein level”). Is compared to the gene expression level or the protein level in the control sputum, and the difference between the two is determined. In the case of a diagnosis of tissue remodeling, there is a statistically significant correlation between the amount of amphiregulin expression and the degree of goblet cell hyperplasia, so the concentration of amphiregulin in the subject's sputum (protein amount) Can be performed by comparing the control and the same subject over time with the protein in the sputum and determining the difference between the two.

  The “remodeling” means that the inflamed part is completely restored without leaving any scars, but is completely repaired, whereas the part is replaced with fibrosis (scar), and the scars are left to regenerate. That is, incomplete repair (remodeling).

  In this case, biological samples (samples containing RNA or protein) collected and prepared from normal tissues are required as controls, and these are collected with biopsies or the like from a part of human tissues not suffering from allergic diseases. Alternatively, a protein prepared according to a conventional method from a sample obtained by collecting cells or the like present in a body fluid or a protein dissolved in a body fluid can be used. As used herein, “a person who does not suffer from allergic disease” means that there is at least no subjective symptom of allergic disease, and the results of other testing methods are preferably, for example, normal IgE (RAST) values, various specifics A person who has a normal IgE (RIST) value, a negative prick test, and is diagnosed as not having an allergic disease. The “person who does not suffer from allergic disease” may be simply referred to as a normal person in the present specification.

  Comparison of gene expression level or protein level between a subject's tissue and a normal tissue can be performed by performing measurements on the subject's biological sample and a normal subject's biological sample in parallel. When not performed in parallel, the expression level of the amphiregulin gene obtained by measuring a plurality of (at least 2, preferably 3 or more) normal tissues under uniform measurement conditions, or the amphiregulin protein The average level or statistical intermediate value can be used for comparison as the level of gene expression or the amount of protein in normal subjects.

  Whether a subject has an allergic disease is determined by determining whether the expression level of an amphiregulin gene in the subject's tissue, or the amphiregulin protein level is at least twice that of a normal subject, Preferably, it can be performed as an index that the number is three times or more. If the gene expression level or protein level of the subject is higher than that in a normal subject, the subject can be judged as having an allergic disease, or suffering from the disease is suspected. The relationship between the expression level or protein level and severity can be determined to be higher as the level is higher. In addition, tissue remodeling may occur even if the subject seems to be cured of symptoms by drugs such as steroids. The presence or absence of remodeling can be determined by comparing the heights.

The screening method for a substance that suppresses the expression of amphiregulin of the present invention includes the following steps (a), (b), and (c):
(A) contacting the test substance with a cell capable of measuring the expression of amphiregulin;
(B) measuring the expression level of amphiregulin in cells contacted with the test substance, and comparing the expression level with the expression level of amphiregulin in control cells not contacted with the test substance;
(C) A step of selecting a test substance that decreases the expression level of amphiregulin based on the comparison result of (b).

  In the step (a), the test substance may be any known substance or new substance, for example, a nucleic acid, a carbohydrate, a lipid, a protein, a peptide, a low molecular weight organic compound, or a combinatorial chemistry technique. Examples include compound libraries, random peptide libraries prepared by solid phase synthesis and phage display methods, and natural components derived from microorganisms, animals and plants, marine organisms, and the like.

  Examples of cells capable of measuring the expression of amphiregulin in the step (a) include general cultured cells that express amphiregulin regardless of endogenous or exogenous, cells containing a reporter gene, and the like. Whether or not these genes are expressed in cultured cells can be easily confirmed by detecting the expression of these genes by a known Northern blot method or RT-PCR method.

  Specifically, for example, a mast cell isolated or prepared from an animal with an allergic disease or a cell line thereof; a cell into which any of the genes of the present invention has been introduced; a cell into which a reporter (luciferase, GFP, etc.) gene has been introduced, etc. Can be mentioned.

  As the animal model, any animal model known as an animal model of allergic disease can be used, and specifically, an albumin-sensitized mouse asthma model and the like can be mentioned.

  Examples of the cell for gene transfer include CHO, MCF-7 Mammary carcinoma cell, H295R adrenal cell, and the like.

  In the step (a), the test substance is contacted with a cell capable of measuring the expression of amphiregulin in a culture medium. As the cells, cells obtained by crosslinking FcεRI on the cell membrane of mast cells with IgE and further stimulating with an anti-IgE antibody can be preferably used. The culture medium is appropriately selected depending on the cells capable of measuring the expression of amphiregulin. For example, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum, Dulbecco's modified minimal essential medium ( DMEM), RPMI 1640 medium, 199 medium, and the like. The culture conditions are appropriately determined in the same manner. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is about 12 to about 72 hours.

  In the step (b), the expression level is measured for mRNA or protein. The expression level of mRNA is measured, for example, by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like. The expression level of protein can be measured, for example, by preparing an extract from cells and immunologically. As the immunological technique, Western blotting, radioisotope immunoassay (RIA method), ELISA, fluorescent antibody method and the like can be used. In addition, when a cell containing a reporter gene (eg, a cell into which a vector to which a reporter gene (eg, luciferase, GFP) is operably linked downstream of an amphiregulin promoter) is used, the expression level is , Measured based on the signal intensity of the reporter gene.

  In step (b), the expression level is compared based on the presence or absence of a significant difference in the expression level of amphiregulin in the presence or absence of the test substance. In addition, the expression level of amphiregulin in the control cells that are not contacted with the test substance is measured at the same time, even if the expression level is measured in advance, compared to the measurement of the expression level of amphiregulin in the cells that are contacted with the test substance. However, the expression level is preferably measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

  In step (c), a test substance that reduces the expression level of amphiregulin is selected. Test substances selected in this way include substances that can vary the expression level of amphiregulin due to the nature of the present screening method. The selected test substance is a therapeutic agent for allergic diseases (for example, an expectorant that suppresses mucus secretion during asthma, an inhibitor of remodeling, an inhibitor of goblet cell hyperplasia, particularly asthma with steroid resistance) It is useful not only as a candidate for an expectorant or a remodeling inhibitor, but also as an immunomodulator or a research reagent.

