Test method for predicting prognosis of a subject with ulcerative colitis in remission

Abstract

To provide a new test method for accurately predicting the prognosis of a subject with ulcerative colitis in remission.SOLUTION: Provided is a test method for predicting the prognosis of a subject with ulcerative colitis in remission. The method comprises the steps of: (1) quantifying a plurality of specific interleukins in a large intestine-derived tissue isolated from the subject with ulcerative colitis in remission; (2) using a pre-derived discriminant function 1 that correlates with LCI, MES and UCEIS and a discriminant function 2 that correlates with LCI to calculate coordinates (a value of the discriminant function 1 and a value of the discriminant function 2) on a two-dimensional plane from the plurality of specific interleukins; and (3) calculating a linear distance between the pre-calculated center of gravity coordinates and the coordinates calculated in the step (2) on the two-dimensional plane of a patient with ulcerative colitis in remission corresponding to LCI-A; and (4) comparing the linear distance with a pre-calculated cutoff value.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a testing method for predicting the prognosis of a subject, comprising quantifying the transcripts of a specific gene in samples isolated from a group of ulcerative colitis patients in remission and subjects in a similar state, deriving a discriminant function, measuring the linear distance between the coordinates of the center of gravity of ulcerative colitis patients in remission corresponding to LCI-A calculated from the discriminant function and the coordinates of ulcerative colitis subjects in remission, and comparing the measured distance with a pre-calculated cutoff value. [Background technology]

[0002] Ulcerative colitis is an intractable disease that causes erosions and ulcers in the mucous membrane of the large intestine, and since its cause is still unknown, there is no fundamental treatment. The number of patients in Japan is rapidly increasing, and many cases occur at a young age, so controlling the disease is essential to maintain the quality of life (QOL) of the patient for the rest of their long lives.

[0003] Treatment of ulcerative colitis is centered on symptomatic treatment, but in recent years, various new treatments have been developed, and many patients have more options for disease control. The ulcerative colitis treatment guidelines recommend that appropriate medications be selected according to the severity and condition of the disease, and the mainstay of treatment are 5-aminosalicylic acid preparations (5-ASA preparations) and corticosteroid preparations. Oral administration of 5-ASA preparations alone or in combination with other topical preparations is the basic treatment, but if this is insufficient, remission is induced with oral corticosteroid treatment. Patients who are resistant to or dependent on corticosteroid treatment are considered to have intractable cases, and treatment options include cytapheresis, oral administration of tacrolimus, azathioprine or 6-mercaptopurine (6-MP), and anti-TNFα antibody preparations.

[0004] Ulcerative colitis patients who have been successfully treated with the above treatment enter a period of remission. Some patients who enter the remission period maintain the remission period, but the remaining patients enter the active period again, and the remission and active periods are often repeated. Although there have been a series of reports in recent years that endoscopic mucosal healing (endoscopic findings close to normal) and histological healing (a state in which there are no inflammatory findings in pathological tissue) are desirable to maintain the remission period, the current situation is that there is still no established definition of endoscopic mucosal healing or histological healing. In this regard, the present inventors reported in 2017 that a new endoscopic image enhancement system (Linked Color Imaging: LCI) is useful for predicting the prognosis of ulcerative colitis (Non-Patent Document 1). According to this, it has been revealed that when observing the mucosa of ulcerative colitis with an endoscope, by using LCI to make a judgment, it is possible to significantly predict the disease and grasp the disease activity compared to conventional endoscopic diagnosis (Mayo Endoscopic Subscore (MES) or Ulcerative Colitis Endoscopic Index of Severity (UCEIS)). However, LCI requires specialized medical equipment, and a simple, rapid method for predicting the prognosis of ulcerative colitis patients in remission has been desired. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Uchiyama K et al., J Crohns Colitis, Vol.11, No.8, page 963-969, 2017 Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to provide a new testing method for accurately predicting the prognosis of a subject with ulcerative colitis in remission. [Means for solving the problem]

[0007] In order to achieve the above object, the inventors collected samples from the colons of ulcerative colitis patients in clinical remission, classified each sample into scores using the LCI, MES, and UCEIS, and simultaneously quantified the amount of mRNA for various cytokines (21 types) for each sample. They derived a discriminant function that correlates with the scores classified using LC, MES, and UCEIS, and a discriminant function that does not correlate with the scores classified using MES and UCEIS but specifically correlates with the scores classified using LCI. They used discriminant functions 1 and 2 to calculate the coordinates of the center of gravity (value of discriminant function 1, value of discriminant function 2) on a two-dimensional plane of ulcerative colitis patients in remission corresponding to LCI-A, and the coordinates (value of discriminant function 1, value of discriminant function 2) on a two-dimensional plane of ulcerative colitis subjects in remission, and calculated the linear distance between both coordinates. They found that the relapse of ulcerative colitis in a subject can be accurately predicted using a certain value of the linear distance as a criterion.

[0008] That is, the present invention comprises the following. [1] A method for predicting the prognosis of a subject with ulcerative colitis in remission, comprising the following steps: (1) quantifying the following (a) to (i) in colon-derived tissue isolated from a subject with ulcerative colitis in remission: (a) IL-1β transcript; (b) IL-4 transcript; (c) IL-17A transcript; (d) IL-22 transcript; (e) IL-33 transcript; (f) IL-10 transcript; (g) IL-6 transcript; (h) IL-2 transcript, and (i) IL-13 transcript; (2) calculating coordinates (values ​​of discriminant function 1 and discriminant function 2) on a two-dimensional plane from (a) to (i) using discriminant function 1 correlated with LCI, MES, and UCEIS and discriminant function 2 correlated with LCI, which have been derived in advance; (3) calculating the straight-line distance between the coordinates of the center of gravity calculated in advance on a two-dimensional plane of a patient with ulcerative colitis in remission corresponding to LCI-A and the coordinates calculated in step (2); (4) comparing the linear distance to a pre-calculated cutoff value. [2] The testing method described in [1], wherein the pre-calculated cutoff value is 4.87. [3] The method according to [1], further comprising quantifying the following (j) to (u) in the step (1): (j) TNF-α transcript; (k) IFN-γ transcript; (l) IL-8 transcript; (m) IL-23 transcript; (n) an IL-12 transcript; (o) IL-17F transcript; (p) IL-5 transcript, (q) IL-15 transcript; (r) IL-18 transcript; (s) an IL-21 transcript; (t) IL-27 transcript, and (u) IL-9 transcript. [4] The inspection method according to [3], characterized in that in step (2), coordinates on a two-dimensional plane (the value of discriminant function 1, the value of discriminant function 2) are calculated from the (a) to (u). [5] The testing method described in [4], wherein the pre-calculated cutoff value is 1.72. [6] A method for predicting the prognosis of a subject with ulcerative colitis in remission, comprising the following steps: (1) quantifying the following (a) to (i) in colon-derived tissues isolated from ulcerative colitis patients in remission and subjects with ulcerative colitis in remission: (a) IL-1β transcript; (b) IL-4 transcript; (c) IL-17A transcript; (d) IL-22 transcript; (e) IL-33 transcript; (f) IL-10 transcript; (g) IL-6 transcript; (h) IL-2 transcript, and (i) IL-13 transcript; (2) deriving the following two discriminant functions from the (a) to (i) derived from ulcerative colitis patients in a remission state: (i) a discriminant function 1 including, as independent variables, the amount of IL-10 transcript, the amount of IL-6 transcript, the amount of IL-2 transcript, the amount of IL-13 transcript, and the amount of IL-17A transcript, and including, as coefficients of each of the independent variables, coefficients correlating with LCI, MES, and UCEIS; and (ii) a discriminant function 2 including an IL-1β transcript amount, an IL-4 transcript amount, an IL-17A transcript amount, an IL-22 transcript amount, and an IL-33 transcript amount as independent variables, and including a coefficient correlating with LCI as a coefficient for each of the independent variables; (3) calculating coordinates (values ​​of discriminant function 1 and discriminant function 2) on a two-dimensional plane from the (a) to (i) derived from ulcerative colitis patients in a state of remission corresponding to LCI-A using the discriminant functions derived in the step (2), and calculating the coordinates of the center of gravity; (4) calculating coordinates (values ​​of discriminant function 1 and discriminant function 2) on a two-dimensional plane from the (a) to (i) derived from a subject with ulcerative colitis in remission using the discriminant functions derived in the step (2); (5) calculating the straight-line distance between the center of gravity coordinates calculated in step (3) and the coordinates calculated in step (4); (6) comparing the linear distance to a pre-calculated cutoff value. [7] The method for testing according to [6], further comprising quantifying at least one selected from the group consisting of the following (j) to (u) in the step (1): (j) TNF-α transcript; (k) IFN-γ transcript; (l) IL-8 transcript; (m) IL-23 transcript; (n) an IL-12 transcript; (o) IL-17F transcript; (p) IL-5 transcript, (q) IL-15 transcript; (r) IL-18 transcript; (s) an IL-21 transcript; (t) IL-27 transcript, and (u) IL-9 transcript. [8] In step (2), (i) discriminant function 1 includes, as independent variables, all of an IL-10 transcript amount, an IL-6 transcript amount, an IL-2 transcript amount, an IL-13 transcript amount, and an IL-17A transcript amount, and an amount of at least one transcript selected from the group consisting of an IL-1β transcript, an IL-4 transcript, an IL-22 transcript, an IL-33 transcript, a TNF-α transcript, an IFN-γ transcript, an IL-8 transcript, an IL-23 transcript, an IL-12 transcript, an IL-17F transcript, an IL-5 transcript, an IL-15 transcript, an IL-18 transcript, an IL-21 transcript, an IL-27 transcript, and an IL-9 transcript, and includes, as coefficients of each independent variable, coefficients correlating with LCI, MES, and UCEIS; (ii) discriminant function 2 includes, as independent variables, all of an IL-1β transcript, an IL-4 transcript amount, an IL-17A transcript amount, an IL-22 transcript amount, and an IL-33 transcript amount, and an amount of at least one transcript selected from the group consisting of a TNF-α transcript, an IFN-γ transcript, an IL-8 transcript, an IL-23 transcript, an IL-12 transcript, an IL-17F transcript, an IL-5 transcript, an IL-15 transcript, an IL-18 transcript, an IL-21 transcript, an IL-27 transcript, an IL-9 transcript, an IL-10 transcript, an IL-6 transcript, an IL-2 transcript, and an IL-13 transcript, and includes, as a coefficient of each independent variable, a coefficient correlating with LCI. The inspection method described in [7]. [9] The testing method according to [8], wherein in step (3), coordinates on a two-dimensional plane (the value of discriminant function 1, the value of discriminant function 2) are calculated from at least one selected from the group consisting of all of (a) to (i) and (j) to (u) derived from a patient with ulcerative colitis in remission corresponding to LCI-A, and the centroid coordinates are calculated.

