WO2018043715A1 - Examination method and examination kit for eosinophilic gastrointestinal disease or food-protein induced enteropathy - Google Patents

Abstract

Provided is a new method for examining eosinophilic gastrointestinal diseases or food-protein induced enteropathies. In other words, the present invention relates to a method for examining eosinophilic gastrointestinal diseases or food-protein induced enteropathies, the method including a measuring step for measuring the expression level of at least one of TSLP gene, IL-33 gene, CCL7 gene, and CCL21 gene in a sample collected from a living human subject or measuring the amount of at least one of TSLP protein, IL-33 protein, CCL7 protein, and CCL21 protein in said sample.

Description

Test method and test kit for eosinophilic gastrointestinal disease or food protein-induced enteropathy

The present invention relates to a test method and test kit for eosinophilic digestive tract disease or food protein-induced enteropathy.

Eosinophilic gastroenteritis (EGE) and eosinophilic esophagitis (EoE) are a group of diseases collectively called eosinophilic gastrointestinal disorders (EGID). It is one of the diseases to which it belongs. Eosinophilic gastrointestinal diseases are characterized by widespread infiltration of eosinophils in the gastrointestinal tract and are often caused by food intake (Non-Patent Document 1). In some studies on EoE, T H ₂ deflection cytokines (IL-13, etc.) and chemokines including (CCL26 / eotaxin-3, etc.) overproduction of a particular molecular involved in the pathology of EoE, cellular and immunological Mechanism has been clarified (Non-Patent Document 2). In contrast, the etiology of EGE remains largely unexplained. Infant EGE often exhibits severe, non-specific gastrointestinal symptoms, such as weight loss, making it difficult to diagnose. Previous studies reported that some cytokines (IL-5 and IL-15) can be detected at increased levels in the blood of adult patients with EGE, as in patients with EoE. (Non-Patent Document 3). In addition, non-IgE mediated gastrointestinal food allergy is known as a disease group having high homology with eosinophilic gastrointestinal diseases.

At present, in eosinophilic gastrointestinal diseases including EGE and EoE, it is necessary to prove eosinophil accumulation by repeated gastrointestinal endoscopy for proper diagnosis. Gastrointestinal endoscopy is an examination technique that places a heavy burden on the subject to be examined. In particular, when the test subject is an infant, since it is an excessively invasive test method, it causes a delay in diagnosis and causes serious complications.

Therefore, a new inspection method that is less burdensome for the inspection object than the gastrointestinal endoscopy is desired. The search for molecular markers associated with specific diseases can pave the way for the development of new testing techniques. However, in eosinophilic gastrointestinal diseases, no molecular marker that can be used for testing the disease has yet been found. Non-Patent Documents 2 and 3 also describe the results that molecules useful for diagnosis of eosinophilic gastrointestinal diseases could not be achieved.

The present invention has been made in view of the above problems, and an object thereof is to provide a molecular marker that can be used for examination of eosinophilic gastrointestinal tract disease or food protein-induced enteropathy, and use thereof. That is, it is to provide a novel test method and test kit for eosinophilic gastrointestinal tract disease or food protein-induced enteropathy using the molecular marker.

As a result of intensive studies in view of the above problems, the present inventors have found that among the cytokine and / or chemokine family, a TSLP gene, IL-33 (hereinafter also referred to as IL33) gene, CCL7 / The expression level of MCP-3 gene and CCL21 / 6CKine gene, or the amount of TSLP protein, IL-33 protein, CCL7 / MCP-3 protein and CCL21 / 6CKine protein in the sample is determined to be eosinophilic gastrointestinal disorders. And EGID) or food protein-induced enteropathy (Food-Protein-Induced-Enteropathy-Syndrome- (Enteropathy)).

That is, the present invention includes any of the following inventions.

1) A method for examining eosinophilic gastrointestinal tract disease or food protein-induced enteropathy, wherein thymic stromal lymphopoietin (TSLP) gene, IL-33 gene, CCL7 gene and CCL21 gene in a sample collected from a human body A test method comprising a measurement step of measuring an expression level of at least one of them, or measuring an amount of at least one of TSLP protein, IL-33 protein, CCL7 protein, and CCL21 protein in the sample.

2) A test kit for testing eosinophilic gastrointestinal disease or food protein-induced enteropathy, TSLP gene expression product, IL-33 gene expression product, CCL7 gene expression in a sample collected from a human body A nucleic acid probe, a nucleic acid primer, a nucleic acid aptamer, an antibody for detecting at least one of a product and an expression product of CCL21 gene, or at least one of TSLP protein, IL-33 protein, CCL7 protein and CCL21 protein in the sample Or a test kit comprising a peptide probe.

The present invention provides a novel test method and test kit for eosinophilic digestive tract disease or food protein-induced enteropathy.

FIG. 5 shows increased levels of TSLP and IL-33 in serum. AE shows the results of a serum multiplex assay (Millipore, St Charles, Mo), including 7 healthy control subjects (CTRL) and 37 immediate FA asymptomatic patients, A comparison of 31 active AD patients, 8 active UC patients, and 9 infant EGE patients is shown. Error bars represent the median and interquartile ranges. A and B in the figure show that the levels of serum TSLP and serum IL-33 are clearly increased (**** P <.0001) in patients with infant EGE, while 3 Only 4 mild AD patients showed a mild increase. C and D in the figure indicate that the levels of MCP3 (Monocyte chemoattractant protein 3) and 6CKine increased in patients with EGE (** P <.01). E in the figure indicates that the level of CTACK (cutaneous T cell-attracting chemokine) was increased particularly in AD patients (*** P <.001). F in the figure indicates that the levels of TSLP in serum and IL-33 in serum are strongly correlated with each other (r = 0.91, P <.0001). G in the figure indicates that there is no correlation between the CTACK level and the TSLP level. Allergic inflammation in the skin and gastrointestinal tract was clearly distinguished by CTACK and TSLP. H and I in the figure indicate that both the serum levels of TSLP (H in the figure) and IL-33 (I in the figure) decrease markedly from the active phase to the recovery phase. Shown (* P <05.05). It is a figure which shows the result of the microarray analysis of the gene specifically expressed in the biopsy sample of the inflamed sigmoid colon, and qPCR analysis. A in the figure is 841 genes (fold change> 2, P <.05) specifically expressed in the EGE group compared to the control group, and compared with the control group. The result shows that 762 genes (fold change> 2, P <.05) specifically expressed in the UC group were analyzed using cluster analysis and shown as a heat map. Up-regulated genes are shown in red, and down-regulated genes are shown in blue. Clusters 5 and 6 highlight the EGE transcriptome, and the enlarged image on the right shows 14 genes, including TSLP, that form part of cluster 5. N / A refers to Not applicable. As shown in B in the figure, EGE patients show markedly increased expression of both TSLP and IL33 mRNA in the sigmoid colon when compared to UC patients (** P = .0018, *** P = .0003) was confirmed by qPCR. Error bars represent median and interquartile region. It is a figure which shows the knowledge regarding the microscopic observation of a GI mucous membrane, and the weight gain in the infant EGE patient after the start of a diet therapy. A in the figure shows the mucosa of the esophagus of patient No. 5. Significant infiltration and degranulation of eosinophils were seen in the stratified flat layer of the esophagus. B in the figure shows the mucosa of the esophagus of patient No. 3. C in the figure indicates the duodenal mucosa of patient No. 5. D in the figure indicates the colonic mucosa of patient No. 10 patient. All of AD are preparations stained with hematoxylin and eosin (magnification x1,000). E in the figure is data on a one-year-old boy with weight loss (-3.8 SD) who was transferred to the hospital of the inventor No. 4 and was milk, soy and chicken eggs There was a dramatic increase in body weight after removal. The boy's motor skills were also normalized later. F in the figure is data relating to a 1-year-old boy who is the patient of No. 5 and whose weight gain has stopped from 5 months of age. The boy was treated with dietary restrictions and his weight normalized.

Hereinafter, embodiments of the present invention will be described in detail.

[Explanation of terms]
In the present specification, “protein” can also be referred to as “polypeptide”. The “protein” includes a structure in which amino acids are peptide-bonded, and may further include a structure such as a sugar chain or an isoprenoid group. “Protein”, unless otherwise specified, encompasses a polypeptide containing a known analog of a naturally occurring amino acid that can function similarly to the naturally occurring amino acid.

In the present specification, the “nucleic acid” includes a polynucleotide comprising any simple nucleotide and / or modified nucleotide, such as cDNA, mRNA, total RNA, hnRNA, and the like. “Modified nucleotides” include inosin, acetylcytidine, methylcytidine, methyladenosine, and methyl guanosine, as well as nucleotides that can be generated by the action of ultraviolet light or chemical substances.

In the present specification, “gene” is used interchangeably with “polynucleotide”, “nucleic acid” or “nucleic acid molecule”. “Polynucleotide” means a polymer of nucleotides.

Therefore, the term “gene” in this specification includes not only double-stranded DNA but also single-stranded DNA or RNA (such as mRNA) such as sense strand and antisense strand constituting the DNA. The antisense strand can be utilized as a probe or as an antisense agent.

“DNA” includes, for example, cDNA or genomic DNA obtained by cloning or chemical synthesis techniques, or a combination thereof. That is, the DNA may be a “genomic” type DNA containing a non-coding sequence such as an intron, which is a form contained in the genome of an animal, or obtained via mRNA using a reverse transcriptase or a polymerase. It may also be a “transcribed” form of DNA that does not contain non-coding sequences such as introns.

As used herein, the term “healthy person” refers to a normal individual who does not suffer from eosinophilic gastrointestinal disease or food protein-induced enteropathy.

In the present invention, “having the risk (risk level) of onset” means that the onset has not yet occurred but the possibility of onset is higher than that of healthy subjects, and “eosinophilic gastrointestinal disease” Or at risk of developing `` food protein-induced enteropathy '' is a pre-stage of eosinophilic digestive tract disease or food protein-induced enteropathy and can lead to eosinophilic digestive tract disease or food protein-induced enteropathy It also includes the state of high nature.