Moreover, the screening method of the substance which suppresses the binding activity of the amphiregulin to the EGF receptor of the present invention includes the following steps (a), (b) and (c):
(a) contacting the test substance with amphiregulin and EGF receptor;
(b) measuring the binding activity of the amphiregulin contacted with the test substance with the EGF receptor, and comparing the activity with the binding activity of the control amphiregulin not contacted with the test substance;
(c) A step of selecting a test substance that suppresses the binding activity of amphiregulin to the EGF receptor based on the comparison result of (b).

  In the step (a), the test substance is as described above.

  In step (a), the test substance is contacted with amphiregulin and the EGF receptor. In the step (a), the contact method is not particularly limited. For example, a method in which amphiregulin and EGF receptor are mixed at a predetermined concentration under physiological conditions of 25 to 37 ° C. and a test substance is added. And a method of immobilizing an EGF receptor on a solid phase and binding to amphiregulin in the presence of the test substance, or a method of adding the test substance to a culture solution of cells expressing amphiregulin and EGF receptor. .

In the step (b), the binding activity between amphiregulin and the EGF receptor can be measured by the method described in the following example.
b-1) Immunoprecipitation with anti-amphiregulin antibody or anti-EGF receptor antibody, and Western blotting with antibody not used in immunoprecipitation to measure the amount of binding of amphiregulin and EGF receptor Method.
b-2) Either amphiregulin or EGF receptor is expressed as a fusion protein with a label such as polyhistidine or GST, or biotinylated, polyhistidine is nickel, GST is glutathione, biotin is Since it binds to avidin, the conjugate is recovered using them and the amount of binding is measured by Western blotting similar to b-1).
b-3) Surface plasmon resonance method (Biacore).
b-4) A method of measuring the binding of fluorescently labeled amphiregulin to EGF receptor-expressing cells with a flow cytometer.

  In step (b), the comparison of the binding activity is performed based on the presence or absence of a significant difference in the amount of binding between amphiregulin and the EGF receptor, for example, in the presence or absence of the test substance.

  In step (c), a test substance that reduces the binding activity of amphiregulin to the EGF receptor is selected. The selected test substance is a therapeutic agent for allergic diseases (for example, an expectorant that suppresses mucus secretion during asthma, an inhibitor of remodeling, an inhibitor of goblet cell hyperplasia, particularly asthma with steroid resistance) It is useful not only as a candidate for an expectorant or a remodeling inhibitor, but also as an immunomodulator or a research reagent.

The screening method for a substance that suppresses sputum secretion of the present invention includes the following steps (a), (b) and (c):
(a) contacting the test substance and amphiregulin with a cell capable of measuring mucin expression;
(b) measuring the expression level of mucin in the cell contacted with the test substance, and comparing the expression level with the expression level of mucin in the control cell not contacted with the test substance;
(c) A step of selecting a test substance that suppresses sputum secretion based on the comparison result of (b).

  In the step (a), the test substance is as described above.

  In the step (a), the test substance is contacted with a cell capable of measuring mucin expression together with amphiregulin. Here, mucin refers to a major glycoprotein of mucus that covers the lumen of the digestive tract such as the trachea, gastrointestinal tract, and gonads. In the present invention, examples include MUC2, MUC5AC, MUC1, MUC6, and the like. And MUC5AC are preferred. Examples of “cells capable of measuring mucin expression” include general cultured cells that express mucin regardless of whether they are endogenous or exogenous, and cells containing a reporter gene. Whether or not these genes are expressed in cultured cells can be easily confirmed by detecting the expression of these genes by a known Northern blot method or RT-PCR method.

  In the step (a), the test substance is contacted with a cell capable of measuring the expression of mucin together with amphiregulin in a culture medium. The culture medium is appropriately selected according to cells capable of measuring mucin expression. For example, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum, Dulbecco's modified minimal essential medium (DMEM) RPMI 1640 medium, 199 medium, and the like. The culture conditions are appropriately determined in the same manner. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is about 12 to about 72 hours.

  In the step (b), the expression level is measured for mRNA or protein. The expression level of mRNA is measured, for example, by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like. The expression level of protein can be measured, for example, by preparing an extract from cells and immunologically. As the immunological technique, Western blotting, radioisotope immunoassay (RIA method), ELISA, fluorescent antibody method and the like can be used. In addition, when a cell containing a reporter gene (eg, a cell into which a reporter (eg, luciferase, GFP) gene operably linked downstream of a mucin gene promoter) is used, the expression level is It is measured based on the signal intensity of the reporter gene.

  In step (b), the expression level is compared based on the presence or absence of a significant difference in the expression level of mucin in the presence or absence of the test substance. Note that the expression level of mucin in the control cells not contacted with the test substance is the expression level measured at the same time, even if the expression level was measured in advance, compared to the measurement level of mucin expression in the cells contacted with the test substance. Although it may be present, it is preferably the expression level measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

  In the step (c), a test substance that reduces the expression level of mucin through the expression of amphiregulin is selected. The test substance thus selected includes substances that can vary the expression level of mucin due to the nature of the present screening method. The selected test substance is a therapeutic agent for allergic diseases (for example, an expectorant that suppresses mucus secretion during asthma, an inhibitor of remodeling, an inhibitor of goblet cell hyperplasia, particularly asthma with steroid resistance) It is useful not only as a candidate for an expectorant or a remodeling inhibitor, but also as an immunomodulator or a research reagent.