[10] The testing method according to [9], wherein in step (4), coordinates on a two-dimensional plane (the value of discriminant function 1, the value of discriminant function 2) are calculated from all of (a) to (i) and at least one selected from the group consisting of (j) to (u) derived from a subject with ulcerative colitis in remission.

[11] The examination method according to any one of [1] to

[10] , wherein the large intestine-derived tissue is the cecum, colon or rectum.

[12] A prognostic test for subjects with ulcerative colitis in remission, comprising the following (a) to (i): (a) a nucleic acid probe or primer capable of specifically detecting an IL-1β transcript; (b) a nucleic acid probe or primer capable of specifically detecting an IL-4 transcript; (c) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-17A transcript; (d) a nucleic acid probe or primer capable of specifically detecting an IL-22 transcript; (e) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-33 transcript; (f) a nucleic acid probe or primer capable of specifically detecting an IL-10 transcript; (g) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-6 transcript; (h) a nucleic acid probe or primer capable of specifically detecting an IL-2 transcript, and (i) A nucleic acid probe or primer capable of specifically detecting an IL-13 transcript.

[13] The test agent according to

[12] , further comprising at least one selected from the group consisting of the following (j) to (u): (j) a nucleic acid probe or primer capable of specifically detecting a TNF-α transcript; (k) a nucleic acid probe or primer capable of specifically detecting an IFN-γ transcript; (l) a nucleic acid probe or primer capable of specifically detecting an IL-8 transcript; (m) a nucleic acid probe or primer capable of specifically detecting an IL-23 transcript; (n) a nucleic acid probe or primer capable of specifically detecting an IL-12 transcript; (o) a nucleic acid probe or primer capable of specifically detecting an IL-17F transcript; (p) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-5 transcript; (q) a nucleic acid probe or primer capable of specifically detecting an IL-15 transcript; (r) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-18 transcript; (s) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-21 transcript; (t) a nucleic acid probe or primer capable of specifically detecting an IL-27 transcript, and (u) A nucleic acid probe or primer capable of specifically detecting an IL-9 transcript. Effect of the Invention

[0009] By measuring the amount of the transcript of a specific gene and measuring the linear distance between the coordinates of the subject and the coordinates of the patient with ulcerative colitis who has not relapsed, which is calculated from the discriminant function, a test can be performed to predict the prognosis of a patient with ulcerative colitis in remission. [Brief description of the drawings]

[0010] [Figure 1] This is a scatter plot of the plot (discriminant function 1 value, discriminant function 2 value) based on the values ​​of 21 cytokines for each LCI classification. LCI-A: No redness. LCI-B: Redness (vascular pattern in reddened area), LCI-C: Redness (no vascular pattern in reddened area) [Diagram 2] FIG. 1 is a graph showing a scatter plot of plots based on the levels of 21 cytokines in patients with and without relapse of ulcerative colitis, and the distance between the center of gravity calculated from the plots of LCI-A patients and the plots of patients with and without relapse of ulcerative colitis. [Diagram 3] Discriminant function 1: Correlates with all LCI, MES and UCEIS classifications. Discriminant function 2: Correlates only with LCI classification. [Figure 4] Discriminant function 1: Correlates with all LCI, MES and UCEIS classifications. Discriminant function 2: Correlates only with LCI classification. [Diagram 5] This is a scatter plot of nine cytokine values ​​for each LCI classification (values ​​of discriminant function 1, values ​​of discriminant function 2). LCI-A: no redness. LCI-B: redness (vascular pattern in reddened area), LCI-C: redness (no vascular pattern in reddened area). [Figure 6] FIG. 1 is a graph showing a scatter plot of plots based on the levels of nine cytokines in patients with and without ulcerative colitis relapses, and the distance between the LCI-A centroid and the plots of patients with and without ulcerative colitis relapses. [Figure 7] FIG. 1 shows a ROC curve generated from logistic regression analysis of the distance between the LCI-A centroid and the plot of patients in remission classified as LCI-C, using 21 cytokine values. [Figure 8] FIG. 1 shows a ROC curve generated from logistic regression analysis of the distance between the LCI-A centroid and the plot of patients in remission classified as LCI-C, using nine cytokine values. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention provides a testing method for predicting the prognosis of a subject with ulcerative colitis in remission (the testing method of the present invention).

[0012] The subject with ulcerative colitis in remission to which the test method of the present invention can be applied is not particularly limited as long as the subject is afflicted with ulcerative colitis and is clinically determined to be in remission, and examples of the subject include subjects diagnosed as being in remission using indices such as the Rachmilewitz index (Clinical activity index: CAI), the Sutherland index (Disease activity index: DAI), the Seo index, and the Lichtiger index. Examples of the subject include humans and non-human mammals (e.g., monkeys, cows, horses, pigs, mice, rats, guinea pigs, hamsters, dogs, cats, rabbits, sheep, goats, etc.). The preferred subject is a human.

[0013] In one embodiment, the testing method of the present invention comprises the following steps (the testing method of the present invention comprising the following steps is referred to as testing method 1 of the present invention). (1) quantifying the following (a) to (i) in colon-derived tissue isolated from a subject with ulcerative colitis in remission: (a) IL-1β transcript; (b) IL-4 transcript; (c) IL-17A transcript; (d) IL-22 transcript; (e) IL-33 transcript; (f) IL-10 transcript; (g) IL-6 transcript; (h) IL-2 transcript, and (i) IL-13 transcript. (2) A process of calculating coordinates (value of discriminant function 1, value of discriminant function 2) on a two-dimensional plane from (a) to (i) using discriminant function 1 correlated with the LCI, MES, and UCEIS, and discriminant function 2 correlated with the LCI, which have been derived in advance. (3) A process for calculating the straight-line distance between the coordinates of the center of gravity calculated in advance on a two-dimensional plane of a patient with ulcerative colitis in a state of remission corresponding to LCI-A and the coordinates calculated in step (2). (4) comparing the linear distance to a pre-calculated cutoff value.

[0014] Testing method 1 of the present invention comprises a step (step (1)) of quantifying the amount of a transcript in colon-derived tissue isolated from a subject with ulcerative colitis in a state of remission.

[0015] In step (1), the colon-derived tissue isolated from the subject with ulcerative colitis in remission is not particularly limited as long as it is collected from the subject to be tested and can contain the transcription product to be quantified. Examples of such colon-derived tissue include the cecum, colon (ascending colon, transverse colon, descending colon, sigmoid colon), and rectum.

[0016] The quantity of transcripts in colon-derived tissue isolated from a subject can be determined by preparing an RNA (e.g., total RNA, mRNA) fraction from the tissue and measuring the amount of transcripts contained in the fraction. Thus, in one embodiment, quantification of transcripts in colon-derived tissue isolated from a subject can be carried out using a nucleic acid probe or nucleic acid primer capable of specifically detecting each transcript.