In the present invention, “diagnosis” means whether or not the subject suffers from the target disease, the severity of eosinophilic gastrointestinal disease or food protein-induced enteropathy that the subject suffers from, and the subject To assess the risk (risk level) of developing a disorder and / or the presence or absence of a predisposition to eosinophilic gastrointestinal disease or food protein-induced enteropathy in the subject, and to identify the disease or condition Say.

As used herein, “inspecting” or “examination” means eosinophilic digestive tract disease in a human subject to be examined (sometimes referred to as “subject”) that does not require identification (diagnosis) by a doctor. Or refers to testing for food protein-induced enteropathy. The test result obtained by the test method of the present invention can be a material for diagnosis made by a doctor. It should be noted that the presence or absence of a predisposition to eosinophilic gastrointestinal disease or food protein-induced enteropathy refers to whether or not an eosinophilic gastrointestinal disease or food protein-induced enteropathy has already occurred. It is also a concept that includes examining the possibility of developing eosinophilic gastrointestinal disease or food protein-induced enteropathy. On the other hand, testing for the presence of eosinophilic gastrointestinal disease or food protein-induced enteropathy refers to testing for the occurrence of eosinophilic gastrointestinal disease or food protein-induced enteropathy. .

In the present invention, “treatment” includes complete cure or alleviation of symptoms of a target disease, suppression of deterioration of symptoms of the target disease, and suppression or delay of onset of the target disease. That is, it includes “prevention” when the subject does not develop the target disease.

[1. Inspection method〕
The test method according to the present invention is a method for testing eosinophilic gastrointestinal tract disease or food protein-induced enteropathy, and TSLP in a sample collected from a human living body (hereinafter sometimes referred to as “biological sample”). At least one of a gene, IL-33 gene, CCL7 (also referred to as MCP-3; hereinafter also referred to as CCL7 / MCP-3) gene and CCL21 (also referred to as 6CKine; hereinafter also referred to as CCL21 / 6CKine) gene It is a method including a measurement step of measuring the expression level or measuring the amount of at least one of TSLP protein, IL-33 protein, CCL7 / MCP-3 protein and CCL21 / 6CKine protein in the sample.

<Eosinophilic digestive tract disease>
Eosinophilic gastrointestinal disorders (EGID) is a general term for eosinophilic inflammatory diseases with the gastrointestinal tract as the main component. Eosinophilic gastrointestinal disorders (EGID) are preferred due to abnormal accumulation and infiltration of eosinophils locally in the gastrointestinal tract. It is a disease that causes dysfunction due to injury of tissues caused by acidophilic inflammation. Eosinophilic esophagitis (Eosinophilic Esophagitis, EoE) in which a large number of eosinophils are infiltrated only within the esophageal mucosal epithelium, depending on the site of eosinophil infiltration, and esophageal lesions Eosinophilic Gastroenteritis (EGE), in which a large number of eosinophilic infiltrations appear in the lamina propria of the stomach or intestinal tract regardless of the presence or absence of Eosinophilic Gastroenteritis (EGE).

(Eosinophilic Gastroenteritis, EGE)
The site of eosinophilic infiltration in eosinophilic gastroenteritis varies from the esophagus to the large intestine, so symptoms may vary depending on the lesion site, but anorexia of eosinophilic gastroenteritis, vomiting, abdominal pain Symptoms such as diarrhea, bloody stool and ascites. Symptoms of the disease also include symptoms other than gastrointestinal symptoms such as poor weight gain or weight loss, and decreased activity. In severe cases, digestive tract obstruction (such as when eosinophils infiltrate the muscle layer), intestinal rupture, and peritonitis may occur. In addition, hypoproteinemia or iron deficiency anemia may occur due to gastrointestinal malabsorption.

Eosinophilic gastroenteritis occurs almost equally between men and women in the ages from newborns (below 28 days after birth) to adults (over 20 years old). The cause of the development of eosinophilic gastroenteritis is considered to be non-IgE-mediated food allergy. Patients with eosinophilic gastroenteritis often have allergic diseases such as bronchial asthma and urticaria.

Eosinophilic gastroenteritis is a disease diagnosed based on the following guidelines 1 to 8 in the conventional diagnosis. Targets satisfying 1 and 2 or 3. Items other than these are for reference.
1. If you have symptoms (abdominal pain, diarrhea, vomiting, etc.).
2. Stomach, small intestine, and large intestine biopsies have eosinophil-based inflammatory cell infiltration in the mucosa (20 / high-power field (HPF)) or higher eosinophil infiltration, biopsy It should be done in several places and exclude other inflammatory bowel diseases). In addition, since infiltration of eosinophils into the lamina propria of the stomach or intestinal tract is also observed in healthy subjects, comparison with the number of normal eosinophils by site is necessary for accurate diagnosis.
3. Ascites exists and many eosinophils are present in the ascites.
4). Has a history of allergic diseases such as asthma.
5). Eosinophilia is observed in peripheral blood.
6). CT scan shows thickening of stomach or intestinal wall.
7). Endoscopy shows edema, redness or erosion in stomach, small intestine or large intestine.
8). Glucocorticoids are effective.

(Eosinophilic Esophagitis, EoE)
The site of eosinophil infiltration in eosinophilic esophagitis is limited to the esophagus.

Eosinophilic esophagitis develops at the age from newborn to adult, but it occurs most often in men in their 30s and 50s. Among eosinophilic esophagitis, cases where proton pump inhibitors (Proton 薬 Pump Inhibitor, PPI) are effective should be classified as PPI-REproton-pump inhibitor (PPI) -responsive esophageal eosinophilia (PPI-RE). There is also.

∙ Symptoms vary with age. Infants (1 year old and younger) and infants (6 years old and younger) have suckling disorders, infants to children (18 years old and younger) have vomiting, abdominal pain and dysphagia. Furthermore, esophageal stenosis may occur if the disease becomes longer or more severe.

Eosinophilic esophagitis is a disease diagnosed based on the following guidelines 1 to 7 in the conventional diagnosis. Targets satisfying 1 and 2. Items other than these are for reference.
1. Symptoms due to esophageal dysfunction (dysphagia, feeling of gripping, etc.).
2. In esophageal mucosa biopsy, eosinophils of 20 / HPF (high-power field) or more are present in the epithelium.

(Biopsy should be done in several places in the esophagus)
3. Endoscopy shows vitiligo, longitudinal grooves or tracheal stenosis in the esophagus.
4). CT scan or endoscopy reveals thickening of the esophageal wall.
5). Eosinophilia is observed in peripheral blood.
6). Male 7 Proton pump inhibitors are ineffective and glucocorticoid preparations are effective.

<Food protein-induced enteropathy>
Food protein-induced enteropathy is a type of non-IgE-mediated gastrointestinal food allergy, and is mainly characterized by chronic diarrhea and poor weight gain in infants. Diagnosis is made mainly by inflammatory cell infiltration in the pathological tissue.

<Subject>
The inspection method according to the present embodiment can be applied to subjects of all ages. In some embodiments, the age of the subject is preferably as low as possible. For example, it is preferably up to 6 years old or younger, more preferably up to 2 years old or younger, more preferably up to 1 year old or younger. In some cases, it is more preferable. By examining at a lower age, growth disorder or growth failure due to malabsorption from the gastrointestinal tract, weight loss, hypoproteinemia, and the like can be prevented at an early stage. In addition, newborn infants who are difficult to perform endoscopy of the digestive tract can be diagnosed at an earlier stage. It can be prevented from occurring. In one embodiment, the subject may be a prenatal fetus.

<Biological sample>
The biological sample is collected from the subject's living body. There are no particular limitations on the type of biological sample, and it is sufficient that it contains at least one of the subject's protein and nucleic acid (mRNA as a gene expression product). Examples of the biological sample include a cell sample, a tissue sample, and a body fluid sample, and among them, a body fluid sample is preferable. Examples of the body fluid sample include a blood sample, a lymph fluid sample, a cerebrospinal fluid sample, and the like, but a blood sample and ascites are preferable, and a peripheral blood sample is particularly preferable among the blood samples. The peripheral blood sample can be easily collected, for example, by puncturing a fingertip or the like, so that the burden on the subject is small, and in addition, the peripheral blood sample sufficiently contains the molecular marker that is the subject of the test method of the present invention. Among blood samples, an umbilical cord blood sample has the advantage that it can be easily collected from the umbilical cord at the time of birth and can be diagnosed very early.

Also, if necessary, a biological sample is also collected from a healthy person who does not have eosinophilic gastrointestinal tract disease or food protein-induced enteropathy for use as a control sample. The control sample is preferably the same type as that of the subject (collected from the same site). When control data is prepared in advance when performing the test method of the present invention, it is not necessary to collect a biological sample from a healthy person.

The collected biological sample may be subjected to an operation after performing an operation for extracting a protein or a nucleic acid or an operation for removing an unnecessary component as necessary. For example, when a blood sample is used, it is preferable to use serum or plasma prepared from the collected blood for the test.

In addition, the obtained biological sample may be stored by a method suitable for the type of biological sample, such as cryopreservation as necessary. A biological sample can be stored to measure a molecular marker that is an object of an inspection method at a desired time. At the time of storage, a sample as it is collected may be stored, or a sample (for example, serum or plasma) prepared after collection may be stored.

<Measurement process>
The inspection method of the present invention includes the measurement steps shown in the following a) or b).
Step a) Measuring the expression level of at least one of the TSLP gene, IL-33 gene, CCL7 / MCP-3 gene and CCL21 / 6CKine gene in the biological sample b) TSLP protein in the biological sample, IL-33 The amount of at least one of protein, CCL7 / MCP-3 protein and CCL21 / 6CKine protein is measured.