  Among the disease markers of the present invention, a disease marker containing the above antibody can be applied to animals to prevent or treat changes in the state of allergic diseases, and the present invention provides such a method. Among the antibodies described above, neutralizing antibodies and antibodies that can inhibit the binding of amphiregulin and EGF receptor are particularly preferable.

  The animals are preferably humans and vertebrates other than humans, and in particular, domestic animals such as cows, horses, pigs, sheep, goats, chickens, dogs, cats, and pets are preferred.

  The present invention provides a therapeutic agent for an allergic disease containing a substance that reduces the amount or activity of the disease marker as an active ingredient.

  The therapeutic agent of the present invention is not particularly limited as long as it reduces the amount or activity of amphiregulin, and contains various compounds that specifically bind to amphiregulin or EGF receptor as active ingredients. Things can be raised.

  Examples of the compound include the above-mentioned various antibodies, known receptor tyrosine kinase inhibitors (eg, AG1478, gefitinib (ZD1839)), anti-EGF receptor antibody, and the like, preferably neutralizing antibodies to amphiregulin or dominant negative Mutant or anti-EGF receptor antibody. Here, “dominant negative mutant” refers to a substance whose activity is reduced by introducing a mutation to amphiregulin. The dominant negative mutant can indirectly inhibit its activity by competing with natural amphiregulin. A dominant negative mutant of amphiregulin can be produced by introducing a mutation into amphiregulin. Examples of the mutation include deletion, substitution, addition and the like of one or more amino acids in the N-terminal region.

  The therapeutic agent of the present invention is a therapeutic agent for allergic diseases, preferably an expectorant that suppresses mucus secretion during asthma, an inhibitor of remodeling, an inhibitor of goblet cell hyperplasia, particularly steroid-resistant asthma, etc. Used as expectorant or remodeling inhibitor.

  The therapeutic agent of the present invention may be the active ingredient as it is or may contain a known pharmaceutically acceptable carrier. Examples of the carrier include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, arabic Binders such as rubber, polyethylene glycol, sucrose, starch, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate, aerosil, talc , Lubricants such as sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyllysine / ammonium salt, glycine, orange powder, sodium benzoate, sulfurous acid Preservatives such as sodium hydrogen, methylparaben, propylparaben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants, water, physiological Examples include diluents such as saline, base waxes such as cacao butter, polyethylene glycol, and white kerosene, but are not limited thereto.

  In another embodiment, the therapeutic agent of the present invention contains a substance that suppresses the expression of the amphiregulin gene as an active ingredient.

  The substance is preferably an antisense nucleic acid, ribozyme, decoy nucleic acid or siRNA against an amphiregulin gene.

  “Antisense nucleic acid” means a nucleotide sequence that can hybridize to the target mRNA (initial transcript) under physiological conditions of a cell that expresses the target mRNA (early transcript), and A nucleic acid capable of inhibiting translation of a polypeptide encoded by a target mRNA (early transcript). The type of the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera.

  “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligo DNA having the base sequence of the enzyme active site also has a nucleic acid cleaving activity. In the invention, as long as it has a sequence-specific nucleic acid cleavage activity, it is used as a concept including DNA.

  “Decoy nucleic acid” refers to a nucleic acid molecule that mimics a region to which a transcriptional regulatory factor binds. Decoy nucleic acid as a substance that suppresses the expression of amphiregulin refers to a region to which a transcriptional activator for amphiregulin binds. It can be a nucleic acid molecule that mimics. In the present invention, as the decoy nucleic acid, an oligonucleotide (S-oligo) having a thiophosphate diester bond in which the oxygen atom of the phosphodiester bond portion is substituted with a sulfur atom, or a methyl phosphate group having no phosphodiester bond as a charge And oligonucleotides modified to make the oligonucleotide less susceptible to degradation in vivo. The decoy nucleic acid may completely coincide with the region to which the transcriptional activator binds, but it is sufficient that the decoy nucleic acid retains the identity that allows the transcriptional activator to bind to amphiregulin. The length of the decoy nucleic acid is not particularly limited as long as a transcriptional activator binds. The decoy nucleic acid may contain the same region repeatedly.

  “SiRNA” is a double-stranded oligo RNA complementary to amphiregulin mRNA or a partial sequence in the coding region of the initial transcript (including an intron in the case of the initial transcript). By introducing siRNA into a cell, a phenomenon called so-called RNA interference (RNAi) occurs, and the same effect as the ribozyme is expected.

  The antisense nucleic acid, ribozyme, decoy nucleic acid, and siRNA can be prepared according to known methods.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.

[Cytokines and antibodies]
Recombinant (r) human (h) IL-3 was purchased from Intergen. rhIL-6 and rhSCF were purchased from Pepro Tech EC. rhAREG, rhEGF and mouse anti-human AREG monoclonal antibody (mAG) were purchased from Genzyme Techne. Mouse anti-human tryptase mAb (clone AA1) was purchased from Dako.

[Generation of cord blood (CB) -derived mast cells and adult peripheral blood (PB) -derived mast cells]
All human specimens in this study were provided with written informed consent, and the study was approved by each hospital's Ethics Review Committee. Human CB mononuclear cells (hereinafter sometimes abbreviated as MNC) and PB mononuclear cells were isolated by density gradient centrifugation using Ficoll-Isopaque (Nycomed). After selecting non-lineage mononuclear cells from CB and PB mononuclear cells, serum-free Iscove methylcellulose medium (Stem Cell Technologies) and Iscove modified Dulbecco medium (SCF 200 ng / mL, IL-6 50 ng / mL) (Blood 2003; 102: 2547-2554). On the 42nd day of culture, methylcellulose was dissolved in PBS, the cells were resuspended, and cultured in Iscove modified Dulbecco medium supplemented with 2% FCS (containing SCF 100 ng / mL, IL-6 50 ng / mL).