[0017] The RNA fraction can be prepared using known techniques such as guanidine-CsCl ultracentrifugation and AGPC, but high-purity total RNA can be prepared quickly and easily from a trace amount of sample using a commercially available RNA extraction kit (e.g., RNeasy Mini Kit; manufactured by QIAGEN, etc.). Means for detecting transcription products in the RNA fraction include, for example, a method using hybridization (Northern blot, dot blot, etc.) or a method using PCR (RT-PCR, competitive PCR, real-time PCR, etc.). Quantitative PCR methods such as competitive PCR and real-time PCR are preferred because they can detect gene expression changes quickly, easily, and quantitatively from a trace amount of sample.

[0018] In the case of Northern blot or dot blot hybridization, the quantity of the transcription products can be quantified, for example, using a nucleic acid probe capable of specifically detecting each transcription product. Such a nucleic acid probe is a polynucleotide that contains a continuous nucleotide sequence of about 15 bases or more, preferably about 18 to about 500 bases, more preferably about 18 to about 200 bases, and even more preferably about 18 to about 50 bases, or a complementary sequence thereof, among the polynucleotides that are each transcription product. The nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. DNA is preferred. The nucleic acid used as a probe may be double-stranded or single-stranded. In the case of a double-stranded nucleic acid, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. In the case of a single-stranded nucleic acid, an antisense strand may be used.

[0019] The nucleic acid probe used in the test method 1 of the present invention is a polynucleotide containing a nucleotide sequence that hybridizes to each polynucleotide, which is a transcription product, under stringent conditions. Hybridization can be performed according to a method known per se or a method similar thereto, for example, the method described in Molecular Cloning, 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Examples of stringent conditions include hybridization reaction in 6×SSC (sodium chloride / sodium citrate) at 45° C., followed by one or more washings in 0.2×SSC / 0.1% SDS at 65° C. Those skilled in the art can easily adjust the stringency to a desired level by appropriately changing the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc.

[0020] The nucleic acid functioning as a probe is a nucleic acid that is amplified by using a primer set capable of amplifying a part or the whole of a transcription product, and is capable of amplifying any cell (e.g., hepatocyte, spleen cell, nerve cell, glial cell, pancreatic β cell, bone marrow cell, mesangial cell, Langerhans cell, epidermal cell, epithelial cell, goblet cell, endothelial cell, smooth muscle cell, fibroblast, fibrocyte, muscle cell, adipocyte, immune cell (e.g., macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, eosinophil, monocyte), megakaryocyte, synovial cell, chondrocyte, osteocyte, osteoblast, osteoclast, mammary cell, or stromal cell, or a precursor cell, stem cell, or cancer cell of these cells) or The nucleic acid of the desired length can be amplified by PCR using cDNA or genomic DNA derived from any tissue in which the cells exist [for example, brain, each part of the brain (e.g., olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonads, thyroid gland, gallbladder, bone marrow, adrenal gland, skin, lung, digestive tract (e.g., large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue, skeletal muscle, etc.] as a template, or the gene or cDNA can be cloned from the cDNA or genomic DNA library derived from the above-mentioned cells or tissues by colony or plaque hybridization, etc., and, if necessary, can be obtained by making it into a fragment of an appropriate length using a restriction enzyme, etc. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd Edition (mentioned above), etc. Alternatively, the nucleic acid can be obtained by chemically synthesizing a part or all of the base sequence and / or its complementary strand sequence based on known base sequence information using a commercially available automatic DNA / RNA synthesizer or the like.

[0021] The nucleic acid is preferably labeled with a labeling agent to enable quantification of the transcription product. Examples of the labeling agent include radioisotopes, enzymes, fluorescent substances, and luminescent substances. Examples of the radioisotopes include 32 P], [ 3 H], [ 14 C], etc. are used. As the enzyme, those that are stable and have a large specific activity are preferred, for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc. are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin, etc. are used. Furthermore, biotin-(strept)avidin can also be used to bind the probe and the labeling agent.

[0022] In the case of Northern hybridization, the RNA fraction prepared as described above is separated by gel electrophoresis, then transferred to a membrane such as nitrocellulose, nylon, polyvinylidene difluoride, etc., and specifically hybridized in a hybridization buffer containing the labeled probe prepared as described above, and the amount of the label bound to the membrane is measured for each band by an appropriate method, thereby measuring the amount of the transcript. In the case of dot blot, the membrane spotted with the RNA fraction is subjected to a hybridization reaction in the same manner, and the amount of the transcript can be measured by measuring the amount of the label on the spot.

[0023] In another preferred embodiment, the transcription product is quantified by quantitative PCR, such as competitive PCR or real-time PCR, preferably real-time PCR. An example of a set of oligonucleotides used as primers in PCR is a nucleic acid primer capable of specifically detecting a transcription product. In one preferred embodiment, the nucleic acid primers used in the testing method of the present invention include a set of oligonucleotides of a polynucleotide that is a transcription product, which has a length of a continuous nucleotide sequence of about 15 bases or more, preferably about 15 to about 50 bases, more preferably about 15 to about 30 bases, and even more preferably about 15 to about 25 bases, and is designed to amplify a DNA fragment of about 100 bp to several kbp, and which is complementary to a polynucleotide (sense strand) sequence, and a polynucleotide that can hybridize to a polynucleotide (antisense strand) having a nucleotide sequence complementary to the polynucleotide sequence.

[0024] The value of the transcript quantified according to the above quantification method may be expressed in any manner, including concentration, number of molecules, the number of amplification cycles required for the initial transcript amount to reach a specified amount by real-time PCR (if there are multiple transcripts to be quantified, the value is corrected by the number of amplification cycles required for the initial transcript amount of the internal control gene to reach a specified amount), etc.

[0025] In step (1), the transcription products to be quantified include the following (a) to (i): (a) IL-1β transcript; (b) IL-4 transcript; (c) IL-17A transcript; (d) IL-22 transcript; (e) IL-33 transcript; (f) IL-10 transcript; (g) IL-6 transcript; (h) IL-2 transcript, and (i) IL-13 transcript. Furthermore, in the step (1), in addition to the above (a) to (i), the following (j) to (u) may be further quantified. (j) TNF-α transcript; (k) IFN-γ transcript; (l) IL-8 transcript; (m) IL-23 transcript; (n) an IL-12 transcript; (o) IL-17F transcript; (p) IL-5 transcript, (q) IL-15 transcript; (r) IL-18 transcript; (s) an IL-21 transcript; (t) IL-27 transcript, and (u) IL-9 transcript.

[0026] The above transcription products (a) to (u) are known transcription products, and are identified as Genbank Accession No.: NM_000576 (IL-1β), Genbank Accession No.: M13982 (IL-4), Genbank Accession No.: NM_002190 (IL-17A), Genbank Accession No.: NM_020525 (IL-22), Genbank Accession No.: AY905581 (IL-33), Genbank Accession No.: NM_000572 (IL-10), Genbank Accession No.: M54894 (IL-6), Genbank Accession No.:NM_000586 (IL-2), Genbank Accession No.: L06801 (IL-13), Genbank Accession No.: NM_000594 (TNF-α), Genbank Accession No.: NM_000619 (IFN-γ), Genbank Accession No.: BT007067 (IL-8), Genbank Accession No.: NM_016584 (IL-23), Genbank Accession No.: AF180562 (IL-12), Genbank Accession No.: NM_052872 (IL-17F), Genbank Accession No.: NM_000879 (IL-5), Genbank Accession No.: U14407 (IL-15), Genbank Accession No.: AY044641 (IL-18), Genbank Accession No.: AF254069 (IL-21), Genbank Accession No.: NM_145659 (IL-27), Genbank Accession No.: NM_000590 The base sequence has been disclosed as (IL-9).In the testing method 1 of the present invention, the transcription products of IL-1β, IL-4, IL-17A, IL-22, IL-33, IL-10, IL-6, IL-2, IL-13, TNF-α, IFN-γ, IL-8, IL-23, IL-12, IL-17F, IL-5, IL-15, IL-18, IL-21, IL-27 and IL-9 are SEQ ID NO: 1 (IL-1β), SEQ ID NO: 2 (IL-4), SEQ ID NO: 3 (IL-17A), SEQ ID NO: 4 (IL-22), SEQ ID NO: 5 (IL-33), SEQ ID NO: 6 (IL-10), SEQ ID NO: 7 (IL-6), SEQ ID NO: 8 (IL-2), SEQ ID NO: 9 (IL-13), SEQ ID NO: 10 (TNF-α), SEQ ID NO: 11 (IFN-γ), SEQ ID NO: 12 (IL-8), SEQ ID NO: 13 (IL-23), SEQ ID NO: 14, respectively. (IL-12), SEQ ID NO: 15 (IL-17F), SEQ ID NO: 16 (IL-5), SEQ ID NO: 17 (IL-15), SEQ ID NO: 18 (IL-18), SEQ ID NO: 19 (IL-21), SEQ ID NO: 20 (IL-27) and SEQ ID NO: 21 (IL-9). Examples of nucleic acids containing a nucleotide sequence substantially identical to the nucleotide sequences represented by SEQ ID NOs:1 to 21 include nucleic acids that contain a nucleotide sequence having a homology of about 85% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 98% or more to the nucleotide sequences represented by SEQ ID NOs:1 to 21 and encode a protein having substantially the same activity as IL-1β (or IL-4, IL-17A, IL-22, IL-33, IL-10, IL-6, IL-2, IL-13, TNF-α, IFN-γ, IL-8, IL-23, IL-12, IL-17F, IL-5, IL-15, IL-18, IL-21, IL-27, or IL-9). The homology of the base sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3). Alternatively, examples of nucleic acids containing a nucleotide sequence substantially identical to the nucleotide sequences represented by SEQ ID NOs:1 to 21 include nucleic acids that contain a nucleotide sequence having an identity of about 85% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 98% or more to the nucleotide sequences represented by SEQ ID NOs:1 to 21 and encode a protein having substantially the same activity as IL-1β (or IL-4, IL-17A, IL-22, IL-33, IL-10, IL-6, IL-2, IL-13, TNF-α, IFN-γ, IL-8, IL-23, IL-12, IL-17F, IL-5, IL-15, IL-18, IL-21, IL-27, or IL-9).