Whether to use step a) or step b) is selected based on conditions such as the type of biological sample or the type of subject (age, disease to be examined), but in the case of a serum sample, step b) Is preferred. In the case of step a), it is preferable to measure the expression level of at least one of the TSLP gene and IL-33 gene. For the determination of eosinophilic gastroenteritis, at least the TSLP gene and IL-33 More preferably, the expression level of the gene is measured, and the expression level of the TSLP gene and the IL-33 gene and the expression level of at least one of the CCL7 / MCP-3 gene and the CCL21 / 6CKine gene are further measured. Preferably, the expression levels of TSLP gene, IL-33 gene, CCL7 / MCP-3 gene and CCL21 / 6CKine gene are most preferably measured. In the case of step b), it is preferable to measure at least one of the amounts of TSLP protein and IL-33 protein. For the determination of eosinophilic gastroenteritis, at least the amount of TSLP protein and IL-33 protein More preferably, the amount of TSLP protein and IL-33 protein and the amount of at least one of CCL7 / MCP-3 protein and CCL21 / 6CKine protein are measured, and TSLP protein, IL Most preferably, the amount of −33 protein, CCL7 / MCP-3 protein and CCL21 / 6CKine protein is measured.

In addition, in order to more accurately determine eosinophilic gastroenteritis among eosinophilic gastrointestinal diseases, CCL5, CCL20, CCL22 and neurofilament medium polypeptide (NEFM) in addition to the above-mentioned cytokines and / or chemokine proteins. ) At least one gene expression level or protein level may be further included.

Step a)
Step a) is a step of measuring the expression level of at least one of TSLP gene, IL-33 gene, CCL7 / MCP-3 gene and CCL21 / 6CKine gene in the sample using the biological sample. TSLP gene is a general term for nucleic acids encoding TSLP, IL-33 gene is a general term for nucleic acids encoding IL-33, and CCL7 / MCP-3 gene is CCL7 / MCP-. 3 is a generic term for nucleic acids that encode C3, and the CCL21 / 6CKine gene is a generic term for nucleic acids that encode CCL21 / 6CKine.

The method for measuring the expression level of at least one of the TSLP gene, IL-33 gene, CCL7 / MCP-3 gene, and CCL21 / 6CKine gene is not particularly limited, but may be obtained using a nucleic acid amplification technique such as a PCR method. A method including a method of amplifying a nucleic acid (for example, mRNA which is a transcript) may be used. Examples of the method using the nucleic acid amplification technique include quantitative RT-PCR, and examples of the method for directly detecting mRNA include the Northern blot method. Alternatively, it may be a method for measuring the expression level of a gene using a nucleic acid chip such as a microarray. Note that “measuring the expression level of a gene” can be used interchangeably with “measuring the amount (concentration, etc.) of a gene expression product (described later)”.

In measuring the gene expression level, cDNA may be prepared using mRNA contained in a biological sample as a template. Amplification of TSLP gene, IL-33 gene, CCL7 / MCP-3 gene and CCL21 / 6CKine gene as mRNA can be performed based on nucleotide sequence information available from public databases such as NCBI. For example, in the NCBI database, the registration number of the TSLP gene is NM_033035.4. The registration number of the IL-33 gene is NM_033439.3. The registration number of the CCL7 / MCP-3 gene is NM_006273.3. The registration number of the CCL21 / 6CKine gene is NM_002989.3. Based on the above information, those skilled in the art can easily design appropriate primers for amplifying the TSLP gene, IL-33 gene, CCL7 / MCP-3 gene or CCL21 / 6CKine gene.

Step b)
Step b) is an amount of at least one protein of TSLP protein, IL-33 protein, CCL7 / MCP-3 protein and CCL21 / 6CKine protein in the biological sample, more specifically, per unit amount of the biological sample. Is a step of measuring the amount of protein (for example, protein concentration) contained in. However, the concept of measuring the amount of protein contained in a unit amount of a biological sample includes both quantitative and qualitative measurements. To present. More specifically, for example, a data comparison at the time of acquisition before concentration conversion using a calibration curve or the like, or a result in a format indicating whether the amount of protein exceeds a certain threshold value. This also includes the presentation of

A method for measuring the amount of at least one protein of TSLP, IL-33, CCL7 / MCP-3 and CCL21 / 6CKine is not particularly limited. For example, TSLP, IL-33, CCL7 / MCP-3 or CCL21 / Examples thereof include a method using an immunological technique using an antibody specific for 6CKine, liquid column chromatography, and mass spectrometry.

In one embodiment, the test method according to the present invention comprises 1) at least one of antibody proteins specific for TSLP, IL-33, CCL7 / MCP-3 or CCL21 / 6CKine according to the present invention, and a subject. And 2) an antibody level measurement step of detecting the antibody and measuring the antibody level after the contact step.

<Contact process>
The contact step is performed by mixing the antibody protein specific for TSLP, IL-33, CCL7 / MCP-3 or CCL21 / 6CKine and a biological sample collected from the subject, etc. Is a step of contacting the.

<Antibody level measurement process>
The antibody level measurement step is a step that is performed after the contact step, detects an anti-protein antibody, and measures the antibody level of the antibody. In the present invention, “antibody level” refers to the amount or antibody titer of the antibody contained in a biological sample. These values can be measured using a known method.

Examples of the method using an antibody include ELISA (Enzyme-Linked Immuno Immunosorbent Assay), quantitative Western blotting, dot blot assay, immunoprecipitation, and the like. Is preferably used. The type of ELISA method is not particularly limited, but is a so-called antigen measurement system (measurement of the amount of antigen contained in a biological sample), ELISA by direct adsorption method, ELISA by competition method, ELISA by sandwich method, and micro flow Examples include ELISA specialized for the measurement of a small amount of sample using a road system or microbeads. Alternatively, it may be detected by an assay method using an in vitro immunohistological method such as radioimmunoassay (RIA) and immunodiffusion assay. It can also be detected by image analysis in vivo.

The amount of antibody present in a biological sample can be determined using standard preparations (eg, standard samples of healthy individuals or typical eosinophilic gastrointestinal diseases or food using, for example, a linear regression computer algorithm). It can be easily calculated by comparison with the amount present in the standard sample of patients with protein-induced enteropathy.

The antibody specific to TSLP, IL-33, CCL7 / MCP-3 or CCL21 / 6CKine may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. For example, the amino acid sequences of human TSLP, IL-33, CCL7 / MCP-3, and CCL21 / 6CKine are available from public databases such as NCBI. For example, in the NCBI database, the registration numbers of TSLP, IL-33, CCL7 / MCP-3, and CCL21 / 6CKine are NP_14924.1, NP_2542744.1, NP_006264.2, and NP_002980.1, respectively. Based on the above information, those skilled in the art can easily determine an amino acid sequence suitable as an antigen for preparing antibodies specific for TSLP, IL-33, CCL7 / MCP-3 and CCL21 / 6CKine.

In the present invention, “antibody” is intended to mean a form including all classes and subclasses of immunoglobulins and functional fragments of antibodies. The antibody is a concept including both a natural antibody of a polyclonal antibody and a monoclonal antibody, and additionally includes an antibody produced using a gene recombination technique, and a functional fragment of the antibody. The “functional fragment of an antibody” refers to one having a partial region of the above-described antibody and having an antigen-binding ability (synonymous with a binding fragment). Natural antibodies can be derived from any species including, but not limited to, humans, mice, rats, goats, rabbits, camels, llamas, cows, chickens, sharks, and fish. The antibody produced using gene recombination technology is not particularly limited, but chimeric antibodies such as humanized antibodies and primatized antibodies obtained by genetic modification of natural antibodies, synthetic antibodies, recombinant antibodies, Mutagenized antibodies and graft-bound antibodies (for example, antibodies to which other proteins and radioactive labels are conjugated or fused), and antibodies already produced using genetic recombination techniques are described above. These also include antibodies that have been modified in the same manner as when genetically modifying natural antibodies. Specific examples of functional fragments of antibodies include F (ab ′) ₂ , Fab ′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv), and dAb (single domain antibody). (George et al, Exp. Opin. Ther. Patents, Vol. 6, No. 5, p.441-456, 1996).

Further, in the present invention, the binding fragment includes an antibody fragment mutated in a range that maintains reactivity with the target protein as a concept of the binding fragment. The above-described mutation introduction is performed using a known technique such as a gene modification technique, which is appropriately selected by those skilled in the art.

<Inspection process>
The test method according to the present invention may further include a test process for testing eosinophilic gastrointestinal tract disease or food protein-induced enteropathy based on the measurement result in the measurement process, if necessary. The examination is intended to be performed by any examiner and does not include a diagnosis by a doctor or the like. The test for eosinophilic gastrointestinal tract disease or food protein-induced enteropathy is performed using the TSLP gene, IL-33 gene, CCL7 / MCP-3 gene, and The amount of expression of at least one of the CCL21 / 6CKine genes or the amount of at least one of the TSLP protein, IL-33 protein, CCL7 / MCP-3 protein, and CCL21 / 6CKine protein is compared between the subject and the control. By doing.

In another embodiment, the test step for eosinophilic gastrointestinal tract disease or food protein-induced enteropathy is a TSLP gene, IL-33 gene obtained by the measurement step shown in the above step a) or step b). An expression level of at least one of CCL7 / MCP-3 gene and CCL21 / 6CKine gene, or an amount of at least one protein of TSLP protein, IL-33 protein, CCL7 / MCP-3 protein, and CCL21 / 6CKine protein Is performed by comparing samples at different times of the subject over time. This process is effective for objectively observing and evaluating the progress of disease or the progress of treatment or the effect of treatment in one subject.