[Purification of white blood cells]
Granulocytes and mononuclear cells were isolated from venous blood of normal volunteers. Eosinophils were isolated by Percoll (1.090 g / mL) density centrifugation and then further purified by negative selection using anti-CD16 coupled micromagnetic beads (Blood 1995; 86: 1437-1443). After this negative selection, neutrophils were isolated by Percoll (1.085 g / mL) density centrifugation. Isolated neutrophils were further purified by negative selection using an anti-CD81 antibody to remove mixed eosinophils.

[Activation of human mast cells]
Mast cells were first sensitized with 1 μg / mL human myeloma IgE (CosmoBio) for 24 hours at 37 ° C. for activation by aggregation of FcεRI on the surface of human mast cells. After washing, the cells were challenged with rabbit anti-human IgE Ab (Dako) or medium only for a predetermined time at 37 ° C. To examine the effect of dexamethasone (Pepro Tech EC; glucocorticoid) on IgE-mediated gene expression profiles and AREG production by mast cells, mast cells were pretreated with ^10-6 M dexamethasone 24 hours prior to activation. . In all conditions, cells were suspended in complete Iscove modified Dulbecco medium containing SCF and IL-6. It was confirmed that treatment of mast cells with 10 ⁻⁶ M dexamethasone and / or IgE / anti-IgE did not significantly change cell viability or cell number.

[patient]
Based on the criteria of the Japanese Society of Allergology, 40 patients who defined the severity of the disease and 6 normal controls without asthma were studied by combining the stage of asthma symptoms and the frequency of symptoms (Table 1). ). None of the patients smoked today. In addition, no patient had smoked for the past two years. No patient had bronchial or respiratory infection during the month prior to the examination. The study was approved by the Ethics Committee of Dokkyo Medical University, and all patients agreed informed consent in writing. The total thickness of the basement membrane in each asthma patient and normal control was evaluated according to previous reports (Chest 1997; 111: 852-857). Airway hypersensitivity was measured as the minimum cumulative dose of acetylcholine at the time point when the breath resistance began to increase during continuous inhalation at a concentration increasing in acetylcholine.

[Bronchial biopsy]
Tissue samples from asthmatic patients were subcarina between the right lower lobe bronchus and the middle lower lobe bronchus using standard forceps during fiber optic bronchoscopy according to a previous report (Chest 1997; 111: 852-857). ) (Right B6 bronchi origin). Each biopsy specimen was immediately placed in OCT medium, instantly frozen in liquid nitrogen, and stored at −80 ° C. until the section was prepared with a cryostat.

[Statistical analysis]
Deviations between the two paired groups were analyzed by one-sided Student's t-test and P <0.05 was considered significant. Values are expressed as mean ± SEM.

[Reference Example 1]
RNA isolation, RT-PCR, real-time quantitative RT-PCR and gene chip expression analysis Total cellular RNA was isolated from mast cells using RNeasy Mini Kits (Qiagen) according to manufacturer's specifications. The yield of RNA per 10 ⁶ mast cells was 2.4 (2.0-2.8) μg (median and range, n = 6). An equal amount of RNA (50 ng) was used for RT-PCR analysis. PCR is performed in a thermocycler under the following conditions:
1 minute at 94 ° C followed by 40 amplification cycles (1 minute at 94 ° C; 30 seconds at 60 ° C; 2 minutes at 72 ° C)
I went there. The primer sequence for AREG is:
5 ′ primer: 5′-AGAGTTGAACAGGTAGTTAAGCCCC-3 ′ (SEQ ID NO: 3) and
3 ′ primer: 5′-GTCGAAGTTTCTTTCGTTCCTCAG-3 ′ (SEQ ID NO: 4). Equal amounts of PCR amplification products were visualized with ethidium bromide. MRNA for GAPDH was detected as a positive control. PCR without cDNA was performed to exclude contamination.

Next, the expression of MUC2 and MUC5AC mRNA was measured using GeneAmp 7900 sequence detection system (Applied Biosystems) for real-time RT-PCR. Total cell RNA extraction and cDNA synthesis were performed as described above. PCR was performed using TaqMan Universal PCR Master Mix Kit (Applied Biosystems) and fluorescent probe (Applied Biosystems) according to the manufacturer's protocol. The primer sequences used for MUC5AC detection are:
forward: 5'-TCAACGGAGACTGCGAGTACAC-3 '(SEQ ID NO: 5)
reverse: 5'-TCTTGATGGCCTTGGAGCA-3 '(SEQ ID NO: 6), FAM reporter dye-labeled hybridization probe: 5'-FAM-ACTCCTTTCCTGTTGTCACCGAGAACGTC-TAMRA-3' (SEQ ID NO: 7). The primer sequence used for MUC2 detection is
forward: 5'-GCCCTGGCTTCGAACTCAT-3 '(SEQ ID NO: 8)
reverse: 5'-TCTTCGGGTCGCTCTTGAA-3 '(SEQ ID NO: 9), FAM reporter dye-labeled hybridization probe: 5'-FAM-CACTGTATCATCAAACGGCCCGACAA-TAMRA-3' (SEQ ID NO: 10). The threshold cycle C _t , which inversely correlates with the level of target mRNA, was defined as the number of cycles when reporter fluorescence emission increased beyond the midpoint of the logarithmic growth phase along the amplification curve. Relative expression levels were determined using cycle thresholds and the competitive Ct method and adjusted for co-amplifying housekeeping gene levels, 2-fold amplification / cycle rates, and reference expression levels for control samples.