[0027] Test method 1 of the present invention includes a step (step (2)) of calculating coordinates (values ​​of discriminant function 1, value of discriminant function 2) on a two-dimensional plane from (a) to (i) (or (a) to (u)) using discriminant function 1 correlated with LCI, MES, and UCEIS, and discriminant function 2 correlated with LCI, which have been derived in advance. In test method 1 of the present invention, all analyses can be performed using publicly available statistical analysis software, and for example, SPSS or the like can be used.

[0028] In step (2), the discriminant function 1 and discriminant function 2 derived in advance are discriminant equations constructed using cytokine parameters for Linked Color Imaging (LCI) classification (A, B, C) by discriminant analysis in multidimensional scaling based on the above (a) to (i) (or the above (a) to (u)) of colon-derived tissues isolated from ulcerative colitis patients in a remission state. More specifically, an analytical model is constructed in which the LCI classification (A, B, C) is a grouping variable and each cytokine parameter value is an independent variable. Here, the cytokine parameter value is calculated by multiplying each ΔCt value of the above (a) to (i) (or the above (a) to (u)) by 10. 3 The value obtained by multiplying the value by natural logarithmic transformation is adopted. The Ct value is defined as the number of amplification cycles at which a certain fluorescence intensity is reached by monitoring the amount of amplification of each of the above (a) to (i) (or the above (a) to (u)) by PCR in real time using the fluorescence intensity as an index, and the ΔCt value is the Ct value of the above (a) to (i) (or the above (a) to (u)) corrected by the Ct value of the internal standard gene. The value obtained by natural logarithmic transformation is set to a lower limit of -4, and values ​​less than -4 are treated as -4. In the above analysis model, the canonical discriminant function coefficients are calculated, and two-dimensional discriminant function 1 and discriminant function 2 are constructed.

[0029] The pre-derived discriminant function 1 is a discriminant function that correlates with the LCI, MES, and UCEIS. The pre-derived discriminant function 2 is a discriminant function that does not correlate with the MES and UCEIS, but only correlates with the LCI. As the pre-derived discriminant functions 1 and 2, for example, the discriminant functions 1 and 2 described in the examples described later can be used.

[0030] LCI (Linked Color Imaging) is a prognostic criterion for ulcerative colitis using a new endoscopic image enhancement system reported by the inventors (Uchiyama K et al., J Crohns Colitis, Vol.11, No.8, page 963-969, 2017), and ulcerative colitis can be classified into three scores (A, B, C) based on this criterion. In endoscopic images of ulcerative colitis classified as LCI-A, redness is not observed. In endoscopic images of ulcerative colitis classified as LCI-B, redness is observed, but vascular patterns are seen within the reddened area. In endoscopic images of ulcerative colitis classified as LCI-C, redness is observed, and no vascular patterns are seen within the reddened area. Ulcerative colitis classified as LCI-A continues to remain in remission, but some cases of ulcerative colitis classified as LCI-B will relapse, and the relapse rate is even higher in cases classified as LCI-C.

[0031] MES (Mayo Endoscopic Subscore) is a known endoscopic diagnostic criterion that has been most commonly used, and ulcerative colitis can be classified into four scores (0, 1, 2, 3) based on this criterion. In the endoscopic image of ulcerative colitis classified as MES-0, it is recognized as normal or inactive. In the endoscopic image of ulcerative colitis classified as MES-1, redness is confirmed, the vascular visibility is unclear, and mild bleeding tendency is observed, and it is classified as mild. In the endoscopic image of ulcerative colitis classified as MES-2, marked redness is confirmed, the vascular visibility is lost, bleeding tendency and erosion are observed, and it is classified as moderate. In the endoscopic image of ulcerative colitis classified as MES-3, spontaneous bleeding and ulcers are observed, and it is classified as severe. Ulcerative colitis classified as MES-0 or MES-1 has traditionally been judged to be in mucosal remission or mucosal healing, but long-term monitoring of patients has shown that even MES-0 patients, who are considered to be normal, can experience relapse.

[0032] The Ulcerative Colitis Endoscopic Index of Severity (UCEIs), like the MES, is a known endoscopic diagnostic criterion that has been used for a long time and is the sum of scores for each of the three items: vascular visibility, bleeding, and erosion and ulcer. Vascular visibility is defined as normal (0), partially disappeared (1), or completely disappeared (2). Bleeding is defined as no bleeding (0), dot-like or linear coagulation is observed on the mucosal surface but can be removed by washing (1), mild intraluminal bleeding (2), moderate or severe intraluminal bleeding (3). Erosion and ulcer are defined as no erosion or ulcer (0), small mucosal defect (erosion) of 5 mm or less (1), shallow ulcer of more than 5 mm (2), or deep ulcer (3).

[0033] Using the discriminant function 1 and discriminant function 2, coordinates (value of discriminant function 1, value of discriminant function 2) on a two-dimensional plane are calculated from (a) to (i) (or (a) to (u)). For example, for discriminant function 1 described in the Examples below, the amounts of IL-10 transcripts, IL-6 transcripts, IL-2 transcripts, IL-13 transcripts, and IL-17A transcripts in colon-derived tissues isolated from subjects with ulcerative colitis in remission are measured, and the obtained values ​​are applied to calculate a value on the horizontal axis on the two-dimensional plane. Similarly, for discriminant function 2, the amounts of IL-1β transcripts, IL-4 transcripts, IL-17A transcripts, IL-22 transcripts, and IL-33 transcripts in colon-derived tissues isolated from subjects with ulcerative colitis in remission are measured, and the obtained values ​​are applied to calculate a value on the vertical axis on the two-dimensional plane. The obtained values ​​on the horizontal axis and vertical axis can be treated as coordinates on the two-dimensional plane.

[0034] Testing method 1 of the present invention includes a step (step (3)) of calculating the straight-line distance between the coordinates of the center of gravity calculated in advance on a two-dimensional plane of an ulcerative colitis patient in a state of remission corresponding to LCI-A (hereinafter, an LCI-A patient) and the coordinates calculated in step (2).

[0035] In step (3), the centroid coordinates on the two-dimensional plane of the LCI-A patients can be derived in advance by calculating each coordinate (the value of discriminant function 1, the value of discriminant function 2) on the two-dimensional plane from the above (a) to (i) (or the above (a) to (u)) in the large intestine-derived tissue isolated from a population of LCI-A patients using the discriminant function 1 and the discriminant function 2, and calculating the centroid coordinates from each of the coordinates. For example, for discriminant function 1 described in the Examples below, the amounts of IL-10 transcripts, IL-6 transcripts, IL-2 transcripts, IL-13 transcripts, and IL-17A transcripts in the large intestine-derived tissue isolated from a population of LCI-A patients can be measured, and the obtained values ​​can be applied to calculate the value on the horizontal axis on the two-dimensional plane. Similarly, for the discriminant function 2, the amounts of IL-1β transcripts, IL-4 transcripts, IL-17A transcripts, IL-22 transcripts, and IL-33 transcripts in colon-derived tissues isolated from a group of LCI-A patients are measured, and the obtained values ​​are applied to calculate the value on the vertical axis on a two-dimensional plane. The obtained horizontal and vertical axis values ​​are treated as coordinates on a two-dimensional plane, and their centroid coordinates can be calculated from the average sum of the horizontal and vertical axis values ​​of each coordinate. As described in the examples below, when the above (a) to (i) are quantified, the centroid coordinates on the two-dimensional plane of the LCI-A patient include, but are not limited to, (0.7596, 0.7645). Alternatively, when the above (a) to (u) are quantified, the centroid coordinates on the two-dimensional plane of the LCI-A patient include, but are not limited to, (-0.445, -0.517).

