WO2018043715A1 - Procédé d'examen et kit d'examen pour une maladie gastro-intestinale éosinophile ou une entéropathie induite par des protéines alimentaires - Google Patents

Procédé d'examen et kit d'examen pour une maladie gastro-intestinale éosinophile ou une entéropathie induite par des protéines alimentaires Download PDF

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WO2018043715A1
WO2018043715A1 PCT/JP2017/031636 JP2017031636W WO2018043715A1 WO 2018043715 A1 WO2018043715 A1 WO 2018043715A1 JP 2017031636 W JP2017031636 W JP 2017031636W WO 2018043715 A1 WO2018043715 A1 WO 2018043715A1
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protein
gene
tslp
ccl21
ccl7
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Japanese (ja)
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伊知郎 野村
松本　健治
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国立研究開発法人国立成育医療研究センター
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • 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).
  • Non-Patent Document 2 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).
  • 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).
  • non-IgE mediated gastrointestinal food allergy is known as a disease group having high homology with eosinophilic gastrointestinal diseases.
  • Rothenberg ME Eosinophilic gastrointestinal disorders (EGID). J Allergy Clin Immunol 2004; 113: 11-28. 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.
  • Gastrointestinal endoscopy is an examination technique that places a heavy burden on the subject to be examined.
  • the test subject is an infant, since it is an excessively invasive test method, it causes a delay in diagnosis and causes serious complications.
  • 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.
  • 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.
  • EGID or food protein-induced enteropathy (Food-Protein-Induced-Enteropathy-Syndrome- (Enteropathy)).
  • the present invention includes any of the following inventions.
  • TSLP thymic stromal lymphopoietin
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 encompasses a polypeptide containing a known analog of a naturally occurring amino acid that can function similarly to the naturally occurring amino acid.
  • 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.
  • “gene” is used interchangeably with “polynucleotide”, “nucleic acid” or “nucleic acid molecule”. “Polynucleotide” means a polymer of nucleotides.
  • 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.
  • the term “healthy person” refers to a normal individual who does not suffer from eosinophilic gastrointestinal disease or food protein-induced enteropathy.
  • “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.
  • 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.
  • “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.
  • 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.
  • 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. .
  • 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.
  • 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 gastrointestinal disorders 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 (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
  • EGE Eosinophilic Gastroenteritis
  • EGE Erysinophilic Gastroenteritis
  • 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).
  • HPF high-power field
  • EoE Esophagitis
  • Eosinophilic esophagitis develops at the age from newborn to adult, but it occurs most often in men in their 30s and 50s.
  • PPI proton pump inhibitors
  • PPI-RE PPI-REproton-pump inhibitor
  • PPI-RE PPI-responsive esophageal eosinophilia
  • 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.
  • 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.
  • the inspection method according to the present embodiment can be applied to subjects of all ages.
  • 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.
  • 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.
  • 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.
  • the subject may be a prenatal fetus.
  • 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).
  • the biological sample include a cell sample, a tissue sample, and a body fluid sample, and among them, a body fluid sample is preferable.
  • 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.
  • 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.
  • 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).
  • 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.
  • an operation for extracting a protein or a nucleic acid or an operation for removing an unnecessary component as necessary.
  • serum or plasma prepared from the collected blood for the test.
  • 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.
  • a sample as it is collected may be stored, or a sample (for example, serum or plasma) prepared after collection may be stored.
  • the inspection method of the present invention includes the measurement steps shown in the following a) or b).
  • Step 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.
  • 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.
  • step a) it is preferable to measure the expression level of at least one of the TSLP gene and IL-33 gene.
  • 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.
  • the expression levels of TSLP gene, IL-33 gene, CCL7 / MCP-3 gene and CCL21 / 6CKine gene are most preferably measured.
  • 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.
  • CCL5 CCL20, CCL22 and neurofilament medium polypeptide (NEFM) in addition to the above-mentioned cytokines and / or chemokine proteins.
  • NEFM neurofilament medium polypeptide
  • 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
  • CCL7 / MCP-3 gene is CCL7 / MCP-.
  • 3 is a generic term for nucleic acids that encode C3
  • 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
  • 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.
  • 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)”.
  • 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.
  • 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.
  • 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) 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.
  • a step of measuring the amount of protein for example, protein concentration
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RIA radioimmunoassay
  • 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.
  • 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.
  • the amino acid sequences of human TSLP, IL-33, CCL7 / MCP-3, and CCL21 / 6CKine are available from public databases such as NCBI.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the amount is significantly high may be a result of quantitative measurement or a result of qualitative measurement.
  • 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.
  • 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.
  • a person skilled in the art can easily determine a threshold 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.
  • 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.
  • 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.
  • diagnosis 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).
  • diagnosis 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.
  • 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.
  • the cutoff value As a calculation method of the cutoff value, a method known in the technical field can be used.
  • 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 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.
  • ROC Receiveiver Operating Characteristic
  • 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.
  • 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.
  • 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.
  • diseases differentiated from eosinophilic esophagitis include gastroesophageal reflux disease, Candida infection
  • irritable bowel syndrome and eosinophilic gastroenteritis have heretofore been difficult to distinguish from symptoms, and histopathological examination was necessary.
  • histopathological examination was necessary.
  • eosinophilic gastroenteritis and eosinophilic esophagitis having esophageal lesions had to be differentiated by performing biopsy of the stomach and duodenum.
  • 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).
  • FPIES Food Protein-Induced Enterocolitis Syndrome
  • Proctocolitis Food Protein Induction
  • enteropathy Food Protein-Induced Enteropathy Syndrome (Enteropathy)
  • celiac disease celiac disease
  • 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.
  • 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.
  • 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.
  • 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].
  • 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.
  • 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.
  • 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].
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 any one of 1) to 6).
  • the inspection method according to any one of 1) to 7).
  • 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.
  • 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.
  • NCCHD National Center for Child Health and Development
  • This database is diagnosed as meeting three Powell criteria (Powell's criteria: reference E1) and having non-IgE-mediated gastrointestinal food allergy (reference E2)
  • reference E1 Powell criteria
  • reference E2 non-IgE-mediated gastrointestinal food allergy
  • 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.
  • GI gastrointestinal
  • RNA 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.
  • 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.
  • HPF high-power field
  • 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®.
  • 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).
  • Matts grade was used to classify the severity of UC patients ( ⁇ 3 grades for 3 patients; ⁇ 2 grades for 5 patients).
  • 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 TNF ⁇ , G-CSF, GM-CSF, IFN ⁇ , IL1 ⁇ , IL3, IL5, IL6, IL8, IL9, IL12p70, IL13, IL17, IL33, TSLP, CCL1 / I309, CCL2 / MCP1, CCL3 / MIP1 ⁇ , CCL4 / MIP1 ⁇ , CCL7 / MCP3, CCL8 / MCP2, CCL11 / eotaxin, CCL13 / MCP4, CCL15 / MIP1 ⁇ , CCL17 / TARC, CCL21 / 6CKine, CCL22 / MDC, CCL24 / eotaxin2, CCL26 / CCL27 / ACK27 CXCL1-3 / GRO, CXCL5 / ENA78, CXCL10 / IP10, CXCL12 / SDF1, CXCL13 / BCA1, and
  • RNAlater® solution QIAGEN, Valencia, CA, USA
  • Microarray analysis 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).
  • the inventors have attempted to use microarrays to find genes that are up-regulated in lesions of the gastrointestinal tract in infant EGE patients.
  • 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.
  • EGE histological diagnosis 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.
  • 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).
  • 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 and IL-33 levels in serum decreased with improvement in symptoms and signs (H and I in FIG. 1).
  • TSLP and IL33 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.
  • a polymorphism located in the vicinity of TSLP has been reported to be related to EoE (reference document E15).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne un nouveau procédé d'examen de maladies gastro-intestinales éosinophiles ou d'entéropathies induites par des protéines alimentaires. En d'autres termes, la présente invention concerne un procédé d'examen de maladies gastro-intestinales éosinophiles ou d'entéropathies induites par des protéines alimentaires comprenant une étape de mesure pour mesurer le niveau d'expression d'au moins un gène parmi un gène TSLP, un gène IL-33, un gène CCL7 et un gène CCL21 dans un échantillon prélevé chez un sujet humain vivant ou pour mesurer la quantité d'au moins une protéine parmi la protéine TSLP, la protéine IL-33, la protéine CCL7 et la protéine CCL21 dans ledit échantillon.
PCT/JP2017/031636 2016-09-01 2017-09-01 Procédé d'examen et kit d'examen pour une maladie gastro-intestinale éosinophile ou une entéropathie induite par des protéines alimentaires WO2018043715A1 (fr)