Gene chip expression analysis Human genome-wide gene expression is performed using the Human Genome U95A and U133A probe arrays (GeneChip, Affymetrix) with oligonucleotide probe sets for approximately 23,000 full-length genes and expressed sequence tags (EST). The examination was conducted according to the protocol (Expresion Analysis Technical Mannual) and the previous report. Total RNA (3-10 μg) was extracted from approximately 10 ⁷ cells. Double stranded cDNA was synthesized and subjected to in vitro transcription in the presence of biotinylated nucleoside triphosphates. The biotinylated cRNA was hybridized with the probe array at 45 ° C. for 16 hours, and the hybridized biotinylated cRNA was stained with streptavidin-PE (Phycoerythrin) and then scanned with a Hewlett-Packard Gene Array Scanner. The fluorescence intensity of each probe was quantified using the computer program GeneChip Analysis Suite 5.0 (Affymetrix). The expression level of single-stranded mRNA was determined as the average fluorescence intensity among the intensities obtained with 11 pairs of (perfect match and single nucleotide mismatch) probes consisting of 25-mer oligonucleotides. If the intensity of the mismatch probe was very high, it was determined that no gene expression was present even though high average fluorescence was obtained with the GeneChip Analysis Suite 5.0 program. The level of gene expression was determined as mean deviation (AD) using GeneChip software. The ratio of specific AD level to the average AD level of the six probe sets of housekeeping genes (β-actin and GAPDH) was then calculated.

Furthermore, data analysis was performed using GeneSpring software version 6 (Silicon Genetics). To normalize the change in staining intensity between chips, the AD value for all genes on a given chip was divided by the median of all measurements on that chip. To exclude changes within the background noise range and select the most differentially expressed gene, the data is only available if the raw data value exceeds 100 AD and the gene expression is determined to be present by Affymetrix data analysis It was used. Hierarchical clustering analysis with standard correlations was used to identify gene clusters. Specific samples were normalized to each other, i.e., samples were normalized to the median of the control sample. Each measurement for each gene in these specific samples was divided by the median of that gene's measurement in the corresponding control sample. The split ratio was set to 0.5. Normal values of 0 or less were set to 0. Data is,
(i) at least 2-fold (activation program) expression change and
(ii) Data was considered significant if it contained a call (Affymetrix algorithm) with at least one increased gene expression. Restrictions to classify as up-regulated or down-regulated genes were applied to normalized values.

Mast cell specific transcripts were identified by comparing gene expression levels based on the following criteria.
The average expression level of each gene in mast cells is more than 5 times greater than the highest level of other cell types.
-Absence or limit call is 1 or less in 3 or 4 independent experiments.
・ Standard deviation is less than average.
If multiple transcripts obtained with different probe sets have the same Genebank or Unigene accession number, select the transcript with the highest expression level in the table.

[result]
Clustering analysis of dexamethasone-controlled gene expression in human mast cells To identify genes that are up-regulated by FcεRI aggregation but not down-regulated by dexamethasone pretreatment, a high-density oligonucleotide probe array (GeneChip) was used. We used to explore gene expression profiles in human mast cells. The inventors divided the genes into the following four sets.
Set I: Contains the following genes
(i) its expression changes at least 2-fold (activation program) after aggregation of FcεRI; and
(ii) The increased gene expression decreases to less than 0.5-fold after dexamethasone pretreatment (FIG. 1, I).
Set II: Includes the following genes
(i) its expression changes at least 2-fold (activation program) after aggregation of FcεRI; and
(ii) The increased gene expression changes more than 0.5 times after dexamethasone pretreatment (FIG. 1, II).
Set III: Consists of the following genes
(i) its expression changes less than 2-fold after aggregation of FcεRI; and
(ii) The increased gene expression changes to less than 0.5-fold after dexamethasone pretreatment (FIG. 1, III).
Set IV: Includes the following genes
(i) its expression changes less than 2-fold after aggregation of FcεRI; and
(ii) The increased gene expression changes more than 0.5 times after dexamethasone pretreatment (FIG. 1, IV).

  In addition, mast cell specific genes were selected based on comparisons with gene expression profiles of human peripheral blood mononuclear cells, eosinophils and neutrophils. As described above, data was considered mast cell specific if the expression level in mast cells was at least 5 times higher than the maximum expression level of human peripheral blood mononuclear cells, eosinophils and neutrophils. In this way, 24 genes were identified as set I mast cell specific. These genes were members of the NF-κB pathway such as cytokines (IL-5 and GM-CSF) and chemokines (MCP-1 and I-309) (Table 2). The 17 mast cell-specific genes identified in Set II include AREG and an adhesion molecule called α1- (E) catenin (Table 3). In set III, 22 genes were identified as mast cell specific and included cathepsin G, chymase and metalloprotease. Finally, in set IV, 236 genes were selected as mast cell specific genes. These genes include tryptase, major basic protein, PGD2 synthase, and c-kit.

Footnotes: MC1 (2) c: untreated control mast cells, MC1 (2) a: anti-IgE stimulated mast cells, MC1 (2) d: cells treated with anti-IgE and dexamethasone, Eo: eosinophils, MNC: single Nucleocytes, Ne: Eosinophils, specif: Mast cell specificity (ratio to other leukocytes), CCL: CC-chemokine ligand, CXCL: CXC-chemokine ligand.

Footnotes: MC1 (2) c: untreated control mast cells, MC1 (2) a: anti-IgE stimulated mast cells, MC1 (2) d: cells treated with anti-IgE and dexamethasone, Eo: eosinophils, MNC: single Nucleocytes, Ne: Eosinophils, specif: Mast cell specificity (ratio to other leukocytes).

  Since AREG is a cytokine of the EGF family, we focused on AREG that is upregulated by aggregation of FcεRI but not downregulated by dexamethasone pretreatment, among mast cell-specific genes.

The reasons for focusing on AREG are as follows.
(i) AREG is involved in the process of lung branch morphogenesis in mice,
(ii) As shown in Examples described later, AREG induced MUC2 and MUC5AC mRNA in mucosal epithelial NCI-H292 cells to the same extent as EGF,
(iii) AREG increased mucin expression in NCI-H292 cells through the release of AREG by human airway trypsin-like protease, and
(iv) Unlike EGF, AREG is highly expressed as a mast cell-specificity and therefore excellent as a molecular target capable of controlling allergic symptoms.