[0036] The centroid coordinates are calculated from a patient who falls under LCI-A and does not experience a relapse of ulcerative colitis, and a subject whose coordinates on a two-dimensional plane are close to the centroid coordinates can be determined to be a patient who is unlikely to experience a relapse of ulcerative colitis, similar to an LCI-A patient. Therefore, in step (3), the linear distance between the centroid coordinates obtained as described above and the coordinates calculated in step (2) is calculated.

[0037] The inspection method 1 of the present invention includes a step (step (4)) of comparing the linear distance with a pre-calculated cutoff value.

[0038] The linear distance calculated in step (3) can be used as an index for predicting the prognosis of ulcerative colitis in a subject. Therefore, in step (3), the barycentric coordinates are calculated from an LCI-A patient who does not relapse into ulcerative colitis, and a subject whose coordinates on a two-dimensional plane are close to the barycentric coordinates can be determined to be a patient who is unlikely to relapse into ulcerative colitis, similar to an LCI-A patient. Therefore, in step (4), the linear distance is compared with a cutoff value calculated in advance. If the linear distance is equal to or greater than a predetermined cutoff value as a result of the comparison, it can be predicted that ulcerative colitis in remission will relapse into ulcerative colitis. On the other hand, if the linear distance is less than a predetermined cutoff value, it can be predicted that ulcerative colitis in remission will not relapse into ulcerative colitis.

[0039] As described in the Examples below, when the above (a) to (i) are quantified, the predetermined cutoff value is, but is not limited to, for example, 4.87. Alternatively, when the above (a) to (u) are quantified, the predetermined cutoff value is, but is not limited to, for example, 1.72.

[0040] In another embodiment, the testing method of the present invention comprises the following steps (the testing method of the present invention comprising the following steps is referred to as testing method 2 of the present invention). (1) quantifying the following (a) to (i) in colon-derived tissues isolated from ulcerative colitis patients in remission and subjects with ulcerative colitis in remission: (a) IL-1β transcript; (b) IL-4 transcript; (c) IL-17A transcript; (d) IL-22 transcript; (e) IL-33 transcript; (f) IL-10 transcript; (g) IL-6 transcript; (h) IL-2 transcript, and (i) IL-13 transcript. (2) deriving the following two discriminant functions from the (a) to (i) derived from ulcerative colitis patients in a remission state: (i) a discriminant function 1 including, as independent variables, the amount of IL-10 transcript, the amount of IL-6 transcript, the amount of IL-2 transcript, the amount of IL-13 transcript, and the amount of IL-17A transcript, and including, as coefficients of each of the independent variables, coefficients correlating with LCI, MES, and UCEIS; and (ii) Discriminant function 2, which includes IL-1β transcript amount, IL-4 transcript amount, IL-17A transcript amount, IL-22 transcript amount, and IL-33 transcript amount as independent variables and includes a coefficient correlating with LCI as the coefficient of each of the independent variables. (3) using the discriminant functions derived in step (2), calculating coordinates on a two-dimensional plane (the value of discriminant function 1, the value of discriminant function 2) from (a) to (i) derived from ulcerative colitis patients in a state of remission corresponding to LCI-A, and calculating the coordinates of the center of gravity. (4) calculating coordinates (the value of discriminant function 1, the value of discriminant function 2) on a two-dimensional plane from (a) to (i) derived from a subject with ulcerative colitis in remission using the discriminant functions derived in step (2). (5) A step of calculating the straight-line distance between the center of gravity coordinates calculated in step (3) and the coordinates calculated in step (4). (6) comparing the linear distance to a pre-calculated cutoff value.

[0041] Testing method 2 of the present invention comprises a step (step (1)) of quantifying the amount of a transcript in colon-derived tissue isolated from an ulcerative colitis patient in remission and a subject in remission.

[0042] In step (1), the ulcerative colitis patient in remission is not particularly limited as long as it is a patient suffering from ulcerative colitis who is clinically judged to be in remission, and examples thereof include patients diagnosed as being in remission by indices such as the Rachmilewitz index (Clinical activity index: CAI), the Sutherland index (Disease activity index: DAI), the Seo index, and the Lichtiger index. Examples of the patient include humans and non-human mammals (e.g., monkeys, cows, horses, pigs, mice, rats, guinea pigs, hamsters, dogs, cats, rabbits, sheep, goats, etc.). A preferred patient is a human. The number of patients is not particularly limited as long as it is a group large enough to carry out the test method 2 of the present invention, and is usually 100 or more, preferably 300 or more, and more preferably 500 or more.

[0043] In step (1), the transcription products to be quantified include the following (a) to (i): (a) IL-1β transcript; (b) IL-4 transcript; (c) IL-17A transcript; (d) IL-22 transcript; (e) IL-33 transcript; (f) IL-10 transcript; (g) IL-6 transcript; (h) IL-2 transcript, and (i) IL-13 transcript. Furthermore, in the step (1), in addition to the above (a) to (i), at least one selected from the group consisting of the following (j) to (u) may be further quantified. (j) TNF-α transcript; (k) IFN-γ transcript; (l) IL-8 transcript; (m) IL-23 transcript; (n) an IL-12 transcript; (o) IL-17F transcript; (p) IL-5 transcript, (q) IL-15 transcript; (r) IL-18 transcript; (s) an IL-21 transcript; (t) IL-27 transcript, and (u) IL-9 transcript.

[0044] Test method 2 of the present invention comprises a step (step (2)) of deriving two discriminant functions (discriminant functions 1 and 2) from the above (a) to (i) (or at least one selected from the group consisting of all of the above (a) to (i) and the above (j) to (u)) derived from a patient with ulcerative colitis in remission. The discriminant functions derived in step (2) can be derived by the same derivation method as the discriminant functions described in test method 1 of the present invention.

[0045] The discriminant function 1 derived in step (2) is not limited as long as it is a discriminant function correlated with LCI, MES, and UCEIS. For example, discriminant function 1 derived from (a) to (i) above from an ulcerative colitis patient in remission may include discriminant function 1 including, as independent variables, the amount of IL-10 transcript, the amount of IL-6 transcript, the amount of IL-2 transcript, the amount of IL-13 transcript, and the amount of IL-17A transcript, and including, as coefficients of the independent variables, coefficients correlated with LCI, MES, and UCEIS. Alternatively, the discriminant function 1 derived from at least one selected from the group consisting of all of (a) to (i) and (j) to (u) may be a discriminant function 1 which includes, as independent variables, all of the amounts of IL-10 transcripts, IL-6 transcripts, IL-2 transcripts, IL-13 transcripts and IL-17A transcripts, and the amount of at least one transcript selected from the group consisting of IL-1β transcripts, IL-4 transcripts, IL-22 transcripts, IL-33 transcripts, TNF-α transcripts, IFN-γ transcripts, IL-8 transcripts, IL-23 transcripts, IL-12 transcripts, IL-17F transcripts, IL-5 transcripts, IL-15 transcripts, IL-18 transcripts, IL-21 transcripts, IL-27 transcripts and IL-9 transcripts, and includes, as coefficients for each of the independent variables, coefficients correlating with LCI, MES and UCEIS. A specific example of the discriminant function 1 is the discriminant function 1 described in the examples below.

[0046] The discriminant function 2 derived in step (2) is not limited as long as it is a discriminant function that does not correlate with MES and UCEIS, but only correlates with LCI. For example, the discriminant function 2 derived from the above (a) to (i) from an ulcerative colitis patient in remission includes a discriminant function 2 that includes an IL-1β transcript amount, an IL-4 transcript amount, an IL-17A transcript amount, an IL-22 transcript amount, and an IL-33 transcript amount as independent variables and includes a coefficient that correlates with LCI as a coefficient of each independent variable. Alternatively, the discriminant function 2 derived from at least one selected from the group consisting of all of the (a) to (i) and the (j) to (u) may include a discriminant function 2 that includes, as independent variables, the amount of all of the IL-1β transcript, the amount of the IL-4 transcript, the amount of the IL-17A transcript, the amount of the IL-22 transcript, and the amount of at least one transcript selected from the group consisting of the TNF-α transcript, the IFN-γ transcript, the IL-8 transcript, the IL-23 transcript, the IL-12 transcript, the IL-17F transcript, the IL-5 transcript, the IL-15 transcript, the IL-18 transcript, the IL-21 transcript, the IL-27 transcript, the IL-9 transcript, the IL-10 transcript, the IL-6 transcript, the IL-2 transcript, and the IL-13 transcript, and includes, as coefficients of the independent variables, coefficients that correlate with LCI. A specific example of the discriminant function 2 is the discriminant function 2 described in the Examples below.