In an example of the determination made in the test in the test process, TSLP, IL-33, CCL7 / MCP-3 or CCL21 / 6CKine (the amount of protein or the amount of gene expression) in the subject's biological sample compared to the control If the amount of at least one of (good) is significantly higher, the subject is determined to have or develop a predisposition to eosinophilic gastrointestinal disease or food protein-induced enteropathy. In another example, the expression level of TSLP gene, the expression level of IL-33 gene, the expression level of CCL7 gene and the expression level of CCL21 gene in the biological sample of the subject, or the amount of TSLP protein in the sample, IL -Predisposition to eosinophilic gastrointestinal disease or food protein-induced enteropathy when two, three or all of the amount of 33 protein, CCL7 protein and CCL21 protein are high compared to the control It is determined that there is or is developing.

In addition, the amount is significantly high (large) may be a result of quantitative measurement or a result of qualitative measurement. In addition to comparison of specific numerical values, comparison of relative amounts (actual It is not necessary to calculate the amount, and it is a concept that also determines whether it is higher or lower than a certain standard.

The above-described inspection of the control sample may be performed simultaneously with the inspection of the subject's sample, or may be performed separately. That is, the numerical value of the control sample compared with the numerical value of the subject may be a value obtained by a test performed when the test sample is different from the test sample. Further, the test of the control sample does not need to be performed by the person who performs the test of the subject. For example, the test value of the control sample that has already been acquired and accumulated in a database or the like can be used as the threshold value.

As for the numerical value of the control sample used for the test, the numerical value of the individual sample of healthy individuals may be directly used, or the average value obtained when the numerical value of the sample of a certain number of healthy individuals is used as a population is used. May be. Further, a cutoff value may be set in advance, and the numerical value of the subject may be compared with this cutoff value. A person skilled in the art can determine a quantitative value (normal value) in a healthy person, a quantitative value (disease value) in a typical eosinophilic gastrointestinal disease or food protein-induced enteropathy patient, or mild, moderate or severe eosinophils. The threshold can be appropriately set with reference to the quantitative value (disease value) of patients with sexually digestive tract disease or food protein-induced enteropathy. That is, in general, the threshold value in a diagnostic agent is appropriately set by a person skilled in the art according to the purpose based on many measured values of a healthy person or patient obtained from a clinical trial (for example, as in a screening test). In addition, when making a definitive diagnosis after the secondary examination with the highest priority not to overlook the disease group, priority is given to sensitivity over specificity and eosinophilic gastrointestinal tract disease with a low cutoff value. Or when diagnosing the degree of symptoms of food protein-induced enteropathy, set a high threshold with reference to quantitative values in patients with mild, moderate or severe eosinophilic gastrointestinal diseases or food protein-induced enteropathy, etc. From the description disclosed herein, a person skilled in the art can easily determine a threshold for diagnosis. For the control values used for diagnosis, the values of samples from healthy individuals, patients with eosinophilic gastrointestinal tract disease or individuals with food protein-induced enteropathy may be used directly. You may utilize the average value obtained when the numerical value of the sample of a patient with a spherical gastrointestinal tract disease or a patient with food protein-induced enteropathy is used as a population. For example, the expression level of at least one of TSLP gene, IL-33 gene, CCL7 / MCP-3 gene and CCL21 / 6CKine gene in a biological sample of a subject or TSLP, IL-33, CCL7 / MCP-3 in the sample And when the amount of at least one protein of CCL21 / 6CKine is greater than or equal to the cut-off value, it is determined that the subject is likely to develop eosinophilic gastrointestinal disease or food protein-induced enteropathy Can do. Further, the expression level of at least one of TSLP gene, IL-33 gene, CCL7 / MCP-3 gene and CCL21 / 6CKine gene in a biological sample of a subject, or TSLP, IL-33, CCL7 / MCP-3 and CCL21 in the sample If the amount of at least one protein of / 6CKine is higher than the cut-off value, it is determined that the subject has the possibility and risk of developing eosinophilic gastrointestinal disease or food protein-induced enteropathy Can do.

“Cutoff value” refers to both diagnosis sensitivity (prevalence of disease diagnosis) and specificity of diagnosis (no disease diagnosis rate) when the presence or absence of a predisposition or onset of disease is determined based on this value. Indicates a sufficiently high value. For example, in individuals who develop eosinophilic gastrointestinal tract disease or food protein-induced enteropathy, and in individuals who do not develop eosinophilic GI disease or food protein-induced enteropathy A value showing a high negative rate can be set as a cutoff value.

Here, “diagnostic sensitivity” is a ratio (true value) indicating a positive (abnormal value) when a test is performed on a population having a predisposition to a specific disease or developing a specific disease. Positive rate). The “diagnostic specificity” refers to a ratio (a ratio of true negative) indicating a negative (normal value) when a test is performed on a population not suffering from a specific disease. In addition, “positive predictive value” refers to the proportion of individuals who actually have a disease among subjects who showed a positive result in the test, and negative predictive value represents the proportion of subjects who showed a negative result in the test. Means the percentage of individuals who do not actually have the disease.

As a calculation method of the cutoff value, a method known in the technical field can be used. For example, the TSLP gene, IL in samples collected from individuals who have developed eosinophilic gastrointestinal disease or food protein-induced enteropathy and individuals who have not developed eosinophilic gastrointestinal disease or food protein-induced enteropathy The expression level of at least one cytokine and / or chemokine gene of -33 gene, CCL7 / MCP-3 gene and CCL21 / 6CKine gene, or TSLP protein, IL-33 protein, CCL7 / MCP-3 protein and The amount of at least one of the CCL21 / 6CKine proteins is calculated, and the diagnostic sensitivity and diagnostic specificity at the calculated values are obtained. Based on the values obtained as described above, a ROC (Receiver Operating Characteristic) curve is created using commercially available suitable analysis software. Subsequently, a value when the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible is obtained from the curve, and the value can be used as a cutoff value. Further, for example, the expression level of at least one of TSLP gene, IL-33 gene, CCL7 / MCP-3 gene and CCL21 / 6CKine gene in a sample prepared from a large number of healthy subjects, or TSLP protein, IL-33 protein in the sample, It is also preferable to use “average value + 2 standard deviation” of the amount of at least one protein of CCL7 / MCP-3 protein and CCL21 / 6CKine protein as a cut-off value, and with this value, good sensitivity and specificity can be obtained. It becomes possible to determine that eosinophilic gastrointestinal tract disease or food protein-induced enteropathy has occurred or is at risk of onset.

For example, an average value (protein concentration) obtained from a plurality of control samples or a slightly higher value (for example, normal values of protein concentration in serum include TSLP: 0 pg / mL to 4.7 pg / mL, IL -33: 0 pg / mL to 8.9 pg / mL, CCL7 / MCP3: 0 pg / mL to 1.5 pg / mL, CCL21 / 6CKine: 0 pg / mL to 458 pg / mL) It indicates that the risk of developing acidophilic gastrointestinal tract disease or food protein-induced enteropathy, that is, the presence or absence of predisposition or the presence or absence of onset can be detected. The cut-off value may be determined by classifying data for each fixed age and creating an ROC curve at each age.

<Other processes>
Differentiation step of eosinophilic gastrointestinal tract disease or food protein-induced enteropathy and diseases other than eosinophilic gastrointestinal tract disease or food protein-induced enteropathy In one embodiment, the test method of the present invention comprises eosinophilic It includes a differentiation step for differentiating gastrointestinal diseases or food protein-induced enteropathy from diseases other than eosinophilic gastrointestinal diseases or food protein-induced enteropathy.

Symptoms are similar to those of eosinophilic gastrointestinal tract diseases, and there are several diseases that need to be differentiated. Diseases that should be differentiated from eosinophilic gastrointestinal diseases include irritable bowel syndrome, Crohn's disease, ulcerative colitis (UC), collagen formation colitis, drug-induced enteritis, idiopathic favor Hypereosinophil syndrome, lymphoma, scleroderma, Churg-Strauss syndrome, and Henoch-Schonlein purpura, necrotizing enterocolitis, closed gastrointestinal bacterial enteritis, pseudomembranous enteritis , Hemolytic uremic syndrome, parasitic diseases, lactose intolerance, neonatal melenamekel diverticulosis, midgut volvulus, intussusception, pyloric stenosis and Hirschsprung's disease. In particular, diseases differentiated from eosinophilic esophagitis include gastroesophageal reflux disease, Candida infection and drug allergy.

For example, irritable bowel syndrome and eosinophilic gastroenteritis have heretofore been difficult to distinguish from symptoms, and histopathological examination was necessary. In addition, when there is no typical intestinal lesion from Crohn's disease, ulcerative colitis and eosinophilic gastroenteritis, it is difficult to differentiate from eosinophilic gastroenteritis even by histopathological examination. Therefore, in the past, it took time to make a discrimination such as making a judgment while observing the clinical course. For this reason, there is a possibility that the diagnosis is delayed or the diagnosis is wrong. In addition, eosinophilic gastroenteritis and eosinophilic esophagitis having esophageal lesions had to be differentiated by performing biopsy of the stomach and duodenum.

Also, for example, diseases that are differentiated from eosinophilic gastrointestinal diseases include non-IgE mediated gastrointestinal food allergy in newborns and infants. Non-IgE-mediated gastrointestinal food allergies include: Food Protein-Induced Enterocolitis Syndrome (FPIES), Food Protein-Induced Proctocolitis Syndrome (Proctocolitis), Food Protein Induction Examples include enteropathy (Food Protein-Induced Enteropathy Syndrome (Enteropathy)) and celiac disease (Celiac Disease). As in the case of eosinophilic gastrointestinal diseases, these diseases have been conventionally diagnosed by tests such as a stress test and observation of inflammatory cell infiltration in a pathological tissue. Eosinophilic gastrointestinal diseases traditionally require pathological diagnosis by performing gastrointestinal endoscopy, but non-IgE-mediated gastrointestinal food allergies are induced by food stress tests Becomes a definitive diagnosis.