[Example 1]
Analysis of AREG expression in human mast cells. Mast cells were sensitized with myeloma IgE, washed, and then challenged with 1.5 μg / mL anti-IgE or without antibody for 24 hours before harvesting the culture supernatant. After treatment with NCI-H292 cells (American Type Culture Collection: ATCC), which have grown until the cells are dense and depleted of serum in the medium, with 10 μg / ml anti-AREG neutralizing antibody or isotype control mIgG1 for 20 minutes The culture supernatant of the mast cells was added. Further, human AREG in the culture supernatant was measured using an ELISA kit (R & D Systems) according to the protocol attached to the kit. The sensitivity of the human AREG assay was 5 pg / mL.

[result]
AREG mRNA was clearly detected in IgE or anti-IgE activated mast cells (FIG. 2A). AREG mRNA appeared to be up-regulated by dexamethasone pretreatment of mast cells after FcεRI aggregation (FIG. 2A). In order to verify the secretion of AREG, the culture supernatant of mast cells activated using an ELISA kit was examined to confirm the presence of AREG. Dexamethasone pretreatment appeared to up-regulate IgE-mediated release of AREG, but was not significant (FIG. 2B). FIG. 2C shows the time course of AREG production by mast cells after FcεRI aggregation. AREG production continued to increase until at least 36 hours after FcεRI cross-linking. With dexamethasone alone, the results were almost the same as with the medium alone. AREG production was up-regulated by dexamethasone pretreatment of mast cells 36 hours after FcεRI cross-linking (FIG. 2C). AREG was released by anti-IgE in a concentration-dependent manner (FIG. 2D).

[Example 2]
Analysis of MUC2 and MUC5A expression by AREG Total RNA was extracted from NCI-H292 cells treated in Example 1, and expression of MUC2 and MUC5A was analyzed by quantitative real-time PCR.

[result]
As shown in FIGS. 3A and 3B, AREG increased MUC2 and MUC5AC gene expression in a dose-dependent manner in NCI-H292 cells. At 10 ng / ml, AREG increased the expression of the MUC2 and MUC5AC genes by 3 and 13 fold, respectively. The increased expression of MUC2 and MUC5AC induced by AREG was almost the same as that induced by EGF.

[Example 3]
Increased MUC2 and MUC5AC gene expression by activated mast cell culture supernatants NCI-292 cells that have been densely grown and serum depleted are pretreated with 1 μg / ml anti-AREG neutralizing antibody or 1 μg / ml mouse IgG1 After that, the cells were incubated with unstimulated mast cell-derived culture supernatant (Unsti MC SN) or activated mast cell culture supernatant (Stim MC SN) for 24 hours. Total RNA was extracted from NCI-292 cells and quantitative real-time RT-PCR analysis was used to determine the amount of MUC2 and MUC5AC mRNA. Fold change in mean mucin mRNA levels of anti-AREG mAb or mIgG ₁ treated cells incubated with activated mast cell culture supernatant compared to cells incubated with unstimulated mast cell culture supernatant (mean ± SEM) As shown in FIG.

[result]
4A and 4B show that activated mast cell culture supernatant further increases AREG-mediated MUC2 and MUC5AC gene expression. These increases were significantly inhibited by neutralizing antibodies against AREG.

[Example 4]
Immunohistochemistry 3 μm serial sections of respiratory mucosa from asthmatic patients and normal subjects listed in Table 1 were stained with anti-AREG mAb and anti-tryptase mAb using ABC kit (Vector Laboratories). Specifically, slides were quenched in 3% H ₂ O ₂ for 10 minutes to block endogenous peroxidase and washed in PBS. Sections were incubated with the primary antibody for 1 hour and then with the biotinylated secondary antibody before using ABC reagent. Color development was performed using diaminobenzidine as a substrate. Slides were counterstained with Mayer's hematoxylin. Preincubation of specific blocking peptide with primary antibody or replacement of primary antibody with irrelevant IgG was used as a negative control. AREG positive cells were counted in at least 6 high power fields of each sample by 3 separate observers. Eosinophils were identified using Hansel's stain (Torii Pharmaceutical). Mucin in secretory cells was clearly visualized by serial Alcian blue staining and periodate-Schiff staining of airway tissue sections. Intracellular mucinous glycoproteins of epithelial secretory cells were recognized as purple ellipsoids of various sizes.

To analyze the secretory response of goblet cells, the viscosity was determined from tissue sections by ranking the amount of mucus in each secreted cell as described below. In stage 1, the vertical distance of the stained area is 1/3 or less of the epithelial layer measured from the basement membrane to the apex of the cells. In stage 2, the vertical distance of the stained area exceeds 1/3 of the epithelial layer. The stained areas were ranked with 20 consecutive high power fields along the two walls of the trachea. In each donor, the mucus score was calculated as n ₁ + 2n ₂ (where n ₁ and n ₂ are the total number of stage 1 and stage 2 cells, respectively). The average score given to each sample by each investigator was first calculated, then the average score for each sample by three researchers was calculated and recorded as data.