[0047] The testing method 2 of the present invention includes a step (3) of calculating coordinates (the value of discriminant function 1, the value of discriminant function 2) on a two-dimensional plane from the (a) to (i) (or from at least one selected from the group consisting of all of the (a) to (i) and the (j) to (u)) derived from an LCI-A patient using the discriminant functions derived in the step (2), and calculating the centroid coordinates thereof.

[0048] The coordinates and centroid coordinates in step (3) can be calculated by the same calculation method as the coordinates and centroid coordinates described in the test method 1 of the present invention. As described in the Examples below, when the above (a) to (i) are quantified, the centroid coordinates on a two-dimensional plane of an LCI-A patient can be, but are not limited to, for example, (0.7596, 0.7645). Alternatively, when the above (a) to (u) are quantified, the centroid coordinates on a two-dimensional plane of an LCI-A patient can be, but are not limited to, for example, (-0.445, -0.517).

[0049] Testing method 2 of the present invention includes a step (4) of calculating coordinates (the value of discriminant function 1, the value of discriminant function 2) on a two-dimensional plane from (a) to (i) (or at least one selected from the group consisting of all of (a) to (i) and (j) to (u)) derived from a subject with ulcerative colitis in remission, using the discriminant functions derived in step (2).

[0050] The coordinates in step (4) can be calculated by the same calculation method as the coordinates described in the inspection method 1 of the present invention.

[0051] Inspection method 2 of the present invention includes a step (step (5)) of calculating the linear distance between the centroid coordinates calculated in step (3) and the coordinates calculated in step (4).

[0052] The centroid coordinates calculated in step (3) are those calculated from LCI-A patients who do not suffer from relapse of ulcerative colitis, and subjects whose coordinates on a two-dimensional plane are close to the centroid coordinates can be determined to be patients who are unlikely to suffer from relapse of ulcerative colitis, similar to LCI-A patients. Therefore, in step (5), the linear distance between the centroid coordinates calculated in step (3) and the coordinates calculated in step (4) is calculated from the linear distance between the two coordinates.

[0053] The inspection method 2 of the present invention includes a step (step (6)) of comparing the linear distance with a pre-calculated cutoff value.

[0054] The linear distance calculated in step (5) can be used as an index for predicting the prognosis of ulcerative colitis in a subject. In step (6), the linear distance is compared with a pre-calculated cutoff value. If the linear distance is equal to or greater than the predetermined cutoff value, it can be predicted that ulcerative colitis in remission will relapse. On the other hand, if the linear distance is less than the predetermined cutoff value, it can be predicted that ulcerative colitis in remission will not relapse.

[0055] As described in the Examples below, when the above (a) to (i) are quantified, the predetermined cutoff value is, but is not limited to, for example, 4.87. Alternatively, when the above (a) to (u) are quantified, the predetermined cutoff value is, but is not limited to, for example, 1.72.

[0056] Furthermore, the present invention provides a test agent for predicting the prognosis of a subject with ulcerative colitis in remission (hereinafter, the test agent of the present invention). The test agent of the present invention may be a test agent for easily carrying out the above-mentioned test method of the present invention, and is not particularly limited. For example, (a) a nucleic acid probe or primer capable of specifically detecting an IL-1β transcript; (b) a nucleic acid probe or primer capable of specifically detecting an IL-4 transcript; (c) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-17A transcript; (d) a nucleic acid probe or primer capable of specifically detecting an IL-22 transcript; (e) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-33 transcript; (f) a nucleic acid probe or primer capable of specifically detecting an IL-10 transcript; (g) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-6 transcript; (h) a nucleic acid probe or primer capable of specifically detecting an IL-2 transcript, and (i) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-13 transcript; Includes.

[0057] The test agent of the present invention may further comprise the following (j) to (u) in addition to the above (a) to (i). (j) a nucleic acid probe or primer capable of specifically detecting a TNF-α transcript; (k) a nucleic acid probe or primer capable of specifically detecting an IFN-γ transcript; (l) a nucleic acid probe or primer capable of specifically detecting an IL-8 transcript; (m) a nucleic acid probe or primer capable of specifically detecting an IL-23 transcript; (n) a nucleic acid probe or primer capable of specifically detecting an IL-12 transcript; (o) a nucleic acid probe or primer capable of specifically detecting an IL-17F transcript; (p) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-5 transcript; (q) a nucleic acid probe or primer capable of specifically detecting an IL-15 transcript; (r) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-18 transcript; (s) a nucleic acid probe or nucleic acid primer capable of specifically detecting an IL-21 transcript; (t) a nucleic acid probe or primer capable of specifically detecting an IL-27 transcript, and (u) A nucleic acid probe or primer capable of specifically detecting an IL-9 transcript.

[0058] When the test agent of the present invention comprises the above-mentioned nucleic acid probe or nucleic acid primer as a component, the nucleic acid probe or nucleic acid primer may be the nucleic acid for probe or the oligonucleotide for primer described above in the test method of the present invention.

[0059] The nucleic acid capable of quantifying the transcription product can be provided as a solid in a dried or alcohol-precipitated state, or can be provided dissolved in water or an appropriate buffer solution (e.g., TE buffer, etc.). When used as a labeled probe, the nucleic acid can be provided in a state in which it has been previously labeled with any of the above-mentioned labeling substances, or can be provided separately from the labeling substances and labeled when used. Alternatively, the nucleic acid can be provided in a state of being immobilized on a suitable solid phase. Examples of the solid phase include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene difluoride, etc. In addition, examples of immobilization methods include, but are not limited to, a method in which a functional group such as an amino group, an aldehyde group, an SH group, or biotin is introduced into the nucleic acid in advance, and a functional group that can react with the nucleic acid (e.g., an aldehyde group, an amino group, an SH group, streptavidin, etc.) is also introduced onto the solid phase, and the solid phase and the nucleic acid are crosslinked by a covalent bond between both functional groups, or, for polyanionic nucleic acid, a method in which the solid phase is coated with polycations and the nucleic acid is immobilized by electrostatic binding.

[0060] It is particularly preferred that the nucleic acid contained in the test agent of the present invention is constructed so that the transcription product can be quantified by the same method (eg, Northern blot, dot blot, DNA array technology, quantitative RT-PCR, etc.).

[0061] The reagent constituting the test agent of the present invention may further contain, in addition to the nucleic acid capable of quantifying the transcription product, other substances necessary in the reaction for quantifying the transcription product, which do not adversely affect the reaction when stored in a coexistent state. Alternatively, the reagent may be provided together with a separate reagent containing other substances necessary in the reaction for quantifying the transcription product. For example, when the reaction for quantifying the transcription product is PCR, the other substances include, for example, a reaction buffer, dNTPs, a heat-resistant DNA polymerase, etc. When competitive PCR or real-time PCR is used, competitor nucleic acid, a fluorescent reagent (such as the above-mentioned intercalator or fluorescent probe), etc. may further be included. EXAMPLES

[0062] The present invention will be described in more detail below with reference to examples, but it is clear that the present invention is not limited to these.

[0063] Sample collection Samples (183 sites in total) were obtained from three locations, the ileocecal region, sigmoid colon, and rectum, from 61 patients with ulcerative colitis in clinical remission (Lichtiger index ≦4). Patient characteristics are shown in Table 1.

[0064] [Table 1]

[0065] Statistical analysis of cytokine expression in collected samples The mRNA expression levels of various cytokines (TNFα, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-18, IL-21, IL-22, IL-23, IL-27, and IL-33) were quantified using real-time PCR in each sample. The cytokine expression levels obtained by real-time PCR were compared according to endoscopic classification (Linked Color Imaging (LCI), Mayo Endoscopic Subscore (MES), and Ulcerative Colitis Endoscopic Index of Severity (UCEIS)) using the ΔΔCt method. The LCI classification can predict relapse of ulcerative colitis more accurately than the conventional MES and UCEIS classifications.