On the other hand, by performing a disease determination by the examination method or diagnosis method of the present invention, without performing an examination such as endoscopy or CT scan, biopsy, etc. as essential, or endoscopy etc. Prior to pathological examination or the like, it is possible to easily and easily diagnose differentiation between these diseases showing symptoms similar to eosinophilic gastrointestinal diseases and eosinophilic gastrointestinal diseases. The physical burden on the subject is significantly reduced. Furthermore, since the diagnosis can be performed quickly and easily, the progression or severity of symptoms due to a delay in diagnosis can be prevented.

In addition, as described in the above item of the test method, eosinophilic gastroenteritis and eosinophilic esophagitis can be achieved by appropriately selecting cytokines and / or chemokines that measure the amount of gene expression or protein in the measurement step. Can be easily determined.

Further, the inspection method according to the present invention includes the above-described conventional diagnosis method (that is, a diagnosis method including endoscopy, etc.), determination of severity by scoring symptom scores, etc., diagnosis based on conventional diagnosis criteria, weight At least one or more of conventional diagnostic methods including a diagnosis based on an indicator such as the presence or absence of decrease, diarrhea, hypoproteinemia and hypereosinophilia can be appropriately combined.

Further, the above-described inspection process is the same as the diagnostic process in the diagnostic method described below. Each process in the inspection method is also applied to a diagnostic process in the diagnostic method described below. The diagnostic method is as follows [2. Diagnostic method of eosinophilic gastrointestinal disease or food protein-induced enteropathy].

[2. (Diagnostic method for eosinophilic gastrointestinal tract disease or food protein-induced enteropathy)
The present invention relates to a method for diagnosing eosinophilic gastrointestinal disease or food protein-induced enteropathy, comprising a TSLP gene, an IL-33 gene, a CCL7 / MCP-3 gene and a CCL21 / in a sample collected from a human body. Measuring the amount of expression of at least one of 6CKine genes, or measuring the amount of at least one of TSLP protein, IL-33 protein, CCL7 / MCP-3 protein and CCL21 / 6CKine protein in the sample; A diagnostic method is also provided.

The diagnostic method in one embodiment includes the step of measuring the antibody level of an anti-protein antibody against at least one of TSLP protein, IL-33 protein, CCL7 protein and CCL21 protein in a biological sample collected from a subject. A method for diagnosing eosinophilic gastrointestinal disease or food protein-induced enteropathy is provided.

According to one embodiment, a method for diagnosing eosinophilic gastrointestinal tract disease or food protein-induced enteropathy in a subject is preferably based on a measurement result of a protein amount or a gene expression level in a test step in a biological sample collected from the subject. A diagnostic step of diagnosing the presence or absence of an acidophilic gastrointestinal disease or food protein-induced enteropathy and / or the predisposition to the development of eosinophilic gastrointestinal disease or food protein-induced enteropathy. The measurement step in the diagnostic method according to the present invention is the above described [1. As described in the item <Measurement process> in [Inspection method].

<Diagnosis process>
The diagnostic process in the diagnostic method according to the present invention includes the above described [1. As described in the section of <Examination process> in [Examination method], the diagnosis includes diagnosis by a doctor or other qualified medical professional.

In the diagnostic process, whether the subject is suffering from eosinophilic gastrointestinal disease or food protein-induced enteropathy, and whether the subject is predisposed to developing eosinophilic gastrointestinal disease or food protein-induced enteropathy Including determining. In another embodiment, the diagnostic method of the present invention may comprise the step of determining whether the allergy is a non-IgE mediated reaction when the subject has a food allergy. Good.

Whether the subject suffers from eosinophilic gastrointestinal disease or food protein-induced enteropathy by measuring the amount of protein or gene expression in a biological sample collected from the subject during the diagnostic process Whether there is food protein-induced enteropathy that does not meet the diagnostic criteria of conventional eosinophilic gastrointestinal diseases, or whether it has a predisposition to non-IgE-mediated gastrointestinal food allergy Can be diagnosed. This means that the subject can be assessed for the risk of developing eosinophilic gastrointestinal disease or food protein-induced enteropathy.

[3. Inspection kit)
The present invention also relates to a test kit for examining eosinophilic gastrointestinal disease or food protein-induced enteropathy, an expression product of a TSLP gene, an expression product of an IL-33 gene in a sample collected from a human body, At least one expression product of the expression product of CCL7 / MCP-3 gene and the expression product of CCL21 / 6CKine gene, or TSLP protein, IL-33 protein, CCL7 / MCP-3 protein and CCL21 / 6CKine protein in the sample A test kit comprising a nucleic acid probe, a nucleic acid primer, a nucleic acid aptamer, an antibody or a peptide probe for detecting at least one is provided.

The expression product refers to mRNA transcribed from the TSLP gene, IL-33 gene, CCL7 / MCP-3 gene or CCL21 / 6CKine gene. The test kit of the present invention also includes a detection kit that detects the mRNA as a form of cDNA obtained by reverse transcription.

The nucleic acid probe refers to a nucleic acid probe that specifically binds to any of the expression products, and more specifically includes a TaqMan probe, an Invador probe, and the like. The nucleic acid primer refers to a nucleic acid primer that can specifically amplify the mRNA as the expression product or cDNA obtained by reverse transcription of the mRNA, and more specifically, a primer used in a nucleic acid amplification method such as RT-PCR method. Is mentioned. The nucleic acid aptamer refers to a nucleic acid construct composed of a nucleic acid that specifically binds to any of TSLP protein, IL-33 protein, CCL7 / MCP-3 protein, or CCL21 / 6CKine protein contained in a biological sample. .

The peptide probe refers to a peptidic probe that specifically binds to any of TSLP protein, IL-33 protein, CCL7 / MCP-3 protein, or CCL21 / 6CKine protein. Specific examples include peptide sequences that specifically bind to TSLP, IL-33, CCL7 / MCP-3 or CCL21 / 6CKine.

The nucleic acid probe, nucleic acid primer, and nucleic acid aptamer included in the kit may include a non-natural nucleic acid (PNA or the like) in addition to the natural nucleic acid. Similarly, a peptide probe may be configured to include non-natural amino acids in addition to natural amino acids.

The test kit according to the present invention further includes various reagents and instruments (polymerase, PCR buffer, each dNTP, pipette, etc.) used for nucleic acid amplification methods such as PCR, and various reagents and instruments for preparing a sample, if necessary. (Test tubes, buffers, etc.), various reagents and instruments for analyzing nucleic acid amplification fragments (electrophoresis gel materials, pipettes, etc.), instruction manuals for test kits, samples used as controls for measurement, and measurement results It is also possible to provide at least one of control data and the like used when analyzing. The instruction manual for the test kit includes the above-mentioned [1. The contents of the inspection method according to the present invention described in the column of “Inspection method” are recorded.

As described above, according to the test method, diagnosis method, and test kit of the present invention, diagnosis of eosinophilic gastrointestinal tract disease or food protein-induced enteropathy, which has so far been based on the subjectivity of doctors, is biological. Since it becomes possible to make a diagnosis based on a test in which a standard is introduced, an improvement in diagnostic technology is expected. Moreover, since it becomes possible to make an early diagnosis, it is possible to prevent a case in which the diagnosis is delayed even though the subject is afflicted with this disease, causing a disorder.

[4. (Use of test results obtained by test methods or diagnostic methods obtained by diagnostic methods)
[1. The test result obtained by performing the test method described in the column of “Test method” can be used as one of the diagnostic materials for diagnosis by a doctor. The above [1. Subjects who have been tested for having a predisposition to developing eosinophilic gastrointestinal tract disease or food protein-induced enteropathy, or eosinophilic gastrointestinal tract About the test subject who obtained the test result of having a disease or food protein-induced enteropathy, it can be treated with the result of diagnosis by a doctor as necessary. Above [2. The test results obtained by conducting the diagnostic method described in the section of `` Diagnosis method '', or the test result that is predisposed to the development of eosinophilic gastrointestinal disease or food protein-induced enteropathy, or the eosinophilic gastrointestinal tract Have a disease or food protein-induced enteropathy, or the gene expression level or protein level of cytokines and / or chemokines associated with the eosinophilic gastrointestinal disease or food protein-induced enteropathy is not in a healthy state, And / or subject's judgment, if necessary, for a subject who had a diagnosis that the subject was determined to be at high risk of developing eosinophilic gastrointestinal disease or food protein-induced enteropathy Above, treatment can be performed.

In addition, according to the test method or diagnosis method of the present invention, determination of a treatment method, determination of a disease and observation of a disease, determination of the presence or absence of a therapeutic effect, prediction of a prognosis, and differentiation from other diseases can be performed. . Here, examples of treatment methods are as follows [5. Details of the method of treatment of eosinophilic gastrointestinal tract disease or food protein-induced enteropathy are described below.

[5. (Method of treating eosinophilic gastrointestinal tract disease or food protein-induced enteropathy)
The present invention further determines that, as a result of the test by the test method described above or as a result of the diagnosis by the diagnostic method described above, has or is predisposed to eosinophilic gastrointestinal disease or food protein-induced enteropathy. Provided is a method for treating eosinophilic gastrointestinal tract disease or food protein-induced enteropathy, characterized in that the subject is given at least one therapy selected from diet therapy, drug therapy and balloon dilatation and surgical therapy. In one example, a subject diagnosed as in need of treatment is given to a subject when the amount of protein or gene expression obtained in the above measurement step is higher than a specified value (for example, a cutoff value calculated from a control). The present invention provides a method for treating eosinophilic gastrointestinal tract disease or food protein-induced enteropathy, characterized by performing at least one or more of the above-mentioned therapies.

(Target of treatment method)
The subject of the treatment method of the present invention has an eosinophilic digestive tract disease or food protein-induced enteropathy, or an eosinophilic digestive tract disease or food protein-induced enteropathy, according to the test method or diagnostic method of the present invention. A subject determined to have a predisposition.