[result]
Expression of AREG in bronchial mast cells of asthmatic patients To identify AREG positive (AREG ⁺ ) cells as mast cells, serial sections were used to stain one section with anti-tryptase mAb and another section anti Stained with AREG mAb. Bronchial bipsi samples from normal control subjects showed little immunoreactivity to AREG. In contrast, the bipsi samples from asthmatic patients showed a clear positive immunoreactivity for AREG in bronchial mast cells (FIG. 5A). Next, the number of AREG ⁺ cells in tryptase positive (tryptase ⁺ ) cells was counted. The proportion of AREG ⁺ mast cells averaged 35%, 52.8% and 52% in mild, moderate and severe asthmatic patients, respectively. The number of AREG ⁺ cells (AREG ⁺ cells / mm ² ) in 1 mm ² of bronchial mucosa of asthma samples and control samples was counted (FIG. 5B). The number of AREG ⁺ cells / mm ² was significantly increased in asthmatic samples compared to control samples (P <0.01). Airway epithelial cells of both asthmatic patients and normal subjects showed little immunoreactivity to AREG. Next, in order to clarify this, the number of AREG ⁺ tryptase ⁺ / mm ² was counted (FIG. 5C). In addition, AREG ⁺ epithelial cells, eosinophils and other proportions of total AREG ⁺ cells were counted in normal and asthmatic lung samples. As a result, it was found that less than 10% of all AREG ⁺ cells were epithelial cells and eosinophils (FIG. 5D). Other cells were less than 1%. Therefore, the number of AREG ⁺ cells was approximately the same as the total number of mast cells. Next, the relationship between the number of AREG ⁺ mast cells and the mucosal score was examined. As a result, it was revealed that there is a significant correlation between these two numerical values (FIG. 5E, P <0.005, r = 0.54). This suggests that AREG induces goblet cell hyperplasia.

  According to the present invention, it is possible to provide an index of allergic diseases such as asthma, atopy, and allergic rhinitis, and it is possible to easily and accurately diagnose the disease, and the mechanism of action of amphiregulin. Development of a novel therapeutic agent for allergic diseases based on the above becomes possible.

FIG. 1 shows mRNA expression levels in control mast cells, mast cells stimulated with anti-IgE and mast cells stimulated with anti-IgE and dexamethasone. Human mast cells were precultured with IgE in the presence or absence of dexamethasone and then activated with anti-IgE for 6 hours. Each row of colored bars represents one gene and each column represents one stimulus. The colored bar indicates the strength of the response of each gene according to the indicated scale. I represents a set of genes that are up-regulated by anti-IgE stimulation but reduced when activated with anti-IgE and then pretreated with dexamethasone. II represents a set of genes that are up-regulated by anti-IgE stimulation but are not affected by pretreatment with dexamethasone after activation with anti-IgE. III shows a set of genes that are not affected by anti-IgE stimulation but are down-regulated by treatment with dexamethasone prior to activation with anti-IgE. IV represents a set of genes that are not affected by anti-IgE stimulation but are up-regulated by pretreatment with dexamethasone. FIG. 2 is a diagram showing the expression of AREG by human mast cells. 2A: Upregulation of AREG expression in human mast cells by FcεRI-mediated activation. Human mast cells pretreated in the presence (+) or absence (−) of dexamethasone were pre-cultured with IgE and then activated with 1.5 μg / ml anti-IgE. Intracellular mRNA for AREG and GAPDH was amplified by RT-PCR. 2B: AREG secretion from mast cells after 1.5 μg / ml anti-IgE stimulation with (+) or without (−) dexamethasone pretreatment. The cell culture supernatant was collected at 24 hours, and ELISA against AREG was performed (n = 3 donor). 2C: Time course of AREG production by human mast cells stimulated with anti-IgE (1.5 μg / ml) with or without dexamethasone pretreatment (dotted bars) or without (black bars). Control cells were incubated with IgE in the presence (hatched bars) or absence (white bars) of dexamethasone, but anti-IgE treatment was excluded. 2D: Concentration-response study of anti-IgE induced AREG production by human mast cells. Mast cells were preincubated with IgE and then activated with 0.1, 0.5, 1.5, or 5 μg / ml anti-IgE for 24 hours. N. D. Means not detected. FIG. 3 is a graph depicting the effect of AREG on MUC2 and MUC5AC expression in NCI-292 cells. NCI-292 cells were incubated with rhAREG or rhEGF, and total RNA extracted from the cells was used to determine the amount of MUC2 (A) and MUC5AC (B) mRNA using quantitative real-time RT-PCR analysis. Results are shown as fold change (mean ± SEM) in mucin mRNA levels of AREG or EGF treated cells compared to AREG or EGF untreated cells (n = 3). * P <0.05 compared to cells not treated with AREG or EGF. FIG. 4 is a graph showing that activated mast cell culture supernatant increases AREG-mediated MUC2 and MUC5AC gene expression. Resting NCI-292 cells were pretreated with anti-AREG neutralizing antibody or mouse IgG1 and then cultured cultures derived from unstimulated mast cells (Unsti MC SN) or activated mast cell culture supernatants (Stim MC SN) for 24 hours. Total RNA was extracted from NCI-292 cells and quantitative real-time RT-PCR analysis was used to determine the amount of MUC2 (A) and MUC5AC (B) mRNA. Results are compared to cells incubated with unstimulated mast cell culture supernatants, fold change in mucin mRNA levels of anti-AREG mAb or mIgG ₁ treated cells incubated with activated mast cell culture supernatants (mean ± SEM) (n = 3). * P <0.05, comparison of cells treated with anti-AREG neutralizing antibody and mIgG ₁ treated cells. FIG. 5 is a diagram showing the correlation between the number of AREG ⁺ tryptase ⁺ cells and the degree of goblet cell hyperplasia in the airways of asthmatic patients. 5A: Coexistence of AREG in tryptase ⁺ mast cells. Two consecutive 3 μm sections of bronchial biopsy specimens from asthmatic patients were immunostained with tryptase (left panel) and AREG (right panel). 5B: Number of AREG ⁺ cells per mm ² of bronchial mucosa of normal control (Control) and asthma (Asthma). AREG ⁺ cells were counted in at least 6 high power fields of each sample by 3 separate observers. ** P <0.01, compare between numbers of AREG ⁺ cells in normal controls and asthmatic patients. 5C: Number of AREG ⁺ tryptase ⁺ cells per 1 mm ² of bronchial mucosa of normal control (Control) and asthma patients (Asthma). ** P <0.01, compare between number of AREG ⁺ tryptase ⁺ cells in normal controls and asthmatic patients. 5D: Percentage of AREG ⁺ epithelial cells and eosinophils between total AREG ⁺ normal cell specimens (open bars) and asthmatic lung specimens (filled bars). * P <0.05 vs. percentage of AREG ⁺ epithelial cells in control subjects and asthmatic patients. 5E: Correlation between the expression of AREG in mast cells and the extent of goblet cell hyperplasia in asthmatic patients. The degree of goblet cell hyperplasia was determined as described in Example 4, and the correlation with the number of AREG ⁺ tryptase ⁺ cells in the airways of asthmatic patients was analyzed.