[0066] Calculation of discriminant functions for LCI classification by cytokine parameters using discriminant analysis in multidimensional scaling (1) The above 21 cytokine values ​​were logarithmically transformed, and discriminant analysis was performed using an analysis model with the LCI classification as a grouping variable and the cytokine value as an independent variable. The canonical discriminant function coefficients were calculated using the following formula, and discriminant function 1, which correlates with the LCI classification, MES classification, and UCEIS classification, and discriminant function 2, which correlates only with the LCI classification, were constructed. The centers of gravity of each LCI classification were calculated and plotted on the first and second axes of the discriminant function. Then, in order to examine the characteristics of the discriminant functions in the MES and UCEIS classifications, a comparison of the canonical discriminant functions between the MES and UCEIS classifications was performed between groups. As a result, a scatter plot by LCI classification was drawn, as shown in Figure 1. Since patients in remission classified as LCI-A do not show relapse of ulcerative colitis, the center of gravity of the plot of patients in remission classified as LCI-A in the scatter plot (LCI-A center of gravity) can be a criterion for determining relapse in ulcerative colitis patients. Therefore, first, among the plots obtained from the three samples of the sigmoid colon and rectum of each ulcerative colitis patient, the plot farthest from the LCI-A centroid was treated as the plot representing that patient. Then, the distance between the plot representing each patient and the LCI-A centroid was calculated. As a result, it was found that the relapse of ulcerative colitis was significantly higher in cases having plots far from the LCI-A centroid, and it was found that the relapse of ulcerative colitis can be predicted by calculating the distance between the plot representing the ulcerative colitis patient and the LCI-A centroid (Figure 2).

[0067] Function Expression Function 1: Function 1 = (0.1672*ln_TNFα_*10^3) + (0.7617*ln_IL-10_*10^3) + (0.8036*ln_IL-6_*10^3) + (-0.1215*ln_IFNγ_*10^3) + (-0.0554*ln_IL-1β_*10^3) + (0.1647*ln_IL-8_*10^3) + (-0.7998*ln_IL23_*10^3) + (0.5548*ln_IL12_*10^3) + (-1.3550*ln_IL17F_*10^3) + (-1.1933*ln_IL2_*10^3) + (0.1310*ln_IL5_*10^3) + (-0.8450*ln_IL13_*10^3) + (0.4299*ln_IL15_*10^3) + (0.7382*ln_IL18_*10^3) + (0.1920*ln_IL21_*10^3) + (-0.1565*ln_IL4_*10^3) + (-0.1200*ln_IL27_*10^3) + (0.0602*ln_IL33_*10^3) + (0.0329*ln_IL9_*10^3) + (-0.1109*ln_IL22_*10^3) + (1.0076*ln_IL17A_*10^3) -1.5295 Function 2: Function 2 = (0.3834*ln_TNFα_*10^3) ​​+ (-0.1385*ln_IL-10_*10^3) ​​+ (0.1570*ln_IL-6_*10^3) ​​+ (-0.2092*ln_IFNγ_*10^3) ​​+ (0.5000*ln_IL-1β_*10^3) ​​+ (0.1547*ln_IL-8_*10^3) ​​+ (0.6801*ln_IL23_*10^3) ​​+ (0.3162*ln_IL12_*10^3) ​​+ (0.7492*ln_IL17F_*10^3) ​​+ (-0.0659*ln_IL2_*10^3) ​​+ (-0.5527*ln_IL5_*10^3) ​​+ (-0.3090*ln_IL13_*10^3) ​​+ (-0.2303*ln_IL15_*10^3) ​​+ (0.2235*ln_IL18_*10^3) ​​+ (0.1802*ln_IL21_*10^3) ​​+ (0.5542*ln_IL4_*10^3) ​​+ (-0.3643*ln_IL27_*10^3) ​​+ (1.0020*ln_IL33_*10^3) ​​+ (0.0515*ln_IL9_*10^3) ​​+ (-0.5224*ln_IL22_*10^3) ​​+ (-1.2609*ln_IL17A_*10^3) -2.4371 *ln: logarithmic transformation (natural logarithm) *Addition and subtraction of each variable: After logarithmic transformation, "-4" will be the lower limit when calculating the function.

[0068] The inventors also extracted the fluctuations of the 21 cytokines measured, and selected cytokines with large fluctuations for discriminant functions 1 and 2 (Figure 3). Note that discriminant function 2 did not show any correlation with the MES classification and UCEIS classification (Figure 4). In Figure 2, the inventors extracted IL-10, IL-6, IL-2, IL-13, and IL-17A as the top five factors significantly correlated with the LCI classification, MES classification, and UCEIS classification in discriminant function 1, and IL-1β, IL-4, IL-17A, IL-22, and IL-33 significantly correlated with the LCI classification in discriminant function 2. Discriminant equations were reconstructed to determine whether the nine extracted cytokines above can be used as factors for predicting the prognosis of ulcerative colitis.

[0069] Calculation of discriminant functions for LCI classification by cytokine parameters using discriminant analysis in multidimensional scaling (2) The nine cytokine values ​​extracted above were logarithmically transformed, and discriminant analysis was performed using an analysis model with the LCI classification as a grouping variable and the cytokine value as an independent variable. The canonical discriminant function coefficients were calculated using the following formula, and discriminant function 1, which correlates with the LCI classification, MES classification, and UCEIS classification, and discriminant function 2, which correlates only with the LCI classification, were constructed. The centers of gravity of each LCI classification were calculated, and the first and second axes of the discriminant functions were plotted. Then, in order to examine the characteristics of the discriminant functions in the MES and UCEIS classifications, a comparison of the canonical discriminant functions between the MES and UCEIS classifications was performed between groups. As a result, a scatter plot based on the LCI classification was drawn, as shown in Figure 5. As in the case of using 21 cytokine values, the distance between the plots showing each patient and the LCI-A center of gravity was calculated. As a result, even when only the values ​​of the above nine cytokines were used, it was found that the incidence of relapse of ulcerative colitis was significantly higher in cases with plots far from the LCI-A centroid, and it was found that the relapse of ulcerative colitis can be predicted by calculating the distance between the plot representing ulcerative colitis patients and the LCI-A centroid (Figure 6).

[0070] Function Expression Function 1: Function 1 = (0.7617*ln_IL-10_*10^3) ​​+ (0.8036*ln_IL-6_*10^3) ​​+ (-1.1933*ln_IL2_*10^3) ​​+ (-0.8450*ln_IL13_*10^3) ​​+ (1.0076*ln_IL17A_*10^3) Function 2: Function 2 = (0.5000*ln_IL-1β_*10^3) ​​+ (0.5542*ln_IL4_*10^3) ​​+ (1.0020*ln_IL33_*10^3) ​​+ (-0.5224*ln_IL22_*10^3) ​​+ (-1.2609*ln_IL17A_*10^3) *ln: logarithmic transformation (natural logarithm) *Addition and subtraction of each variable: After logarithmic transformation, "-4" will be the lower limit when calculating the function.

[0071] In 2017, the inventors reported that a new endoscopic image enhancement system (Linked Color Imaging: LCI) was useful for predicting the prognosis of ulcerative colitis. Among the patients collected in this example, those in remission classified as LCI-A did not relapse (relapse 0% (0 / 12)), while those in remission classified as LCI-C had a high probability of relapse (relapse 71.4% (20 / 28)). However, it was difficult to predict relapse in patients in remission classified as LCI-B (relapse 42.9% (9 / 21)). As mentioned above, it was found that the relapse of ulcerative colitis was significantly higher in cases far from the LCI-A center of gravity. Therefore, using the statistical analysis software JMP Pro14.0.0, ROC curves (Figures 7 and 8) were created from logistic regression analysis for the distance from the LCI-A center of gravity of each patient in remission, and the cutoff value, AUC, sensitivity, and specificity for relapse and non-relapse of ulcerative colitis were calculated. As a result, when 21 cytokine values ​​were used, the cutoff value was calculated as 1.72, and when 9 cytokine values ​​were used, the cutoff value was calculated as 4.87. Here, we verified whether the above cutoff values ​​could be applied to predict relapse in patients in remission classified as LCI-B. Based on the above two types of cutoff values, patients in remission classified as LCI-B were divided into two groups, and the association with the presence or absence of relapse of ulcerative colitis was verified. The results are shown in Tables 2 and 3.

[0072] [Table 2]

[0073] [Table 3]

[0074] For patients in remission classified as LCI-B, relapse prediction using 21 cytokines with a cutoff value of 1.72 and relapse prediction using 9 cytokines with a cutoff value of 4.87 were considered to have similar predictive ability. [Industrial Applicability]

[0075] By employing the testing method of the present invention, it is possible to easily and quickly predict the prognosis of a patient with ulcerative colitis in remission without requiring expensive dedicated medical equipment.