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JP2016171207A JP2019193577A (ja) 2016-09-01 2016-09-01 好酸球性消化管疾患または食物蛋白誘発腸症の検査方法および検査キット
JP2016-171207 2016-09-01

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WO2020179887A1 (fr) * 2019-03-05 2020-09-10 国立大学法人 東京大学 Procédé de distinction entre une maladie intestinale inflammatoire et un lymphome gastro-intestinal, marqueur de distinction et kit

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JP6707704B2 (ja) * 2019-10-24 2020-06-10 東芝映像ソリューション株式会社 放送信号受信装置

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WO2014059178A1 (fr) * 2012-10-10 2014-04-17 Rhode Island Hospital Expression différentielle de nouveaux marqueurs protéiques pour le diagnostic et le traitement de l'œsophagite à éosinophiles
WO2015006571A1 (fr) * 2013-07-11 2015-01-15 Regeneron Pharmaceuticals, Inc. Méthodes de traitement d'une œsophagite à éosinophiles impliquant l'administration d'un inhibiteur des il-4r

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WO2014059178A1 (fr) * 2012-10-10 2014-04-17 Rhode Island Hospital Expression différentielle de nouveaux marqueurs protéiques pour le diagnostic et le traitement de l'œsophagite à éosinophiles
WO2015006571A1 (fr) * 2013-07-11 2015-01-15 Regeneron Pharmaceuticals, Inc. Méthodes de traitement d'une œsophagite à éosinophiles impliquant l'administration d'un inhibiteur des il-4r

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SHODA, T. ET AL.: "Sera of patients with infantile eosinophilic gastroenteritis showed a specific increase in both thymic stromal lymphopoietin and IL -33 levels.", J. ALLERGY CLIN.IMMUNOL., vol. 138, no. 1, 2 March 2016 (2016-03-02), pages 299 - 303, XP029627846, DOI: doi:10.1016/j.jaci.2015.11.042 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020179887A1 (fr) * 2019-03-05 2020-09-10 国立大学法人 東京大学 Procédé de distinction entre une maladie intestinale inflammatoire et un lymphome gastro-intestinal, marqueur de distinction et kit

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