The subject of the treatment method of the present invention is related to the eosinophilic digestive tract disease or food protein-induced enteropathy by the diagnosis method of eosinophilic gastrointestinal disease or food protein-induced enteropathy according to the present invention. A subject whose cytokine and / or chemokine gene expression level or protein expression level is not healthy and / or has a high risk of developing eosinophilic gastrointestinal disease or food protein-induced enteropathy Also good. In one embodiment, the subject does not meet the conventional diagnostic criteria for eosinophilic gastrointestinal disease or food protein-induced enteropathy, and the subject has eosinophilic gastrointestinal disease or food protein-induced enteropathy. It may be a subject diagnosed as having a predisposition to develop eosinophilic gastrointestinal disease or food protein-induced enteropathy by a test method or a diagnostic method. Such subjects are at high risk of developing eosinophilic gastrointestinal disease or food protein-induced enteropathy in the future. Therefore, for example, by performing the treatment method of the present invention including diet therapy on such a subject, it becomes possible to prevent the onset of eosinophilic gastrointestinal disease or food protein-induced enteropathy in advance. .

(Applicable treatment methods)
Dietary therapy applicable in the treatment method of the present invention includes component nutrition therapy in which only the amino acid component nutritional food is fed, and predetermined foods that are likely to become allergens of a plurality of types (for example, four or six types). These include diets such as personalized removal diets, which include removal diets, and selection and removal of ingredients that are likely to be allergens for the subject by performing skin prick tests and skin patch tests. Examples of the six allergens to be removed are wheat, egg, milk, soy, nuts and marine products.

The drug therapy applicable in the treatment method of the present invention includes drug therapy in which drugs such as PPI, local action steroids, systemic steroids such as systemic glucocorticoids or antiallergic drugs are administered. Immunosuppressive drugs are considered when steroid dependence or side effects are strong.

In particular, PPI is preferably selected for pharmacotherapy for eosinophilic esophagitis. An example of PPI resistance may be topical steroid therapy by intraoral spraying and swallowing of locally acting steroids such as fluticasone. Treatment methods other than steroid therapy are preferable from the viewpoint of the recurrence of recurrence after discontinuation of steroid administration and the risk of side effects.

In one embodiment, the drug therapy may be a therapy in which the above-described cytokine and / or chemokine antagonist used as a molecular marker in the test method or diagnostic method of the present invention is administered.

Further, in one embodiment, a subject under treatment using the above-described treatment method may be diagnosed by an examination using the examination method of the present invention or a diagnosis by a diagnostic method. The expression level of TSLP gene, IL-33 gene, CCL7 / MCP-3 gene and CCL21 / 6CKine gene, or the amount of TSLP protein, IL-33 protein, CCL7 / MCP-3 protein and CCL21 / 6CKine protein in the sample, Since it is proportional to the severity of the disease, it is possible to determine the presence or absence of therapeutic effects and the disappearance of inflammation at each stage of treatment.

[6. Illustrative examples of specific embodiments according to the present invention]
The present invention includes any of the following aspects.

1) A method for examining eosinophilic gastrointestinal disorders (EGID) or food protein-induced enteropathy (Food-Protein-Induced Enteropathy-Syndrome (Enteropathy)) in a sample collected from a human body The expression level of at least one of stromal lymphopoietin (TSLP) gene, IL-33 gene, CCL7 gene and CCL21 gene is measured, or at least one of TSLP protein, IL-33 protein, CCL7 protein and CCL21 protein in the sample An inspection method including a measuring step of measuring one quantity.

2) The test method according to 1), wherein in the measurement step, at least the expression levels of TSLP gene and IL-33 gene are measured, or the amounts of at least TSLP protein and IL-33 protein in the sample are measured.

3) In the measurement step, the expression level of TSLP gene, IL-33 gene, CCL7 gene and CCL21 gene is measured, or the amount of TSLP protein, IL-33 protein, CCL7 protein and CCL21 protein in the sample is measured. The inspection method according to 1) or 2).

4) The inspection method according to any one of 1) to 3), wherein the sample is a blood-derived sample.

5) The test method according to any one of 1) to 4), wherein the eosinophilic gastrointestinal disease is eosinophilic gastroenteritis (EGE).

6) The inspection method according to any one of 1) to 5) above, wherein the human is under 2 years old.

7) In the measurement step, in the sample, the expression level of TSLP gene, the expression level of IL-33 gene, the expression level of CCL7 gene and the expression level of CCL21 gene, the amount of TSLP protein, the amount of IL-33 protein, Determining that there is a predisposition or onset of eosinophilic gastrointestinal disease or food protein-induced enteropathy when at least one selected from the amount of CCL7 protein and the amount of CCL21 protein is high compared to a control The inspection method according to any one of 1) to 6).

8) In the measurement step, the expression level of TSLP gene, the expression level of IL-33 gene, the expression level of CCL7 gene and the expression level of CCL21 gene in the sample, or the amount of TSLP protein in the sample, IL A predisposition or development of eosinophilic gastrointestinal disease or food protein-induced enteropathy when the amount of -33 protein, the amount of CCL7 protein and the amount of CCL21 protein are all high compared to the control; The inspection method according to any one of 1) to 7).

9) A test kit for testing eosinophilic gastrointestinal disease or food protein-induced enteropathy,
At least one of TSLP gene expression product, IL-33 gene expression product, CCL7 gene expression product and CCL21 gene expression product in a sample collected from a human organism, or TSLP protein, IL-33 protein in the sample, A test kit comprising a nucleic acid probe, a nucleic acid primer, a nucleic acid aptamer, an antibody or a peptide probe for detecting at least one of CCL7 protein and CCL21 protein.

10) TSLP gene expression product, IL-33 gene expression product, CCL7 gene expression product and CCL21 gene expression product in the above sample, or TSLP protein, IL-33 protein, CCL7 protein and CCL21 in the sample The test kit according to 9), comprising a nucleic acid probe, a nucleic acid primer, a nucleic acid aptamer, an antibody or a peptide probe for detecting all of the protein.

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention. Furthermore, a new technical feature can be formed by combining the technical means disclosed in each embodiment.

Hereinafter, embodiments of the present invention will be described in detail.

[Example 1]
(1. Method etc.)
= Patient selection etc. =
This case-control study was conducted at the National Center for Child Health and Development (NCCHD). Patients with eosinophilic gastroenteritis (EGE) in infants, patients with ulcerative colitis (UC), patients with immediate-type food allergy (FA) In order to identify serum and mucosal biopsy samples from patients with atopic dermatitis (AD) and healthy controls (CTRL), the inventors have developed a clinical database ( NCHHD Allergy-2 “Allergy-2” database). Duplicate samples from the same patient and samples from adult patients (20 years and older) were excluded.

This database is diagnosed as meeting three Powell criteria (Powell's criteria: reference E1) and having non-IgE-mediated gastrointestinal food allergy (reference E2) In addition, 104 infants were included. Twenty-four of the 104 patients experienced neither repeated vomiting nor bloody stool. Of these 24 patients, 13 patients have experienced severe weight loss and / or intractable diarrhea, and gastrointestinal fiberscope examination to establish a clinical diagnosis And histological examination was performed. All 13 patients showed eosinophilia in the gastrointestinal (GI) mucosa and were to be included in this study (Table E1). For patients numbered 1-9, serum samples were successfully stocked at the time of hospitalization prior to the start of treatment for cytokine and chemokine assays. For patients numbered 2-5, 7 and 10-13, mucosal biopsy samples were taken to measure RNA and samples from all 9 were subjected to qPCR. Samples from patients numbered 2-4, 7 and 10 were of appropriate RNA quality and RNA content for microarray analysis.

We have 12 CTRLs (serum n = 7, GI mucosal biopsy sample n = 5), 37 immediate FA patients with no symptoms (serum: n = 37), 31 Of AD active-phase AD (serum: n = 31) and 13 active UC patients (serum n = 8, GI mucosa biopsy sample n = 8) Went. All collected serum samples were used for analysis of cytokine and chemokine levels. All five CTRL mucosal samples were subjected to microarray analysis. For UC patients, all 8 mucosal samples were used for qPCR, of which 5 were of appropriate RNA quality and RNA quantity for microarray analysis.

= Inclusion criteria =
The diagnosis of EGE was made based on clinical symptoms and the accumulation of eosinophils in the GI mucosa, provided that it was not accompanied by any other pathology. There is no published EGE diagnostic guideline, and the diagnostic guideline is very controversial (reference documents E3 to E5). Therefore, eosinophilia in the GI mucosa was reported by DeBrosse et al. Defined according to document E6). That is, when an increased eosinophil count was found in at least one GI organ (listed below), a histological diagnosis of EGE was made.

> 15 eosinophils / high-power field (HPF) in the esophagus,
> 15 eosinophils / HPF in the stomach,
> 25 eosinophils / HPF in the duodenum,
> 25 eosinophils / HPF in the ileum,
> 40 eosinophils / HPF in the ascending colon, or
> 25 eosinophils / HPF, transverse colon-rectum.
Here,> indicates that it exceeds the number shown on the right side.

CTRL subjects were determined to have no illness by a pediatrician based on blood tests or GI endoscopy if illness was suspected. It was also confirmed that the subject who is a CTRL has no history of FA, AD, UC or EGE. Five people were examined by GI endoscopy and all gastrointestinal parts examined showed normal pathological results. All subjects who were CTRLs had not received any oral glucocorticoid treatment.

FA and AD were diagnosed by two pediatric allergists belonging to NCCHD based on “Japanese「 Guideline for Food Allergy 2014 ”(reference E7) and“ Japanese Guideline for Atopic Dermatitis 2014 (reference E8) ”, respectively. . These subjects, FA and AD, confirmed that they had no history of UC or EGE and had not received any oral glucocorticoid treatment.

UC was diagnosed by two pediatric gastroenterologists and pathologists belonging to NCCHD based on a combination of clinical characteristics, endoscopic characteristics, and histological characteristics.