Claims

1. A method for predicting the prognosis of a subject with ulcerative colitis in remission, comprising the steps of: (1) quantifying the following (a) to (i) in colon-derived tissue isolated from a subject with ulcerative colitis in remission: (a) IL-1β transcript; (b) IL-4 transcript; (c) IL-17A transcript; (d) IL-22 transcript; (e) IL-33 transcript; (f) IL-10 transcript; (g) IL-6 transcript; (h) IL-2 transcript, and (i) IL-13 transcript; (2) calculating coordinates (values ​​of discriminant function 1 and discriminant function 2) on a two-dimensional plane from (a) to (i) using discriminant function 1 correlated with LCI, MES, and UCEIS and discriminant function 2 correlated with LCI, which have been derived in advance; (3) calculating the linear distance between the coordinates of the center of gravity calculated in advance on a two-dimensional plane of the patient with ulcerative colitis in remission corresponding to LCI-A and the coordinates calculated in step (2); (4) comparing the linear distance to a pre-calculated cutoff value; wherein the discriminant function 1 and the discriminant function 2 are derived from the (a) to (i) derived from ulcerative colitis patients in remission, the discriminant function 1 includes an IL-10 transcript amount, an IL-6 transcript amount, an IL-2 transcript amount, an IL-13 transcript amount, and an IL-17A transcript amount as independent variables, and includes a coefficient correlating with LCI, MES, and UCEIS as a coefficient of each of the independent variables; The discriminant function 2 includes an IL-1β transcript amount, an IL-4 transcript amount, an IL-17A transcript amount, an IL-22 transcript amount, and an IL-33 transcript amount as independent variables, and includes a coefficient correlating with LCI as a coefficient for each of the independent variables.

2. The method of claim 1, wherein the pre-calculated cutoff value is 4.

87.

3. The method according to claim 1, further comprising quantifying the following (j) to (u) in step (1): (j) TNF-α transcript; (k) IFN-γ transcript; (l) IL-8 transcript; (m) IL-23 transcript; (n) an IL-12 transcript; (o) IL-17F transcript; (p) IL-5 transcript, (q) IL-15 transcript; (r) IL-18 transcript; (s) an IL-21 transcript; (t) IL-27 transcript, and (u) IL-9 transcript; wherein the discriminant function 1 includes, as independent variables, all of an IL-10 transcript amount, an IL-6 transcript amount, an IL-2 transcript amount, an IL-13 transcript amount, and an IL-17A transcript amount, and an amount of at least one transcript selected from the group consisting of an IL-1β transcript, an IL-4 transcript, an IL-22 transcript, an IL-33 transcript, a TNF-α transcript, an IFN-γ transcript, an IL-8 transcript, an IL-23 transcript, an IL-12 transcript, an IL-17F transcript, an IL-5 transcript, an IL-15 transcript, an IL-18 transcript, an IL-21 transcript, an IL-27 transcript, and an IL-9 transcript, and includes, as coefficients of each independent variable, coefficients correlating with LCI, MES, and UCEIS; The discriminant function 2 includes, as independent variables, all of an IL-1β transcript, an IL-4 transcript amount, an IL-17A transcript amount, an IL-22 transcript amount, and an IL-33 transcript amount, and an amount of at least one transcript selected from the group consisting of a TNF-α transcript, an IFN-γ transcript, an IL-8 transcript, an IL-23 transcript, an IL-12 transcript, an IL-17F transcript, an IL-5 transcript, an IL-15 transcript, an IL-18 transcript, an IL-21 transcript, an IL-27 transcript, an IL-9 transcript, an IL-10 transcript, an IL-6 transcript, an IL-2 transcript, and an IL-13 transcript, and includes, as a coefficient of each independent variable, a coefficient correlating with LCI.

4. 4. The inspection method according to claim 3, wherein in the step (2), coordinates on a two-dimensional plane (values ​​of discriminant function 1 and discriminant function 2) are calculated from said (a) to (u).

5. The method of claim 4, wherein the pre-calculated cutoff value is 1.

72.

6. A method for predicting the prognosis of a subject with ulcerative colitis in remission, comprising the steps of: (1) quantifying the following (a) to (i) in colon-derived tissues isolated from ulcerative colitis patients in remission and subjects with ulcerative colitis in remission: (a) IL-1β transcript; (b) IL-4 transcript; (c) IL-17A transcript; (d) IL-22 transcript; (e) IL-33 transcript; (f) IL-10 transcript; (g) IL-6 transcript; (h) IL-2 transcript, and (i) IL-13 transcript; (2) deriving the following two discriminant functions from the (a) to (i) derived from ulcerative colitis patients in a state of remission: (i) a discriminant function 1 including, as independent variables, the amount of IL-10 transcript, the amount of IL-6 transcript, the amount of IL-2 transcript, the amount of IL-13 transcript, and the amount of IL-17A transcript, and including, as coefficients of each of the independent variables, coefficients correlating with LCI, MES, and UCEIS; and (ii) a discriminant function 2 including an IL-1β transcript amount, an IL-4 transcript amount, an IL-17A transcript amount, an IL-22 transcript amount, and an IL-33 transcript amount as independent variables, and including a coefficient correlating with LCI as a coefficient for each of the independent variables; (3) calculating coordinates (values ​​of discriminant function 1 and discriminant function 2) on a two-dimensional plane from the (a) to (i) derived from ulcerative colitis patients in a state of remission corresponding to LCI-A using the discriminant functions derived in the step (2), and calculating the coordinates of the center of gravity; (4) calculating coordinates (values ​​of discriminant function 1 and discriminant function 2) on a two-dimensional plane from the (a) to (i) derived from a subject with ulcerative colitis in remission using the discriminant functions derived in the step (2); (5) calculating a straight-line distance between the center of gravity coordinates calculated in step (3) and the coordinates calculated in step (4); (6) Comparing the linear distance to a pre-calculated cutoff value.

7. The method according to claim 6, further comprising quantifying at least one selected from the group consisting of the following (j) to (u) in the step (1): (j) TNF-α transcript; (k) IFN-γ transcript; (l) IL-8 transcript; (m) IL-23 transcript; (n) an IL-12 transcript; (o) IL-17F transcript; (p) IL-5 transcript, (q) IL-15 transcript; (r) IL-18 transcript; (s) an IL-21 transcript; (t) IL-27 transcript, and (u) IL-9 transcript.

8. In step (2), (i) discriminant function 1 includes, as independent variables, all of an IL-10 transcript amount, an IL-6 transcript amount, an IL-2 transcript amount, an IL-13 transcript amount, and an IL-17A transcript amount, and an amount of at least one transcript selected from the group consisting of an IL-1β transcript, an IL-4 transcript, an IL-22 transcript, an IL-33 transcript, a TNF-α transcript, an IFN-γ transcript, an IL-8 transcript, an IL-23 transcript, an IL-12 transcript, an IL-17F transcript, an IL-5 transcript, an IL-15 transcript, an IL-18 transcript, an IL-21 transcript, an IL-27 transcript, and an IL-9 transcript, and includes, as coefficients of each independent variable, coefficients correlating with LCI, MES, and UCEIS; (ii) discriminant function 2 includes, as independent variables, all of an IL-1β transcript, an IL-4 transcript amount, an IL-17A transcript amount, an IL-22 transcript amount, and an IL-33 transcript amount, and an amount of at least one transcript selected from the group consisting of a TNF-α transcript, an IFN-γ transcript, an IL-8 transcript, an IL-23 transcript, an IL-12 transcript, an IL-17F transcript, an IL-5 transcript, an IL-15 transcript, an IL-18 transcript, an IL-21 transcript, an IL-27 transcript, an IL-9 transcript, an IL-10 transcript, an IL-6 transcript, an IL-2 transcript, and an IL-13 transcript, and includes, as a coefficient of each independent variable, a coefficient correlating with LCI. The inspection method according to claim 7.

9. The method according to claim 8, wherein in the step (3), coordinates on a two-dimensional plane (the value of discriminant function 1, the value of discriminant function 2) are calculated from at least one selected from the group consisting of all of (a) to (i) and (j) to (u) derived from a patient with ulcerative colitis in remission who falls under LCI-A, and the centroid coordinates are calculated.

10. The method according to claim 9, wherein in step (4), coordinates on a two-dimensional plane (the value of discriminant function 1, the value of discriminant function 2) are calculated from all of (a) to (i) and at least one selected from the group consisting of (j) to (u) derived from a subject with ulcerative colitis in remission.

11. The examination method according to any one of claims 1 to 10, wherein the large intestine-derived tissue is the cecum, colon or rectum.

12. A test agent for predicting the prognosis of a subject with ulcerative colitis in remission, comprising the following (a) to (i), wherein the test using the test agent is carried out by the method according to claim 1 or 6: (a) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-1β transcript; (b) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-4 transcript; (c) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-17A transcript; (d) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-22 transcript; (e) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-33 transcription product; (f) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-10 transcript; (g) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-6 transcript; (h) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-2 transcript, and (i) A nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-13 transcript.

13. The test agent according to claim 12, further comprising at least one selected from the group consisting of the following (j) to (u), wherein the test using the test agent is carried out by the method according to claim 3 or 7: (j) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting a TNF-α transcript; (k) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IFN-γ transcript; (l) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-8 transcript; (m) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-23 transcript; (n) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-12 transcript; (o) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-17F transcript; (p) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-5 transcript; (q) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-15 transcript; (r) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-18 transcript; (s) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-21 transcript; (t) a nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-27 transcript, and (u) A nucleic acid probe or a nucleic acid primer set capable of specifically detecting an IL-9 transcript.

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