Claims (24)

  A disease marker for an allergic disease comprising a polynucleotide having a base sequence of at least 15 bases of an amphiregulin gene and / or a polynucleotide complementary to the polynucleotide.   The disease marker according to claim 1, wherein the allergic disease is asthma, atopy or allergic rhinitis, and is used as a probe or primer in the examination of the disease.   The disease marker according to claim 1 or 2, wherein the allergic disease is asthma and used for sputum examination. A method for diagnosing allergic diseases, comprising the following steps (A) and (B):
(A) A step of measuring the expression level of an amphiregulin gene in a biological sample of a subject, and (B) a step of determining the presence or absence of an allergic disease based on the measurement result of (A). A method for diagnosing allergic diseases, comprising the following steps (a), (b) and (c):
(A) a step of binding RNA prepared from a biological sample of a subject or a complementary polynucleotide transcribed from the RNA and the disease marker according to any one of claims 1 to 3,
(B) a step of measuring RNA derived from a biological sample bound to the disease marker or a complementary polynucleotide transcribed from the RNA using the disease marker as an index,
(C) A step of determining the presence or absence of an allergic disease based on the measurement result of (b).   The determination of the presence or absence of allergic disease in the step (c) is performed using the measurement result obtained for the subject as compared with the measurement result obtained for the normal subject as an indicator that the amount of binding to the disease marker is increased. The diagnostic method according to claim 5.   A disease marker for an allergic disease comprising an antibody that recognizes amphiregulin.   The disease marker according to claim 7, wherein the allergic disease is asthma, atopy or allergic rhinitis, and is used as a probe in the examination of the disease.   The disease marker according to claim 7 or 8, wherein the allergic disease is asthma and used for sputum examination. A method for diagnosing allergic diseases, comprising the following steps (A) and (B):
(A) A step of measuring the expression level of amphiregulin in a biological sample of a subject, and (B) a step of determining the presence or absence of an allergic disease based on the measurement result of (A). A method for diagnosing allergic diseases comprising the following steps (a), (b) and (c):
(A) a step of binding a protein prepared from a biological sample of a subject and the disease marker according to any one of claims 7 to 9,
(B) measuring a protein derived from a biological sample bound to the disease marker using the disease marker as an index, and (c) determining the presence or absence of an allergic disease based on the measurement result of (b) .   The determination of the presence or absence of allergic disease in the step (c) is performed using the measurement result obtained for the subject as compared with the measurement result obtained for the normal subject as an indicator that the amount of binding to the disease marker is increased. The method for diagnosing an allergic disease according to claim 11. A screening method for a substance that suppresses the expression of amphiregulin, comprising the following steps (a), (b) and (c):
(a) contacting the test substance with a cell capable of measuring the expression of amphiregulin,
(b) measuring the expression level of amphiregulin in the cell contacted with the test substance, and comparing the expression level with the expression level of amphiregulin in the control cell not contacted with the test substance;
(c) A step of selecting a test substance that decreases the expression level of amphiregulin based on the comparison result of (b). A screening method for a substance that suppresses the binding activity of amphiregulin to the EGF receptor, comprising the following steps (a), (b) and (c):
(a) contacting the test substance with amphiregulin and EGF receptor;
(b) measuring the binding activity of the amphiregulin contacted with the test substance with the EGF receptor, and comparing the activity with the binding activity of the control amphiregulin not contacted with the test substance;
(c) A step of selecting a test substance that suppresses the binding activity of amphiregulin to the EGF receptor based on the comparison result of (b).   The screening method according to claim 13 or 14, which is a method for searching for an active ingredient of a therapeutic agent for allergic diseases. A screening method for a substance that suppresses sputum secretion, comprising the following steps (a), (b) and (c):
(a) contacting the test substance and amphiregulin with a cell capable of measuring mucin expression;
(b) measuring the expression level of mucin in the cell contacted with the test substance, and comparing the expression level with the expression level of mucin in the control cell not contacted with the test substance;
(c) A step of selecting a test substance that suppresses sputum secretion based on the comparison result of (b).   The screening method according to claim 16, wherein the vaginal secretion is associated with an allergic disease.   A therapeutic agent for allergic diseases, comprising an antibody that binds to amphiregulin.   A therapeutic agent for allergic diseases, comprising as an active ingredient a substance that suppresses the expression level of amphiregulin or the binding activity to the EGF receptor.   The therapeutic agent according to claim 19, wherein the substance is an antibody against amphiregulin or a dominant negative mutant or an expression vector comprising a nucleic acid molecule encoding the antibody or dominant negative mutant.   The therapeutic agent according to any one of claims 18 to 20, which has a sputum secretion inhibitory action or a remodeling inhibitory action.   A therapeutic agent for allergic diseases comprising a substance that suppresses the expression of an amphiregulin gene as an active ingredient.   The therapeutic agent according to claim 22, wherein the substance is an antisense nucleic acid, ribozyme, decoy nucleic acid or siRNA against an amphiregulin gene.   The therapeutic agent according to claim 22 or 23, which has a sputum secretion inhibitory action or a remodeling inhibitory action.