= Patient clinical data =
Clinical data for EGE patients is shown in Table E1. In all patients, there was an increase in eosinophils in the gastrointestinal tract (Table E2, AD in FIG. 3).
Serum cytokines / chemokines were examined for patients numbered 1-9. All these nine patients showed a weight loss of less than -2SD. Three patients observed only weight loss and were not accompanied by other GI symptoms. Two patients had hypoproteinemia. All patients gained weight after removal of offending food (E and F in FIG. 3). Eight patients were delayed in developmental milestones, but later caught up with development. The causative food was identified by performing a chronic tolerance test (dose of harmful food every day for 3 weeks) after resolution of symptoms. Harmful food is milk in 8 (89%) patients, breast milk in 1 (11%) patients, soy in 2 (22%) patients, 1 (11%) Patients were chicken eggs, 1 (11%) patients were extensively hydrolyzed formula (New MA-1®), and 1 (11%) patients contained soybean oil Amino acid preparation (Elental-P®. Five of nine patients responded to more than one type of food. Three patients were class 2 or higher titer food-specific IgE. Showed antibodies but did not produce immediate hypersensitivity after ingestion of harmful foods.Several days after initiating ingestion of harmful foods, diarrhea was first manifested as a sign of nonimmediate reaction Only patient No. 5 had an immediate form after ingestion of peanuts, 2 years after EGE disappeared. Resulting in response. This During the study, both patients nine, proton pump inhibitor also steroids has not been administered.

Table E3 compares the basic characteristics of EGE, CTRL, FA, AD, and UC. The severity of AD patients who received cytokine / chemokine analysis in sputum serum ranged from mild to severe. The median value (IQR) of the SCORAD index was 37 (29-57). The severity of UC patients who have been analyzed for serum cytokines / chemokines is between mild and moderate according to “PediatricedUlcerative Colitis Activity Index (reference E9)”. It was. The PUCAI median (IQR) was 32.5 (11.3-38.8). For microarray analysis and qPCR analysis, Matts grade (ref. E10) was used to classify the severity of UC patients (≧ 3 grades for 3 patients; ≧ 2 grades for 5 patients).

= Multiplex analysis =
Serum was separated and a portion was stored frozen at -80 ° C. Serum cytokine and chemokine levels were assayed using Milliplex Human Cytokine / Chemokine Kits (Millipore, St. Charles, Mo.) according to manufacturer's instructions. In total, 36 cytokines and chemokines were assayed. Cytokines and chemokines assayed are TNFα, G-CSF, GM-CSF, IFNγ, IL1β, IL3, IL5, IL6, IL8, IL9, IL12p70, IL13, IL17, IL33, TSLP, CCL1 / I309, CCL2 / MCP1, CCL3 / MIP1α, CCL4 / MIP1β, CCL7 / MCP3, CCL8 / MCP2, CCL11 / eotaxin, CCL13 / MCP4, CCL15 / MIP1δ, CCL17 / TARC, CCL21 / 6CKine, CCL22 / MDC, CCL24 / eotaxin2, CCL26 / CCL27 / ACK27 CXCL1-3 / GRO, CXCL5 / ENA78, CXCL10 / IP10, CXCL12 / SDF1, CXCL13 / BCA1, and , It is a CX3CL1 / Fractalkine.

= Microarray analysis =
After biopsy, at room temperature, samples were placed in RNAlater® solution (QIAGEN, Valencia, CA, USA) and stored at −80 ° C. until gene expression profiling. Microarray analysis (Agilent Technologies, Santa Clara, CA, USA) was performed according to the manufacturer's instructions as previously described (reference E11). Briefly, total RNA was extracted using RNeasy Micro kit (Qiagen), and evaluation was performed using Agilent Bioanalyzer and RNA 6000 Nano kit (Agilent Technologies). The gene expression profile was evaluated using microarray technology using Agilent SurePrint G3 Human GE 8 × 60k. Data analysis was performed using GeneSpring software ver. 12.5 (Agilent Technologies).

In order to normalize the variation in staining intensity between microarrays, the average difference (AD) of all genes on the microarray was divided by the median value of all measured values on the microarray. In EGE patients or UC patients, genes that have a significant difference in signal intensity compared to those in CTRL (P <.05, t test with Benjamini and Hochberg false discovery rate correction), up-regulation (up -regulated) or down-regulated. Hierarchical clustering was performed using gene expression data against EGE, UC, and CTRL.

= QPCR analysis =
For qPCR, total RNA extraction and cDNA synthesis were performed according to previous reports (references E12 and 13). Primer set for human IL33 (sense, 5'-TCA GGT GAC GGT GTT GAT GG-3 '; antisense, 5'-CCT GGT CTG GCA GTG GTT TT-3'), primer set for TSLP (sense, 5 ' -GCCCAGGCTATTCGGAAACT-3 '; antisense, 5'-CGACGCCACAATCCTTGTAATT-3') and primer set for β-actin (sense, 5'-CCCAGCCATGTACGTTGCTAT-3 '; antisense, 5'-TCACCGGAGTCCATCACGAT-3') (Kanagawa, Japan). In each qPCR experiment, an aliquot of cDNA equivalent to 2 ng of total RNA sample was used. The expression level of mRNA was normalized with the level of β-actin in each sample.

= Statistical analysis =
Differences between study groups were determined using the Kruskal-Wallis test with Dunn's multiple comparison test or Mann Whitney U test. Correlation was calculated using Spearman's r value. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, Calif.). P values less than 0.05 were considered significant.

= Ethics =
This study was performed according to a protocol approved by the NCCHD organization evaluation committee (Tokyo, Japan). Informed consent was obtained from each participant or the legal guardian of the participant.

(2. Results and discussion)
The inventors have attempted to use microarrays to find genes that are up-regulated in lesions of the gastrointestinal tract in infant EGE patients. Here we find that serum levels of TSLP (thymic stromal lymphopoietin) and serum IL-33 are particularly increased in infant EGE patients, and TSLP and IL-33. Also proved to be upregulated in the mucosa of the sigmoid colon. The inventors collected 13 patients with active EGE with severe weight loss and / or refractory diarrhea. Blood was collected on admission before the start of treatment. The histological diagnosis of EGE was based on the presence of accumulated eosinophilia in the gastrointestinal mucosa (ie, exceeding the upper limit of the normal range as reported by DeBrosse et al. (Reference E6)). Endoscopic examination of the upper and lower gastrointestinal tract of EGE patients was performed 2 to 4 weeks after the start of treatment, including food elimination. Patients numbered 1-13 showed eosinophilia in the gastrointestinal tract and their weight loss resolved after removal of harmful food (see FIG. 3 and Table E1).

Tables E3 and E4 show a comparison of clinical data and a comparison of serum cytokine / chemokine levels in healthy control subjects and patients with food allergy, AD, UC and EGE. Among the 36 serum cytokines / chemokines analyzed, TSLP, IL-33, CCL7 / monocyte ７ chemoattractant protein 3, and CCL21 / 6 CKine levels were particularly increased in EGE patients (A- in Fig. 1). D and Table E4).

The diagnostic utility of these cytokines / chemokines was examined by setting an appropriate cut-off point, and for EGE, TSLP and IL-33 showed very good sensitivity (77.8%) and specificity ( TSLP, 97.6%; IL-33, 95.2%) (see Table E5). CCL27 / cutaneous T-cell-attracting chemokine is a typical chemokine with increased levels in AD patients (E in FIG. 1). TSLP showed a very strong correlation with IL-33 (r = 0.91, P <.0001), but no correlation with cutaneous T cell-attracting chemokine (F and G in Fig. 1). There was no cross-reactivity between TSLP and IL-33 in this assay kit (data not shown). TSLP and IL-33 levels in serum decreased with improvement in symptoms and signs (H and I in FIG. 1).

Mucosal biopsy samples obtained from the inflamed sigmoid colon of individual patients, together with measurement of serum cytokine / chemokine levels, were subjected to whole genome transcription analysis (EGE patients, UC Control subjects of patients and healthy individuals: n is 5). Compared with the healthy control group, 841 genes specifically expressed in the EGE group (≧ 2-fold change, adjusted P <.05) and activity compared with the healthy control group 762 genes (≧ 2-fold change, adjusted P <.05) that are specifically expressed in the UC group at the stage were analyzed using cluster analysis (A in FIG. 2). The expression of TSLP mRNA was significantly increased in EGE patients (3.3-fold change, adjusted P = 0.02). IL33 expression tended to increase in patients with EGE (1.8-fold change). Table E6 summarizes gene expression of immune system molecules and EoE-related molecules.

In patients with UC, the expression of many inflammation-related genes (eg, IL1B, IFNG, IL6, IL8, CCL11, CCL13, CXCL1, CXCL6, and CXCL11) is upregulated in patients with EGE Expression of a smaller number of similar genes (TSLP, CCL5, CCL20, CCL21, CCL22, and neurofilament medium polypeptide [NEFM]) was up-regulated. qPCR further confirmed that both TSLP mRNA and IL33 mRNA showed significantly increased expression in the sigmoid mucosa of EGE patients compared to UC patients (FIG. 2). B).

The results of microarray analysis and qPCR also showed TSLP and IL33 expression in the mucosa of the sigmoid colon of EGE patients, consistent with the findings obtained with serum. However, the level of gene expression of some cytokines in the microarray did not match the serum data. The cause of this discrepancy may be, for example, that the 2-4 week food removal period provided prior to GI endoscopy has reduced the risk of perforation of the gastrointestinal tract. The inventors determined this period in view of the fact that patients with severe EGE often have malnutrition. However, TSLP and IL33 are up-regulated in the mucosa of EGE patients, demonstrating consistency of results. Serum TSLP and IL-33 expression in patients with EGE may reflect gastrointestinal inflammation and may be at least partially derived from these lesion sites.

TSLP and IL-33 are now recognized as key cytokines in allergic disorders. In particular, a polymorphism located in the vicinity of TSLP has been reported to be related to EoE (reference document E15).

Previous studies have shown increased levels of serum TSLP, IL-33, or both in both UC and AD patients (references E16, 17) In this example, these serum levels were not high in UC patients or AD patients, although the severity of illness and the level of inflammation were considerably higher.

A group of infant EGE patients improved significantly after the beginning of food removal, gained weight, and tested positive in a long-term tolerance test (harmful food taken daily for 2-3 consecutive weeks) Therefore, a diagnosis of non-IgE-mediated gastrointestinal food allergy (reference document E18, 19) was established. Some of these patients had positive IgE levels for multiple foods, but upon ingestion of harmful foods, these patients have any immediate symptoms (eg, urticaria or see-through) Etc.) did not appear. It usually takes several days to elicit a response in a food challenge test. Most patients also showed a negative reaction to food allergy in the skin prick test. Thus, the majority of gastrointestinal inflammation in these patients is believed to be caused by a non-IgE-mediated response to harmful food (not by an IgE-mediated response). Furthermore, unlike patients with food protein-induced gastroenteritis or proctocolitis-syndrome, these patients do not have recurrent vomiting or bloody stool conditions. To date, no significant eosinophilic inflammation has been reported (reference document E19), but food protein-induced enteropathy (Enteropathy) would be an appropriate diagnosis for patients with EGE targeted this time. .

= List of references E =
Reference E1 Powell GK. Food protein-induced enterocolitis of infancy: differential diagnosis and management. Compr Ther 1986; 12: 28-37.
Reference E2 Nomura I, Morita H, Hosokawa S, Hoshina H, Fukuie T, Watanabe M, et al. Four distinct subtypes of non-IgE-mediated gastrointestinal food allergiesin neonates and infants, distinguished by their initial symptoms. J Allergy Clin Immunol 2011; 127: 685-8.
Reference E3 Talley NJ, Shorter RG, Phillips SF, Zinsmeister AR. Eosinophilic gastroenteritis: a clinicopathological study of patients with disease of the mucosa, muscle layer, and subserosal tissues. Gut 1990; 31: 54-8.
Reference E4 Khan S, Orenstein SR. Eosinophilic gastroenteritis. Gastroenterol Clin North Am 2008; 37: 333-48, v.
Reference E5 Collins MH. Histopathologic features of eosinophilic esophagitis and eosinophilic gastrointestinal diseases. Gastroenterol Clin North Am 2014; 43: 257-68.
Reference E6 DeBrosse CW, Case JW, Putnam PE, Collins MH, Rothenberg ME. Quantity and distribution of eosinophils in the gastrointestinal tract of children. Pediatr Dev Pathol 2006; 9: 210-8.
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Reference E9 Turner D, Otley AR, Mack D, Hyams J, de Bruijne J, Uusoue K, et al. Development, validation, and evaluation of a pediatric ulcerative colitis activity index: a prospective multicenter study. Gastroenterology 2007; 133: 423 -32.Reference E10 Matts SG. The value of rectal biopsy in the diagnosis of ulcerative colitis.QJ Med 1961; 30: 393-407.
Reference E11 Shoda T, Morita H, Nomura I, Ishimura N, Ishihara S, Matsuda A, et al. Comparison of gene expression profiles in eosinophilic esophagitis (EoE) between Japan and Western countries. Allergol Int 2015; 64: 260-5 .
Reference E12 Shoda T, Futamura K, Kobayashi F, Saito H, Matsumoto K, Matsuda A. Expression of thymus and activation-regulated chemokine (TARC) by human dermal cells, but not epidermal keratinocytes. J Dermatol Sci 2014; 76:90 -Five.
Reference E13 Shoda T, Futamura K, Kobayashi F, Saito H, Matsumoto K, Matsuda A. Cell type-dependent effects of corticosteroid on periostin production by primary human tissue cells. Allergy 2013; 68: 1467-70.
Reference E14 Wen T, Stucke EM, Grotjan TM, Kemme KA, Abonia JP, Putnam PE, etal. Molecular diagnosis of eosinophilic esophagitis by gene expression profiling. Gastroenterology 2013; 145: 1289-99.
Reference E15 Sherrill JD, Rothenberg ME. Genetic dissection of eosinophilic esophagitis provides insight into disease pathogenesis and treatment strategies. J Allergy Clin Immunol 2011; 128: 23-32.
Reference E16 Pastorelli L, Garg RR, Hoang SB, Spina L, Mattioli B, Scarpa M, et al. Epithelial derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1 / Th2 driven enteritis. Proc Natl Acad Sci USA 2010; 107: 8017-22.
Reference E17 Tamagawa-Mineoka R, Okuzawa Y, Masuda K, Katoh N. Increased serum levels of interleukin 33 in patients with atopic dermatitis. J Am Acad Dermatol 2014; 70: 882-8.
Reference E18 Nomura I, Morita H, Hosokawa S, Hoshina H, Fukuie T, Watanabe M, et al. Four distinct subtypes of non-IgE-mediated gastrointestinal food allergies in neonates and infants, distinguished by their initial symptoms.J Allergy Clin Immunol 2011; 127: 685-8.
Reference E19 Nowak-Wegrzyn A, Katz Y, Mehr SS, Koletzko S. Non-IgE-mediated gastrointestinal food allergy. J Allergy Clin Immunol 2015; 135: 1114-24.

In Table 1, CM: milk; HM: hydrolyzed milk (MA-1); AAM: amino acid milk (containing soybean oil); An: hypersensitivity, ND: not detected; CRP: C-reactive protein.

In Table 2, Tissue eosinophil count (eosinophils / HPF): tissue eosinophil count (eosinophil / HRF) Diagnosis: diagnosis; Esophagus: esophagus; Stomach: stomach; Duodenum: duodenum; Ileum: ileum; Colon: colon, Sigmoid : Sigmoid colon; Rectum: Rectum; NA: Not applicable; HPF: 400X indicates high field magnification. * A typical area of GI biopsy was determined and eosinophil counts at 400X HPF were factored. Bold numbers indicate “increase” according to conventional examination.

In Table 3, Atopic history: Atopic history, Asthma: Asthma, Immediate-type food allergy: Intermediate food allergy, Blood eosinophil ratio: Blood eosinophil ratio, C-reactive protein: C-reactive protein, Total protein : Total protein, Albumin: Albumin, Total IgE, Total IgE, Egg-specific IgE: Egg-specific IgE, Milk-specific IgE: Milk-specific IgE, Wheat-specific IgE: Wheat-specific IgE.

Table 5 shows the area under the ROC curve (AUC), sensitivity and specificity values for the cutoff optimized for the diagnosis of EGE.

The test method and test kit for eosinophilic gastrointestinal disease or food protein-induced enteropathy of the present invention can be used in various fields including clinical use.

Claims (10)

1. A method for testing eosinophilic gastrointestinal disorders (EGID) or food protein-induced enteropathy syndrome (Enteropathy),
   An expression level of at least one of a thymic stromal lymphopoietin (TSLP) gene, an IL-33 gene, a CCL7 gene and a CCL21 gene in a sample collected from a human living body, or a TSLP protein, an IL-33 protein in the sample, A test method comprising a measurement step of measuring the amount of at least one of CCL7 protein and CCL21 protein.
2. 2. The test method according to claim 1, wherein in the measurement step, at least the expression levels of TSLP gene and IL-33 gene are measured, or at least the amounts of TSLP protein and IL-33 protein in the sample are measured.
3. In the measurement step, the expression level of TSLP gene, IL-33 gene, CCL7 gene and CCL21 gene is measured, or the amount of TSLP protein, IL-33 protein, CCL7 protein and CCL21 protein in the sample is measured. 3. The inspection method according to 1 or 2.
4. The inspection method according to any one of claims 1 to 3, wherein the sample is a blood-derived sample.
5. The test method according to any one of claims 1 to 4, wherein the eosinophilic gastrointestinal tract disease is eosinophilic gastroenteritis (EGE).
6. The inspection method according to any one of claims 1 to 5, wherein the human is under 2 years old.
7. In the measurement step, TSLP gene expression level, IL-33 gene expression level, CCL7 gene expression level and CCL21 gene expression level, TSLP protein level, IL-33 protein level, CCL7 protein level in the sample Determining at least one selected from the amount of CCL21 and the amount of CCL21 protein is predisposed to or developing eosinophilic gastrointestinal disease or food protein-induced enteropathy when compared to a control; The inspection method according to any one of claims 1 to 6.
8. In the measurement step, the expression level of TSLP gene, the expression level of IL-33 gene, the expression level of CCL7 gene and the expression level of CCL21 gene in the sample, or the amount of TSLP protein in the sample, IL-33 Determining that the amount of protein, the amount of CCL7 protein, and the amount of CCL21 protein are all predisposed to or developing eosinophilic gastrointestinal disease or food protein-induced enteropathy when compared to the control The inspection method according to any one of claims 1 to 7.
9. A test kit for testing for eosinophilic gastrointestinal disease or food protein-induced enteropathy,
   At least one of TSLP gene expression product, IL-33 gene expression product, CCL7 gene expression product and CCL21 gene expression product in a sample collected from a human organism, or TSLP protein, IL-33 protein in the sample, A test kit comprising a nucleic acid probe, a nucleic acid primer, a nucleic acid aptamer, an antibody or a peptide probe for detecting at least one of CCL7 protein and CCL21 protein.
10. TSLP gene expression product, IL-33 gene expression product, CCL7 gene expression product and CCL21 gene expression product in the sample, or TSLP protein, IL-33 protein, CCL7 protein and CCL21 protein in the sample The test kit according to claim 9, comprising a nucleic acid probe, a nucleic acid primer, a nucleic acid aptamer, an antibody or a peptide probe for detecting all.

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