WO2022202099A1 - Method for testing for inflammatory bowel disease - Google Patents
Method for testing for inflammatory bowel disease Download PDFInfo
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- WO2022202099A1 WO2022202099A1 PCT/JP2022/007951 JP2022007951W WO2022202099A1 WO 2022202099 A1 WO2022202099 A1 WO 2022202099A1 JP 2022007951 W JP2022007951 W JP 2022007951W WO 2022202099 A1 WO2022202099 A1 WO 2022202099A1
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- WIPO (PCT)
- Prior art keywords
- phospholipase
- measuring
- inflammatory bowel
- bowel disease
- intestinal
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- 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
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- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- 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
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- 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
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Definitions
- the present invention relates to the testing and treatment of inflammatory bowel disease.
- IBD Inflammatory bowel disease
- the gut microbiota plays an important role in its host's immune system. Dysbiosis, characterized by decreased diversity and altered composition of the microbiota, is observed in the intestinal microbiota of IBD patients (Non-Patent Document 1). Microbiota digest dietary fiber to produce short chain fatty acids (SCFA). SCFA not only serve as an energy source for the microbiota itself and host intestinal epithelial cells, but also contribute to host intestinal homeostasis by inducing immune cells such as T cells (Non-Patent Document 2). .
- SCFA short chain fatty acids
- Non-Patent Document 3 PS is abundantly present in the lipid bilayer of the cell membrane, and LysoPS is thought to be generated from PS by the action of the enzyme phospholipase A1/A2 (PLA1/A2) and act as a lipid mediator (Non-Patent Document 4).
- PPA1/A2 phospholipase A1/A2
- Non-Patent Document 4 the physiological and pathological functions of LysoPS are unknown.
- the present invention provides a new test method for inflammatory bowel disease, provides a method for screening substances and microorganisms for preventing or treating inflammatory bowel disease, and provides a method for preventing or treating inflammatory bowel disease.
- An object is to provide a composition.
- the present inventors found that lysophosphatidylserine is produced by certain intestinal microorganisms, and that lysophosphatidylserine produced by intestinal microorganisms exacerbates the disease.
- the present invention was completed by discovering that it is directly involved in That is, the present invention includes the following aspects. 1. At least one selected from the group consisting of lysophosphatidylserine, an intestinal microorganism involved in the production of lysophosphatidylserine, a gene encoding phospholipase A derived from intestinal microorganisms, and phospholipase A in a sample obtained from a subject.
- a method of testing for inflammatory bowel disease comprising the step of measuring. 2.
- the intestinal microorganism is an intestinal microorganism that expresses phospholipase A.
- the intestinal microorganism expressing phospholipase A is a microorganism having a gene ECSF — 3660 encoding phospholipase A. 5.
- the method according to the preceding item 1 comprising the step of measuring the concentration of lysophosphatidylserine in a sample obtained from a subject. 10. 10. The method according to item 9, further comprising the step of measuring the concentration of lysophosphatidylcholine. 11. 2. The method according to the preceding item 1, comprising the step of measuring phospholipase A in a sample obtained from a subject. 12. 12. The method according to item 11, wherein the phospholipase A is an intestinal microorganism-derived phospholipase A. 13. 13. The method according to item 12, wherein the intestinal microorganism-derived phospholipase A is phospholipase A encoded by ECSF_3660. 14. 14.
- the step of measuring phospholipase A is a step of measuring the amount of phospholipase A protein or a step of measuring phospholipase A activity.
- the sample is a stool sample.
- a method for screening substances or microorganisms for the prevention or treatment of inflammatory bowel disease comprising the step of measuring the number of intestinal microorganisms involved in lysophosphatidylserine production.
- a method of screening for a substance for prevention or treatment of inflammatory bowel disease comprising the step of measuring phospholipase A activity. 18.
- a composition for preventing or treating inflammatory bowel disease containing a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production. 19.
- a composition for prevention or treatment of inflammatory bowel disease containing a substance that suppresses the activity of phospholipase A.
- 20. 16 The method according to any one of the preceding items 1 to 15, further comprising the step of comparing the test value of the sample obtained from the subject with the test value of the control sample. 21. 21. The method according to item 20, wherein the control sample is a sample obtained from a healthy subject or a sample obtained from the same subject. 22.
- a method for preventing or treating inflammatory bowel disease comprising administering a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production.
- 23. A method for preventing or treating inflammatory bowel disease, comprising administering a substance that suppresses the activity of phospholipase A derived from intestinal microorganisms.
- 24. Use of a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production for the preparation of a composition for the prevention or treatment of inflammatory bowel disease.
- 25. Use of a substance that inhibits the activity of intestinal microorganism-derived phospholipase A for the preparation of a composition for the prevention or treatment of inflammatory bowel disease.
- the method of the present invention can examine intestinal microbial abnormalities associated with inflammatory bowel disease.
- Microorganisms expressing phospholipase A increase due to intestinal microbial dysbiosis, and lysophosphatidylserine (hereinafter also referred to as LysoPS) increases in the intestine. LysoPS is directly involved in the development and exacerbation of inflammatory bowel disease.
- the method of the present invention measures lysophosphatidylserine in a sample, intestinal microorganisms involved in the production of lysophosphatidylserine, or phospholipase A, which is an enzyme that produces lysophosphatidylserine, or its gene. can be inspected.
- the method of the present invention can be used for diagnosing inflammatory bowel disease, testing for determining high-risk individuals with pre-onset inflammatory bowel disease, testing for follow-up of inflammatory bowel disease patients, and the like. It can be used for all examinations related to enteropathy.
- the method of the present invention can anticipate changes in the condition of inflammatory bowel disease patients or high-risk individuals.
- the method of the present invention is a simple examination method that imposes little burden on the patient.
- the screening method of the present invention involves screening for substances or microorganisms that eliminate or reduce lysophosphatidylserine that causes inflammatory bowel disease, phospholipase A that produces it, or intestinal microorganisms involved in lysophosphatidylserine production. It is possible to provide a therapeutic drug, a preventive drug, and a therapeutic or preventive food for inflammatory bowel disease.
- the composition of the present invention eliminates or reduces lysophosphatidylserine that causes inflammatory bowel disease, phospholipase A that produces it, or intestinal microorganisms involved in lysophosphatidylserine production, thereby reducing the development of inflammatory bowel disease. and prevent relapses, enabling radical treatment of inflammatory bowel disease.
- the composition of the present invention suppresses the production of lysophosphatidylserine, which causes inflammatory bowel disease, prevents the onset and relapse of inflammatory bowel disease, and enables radical treatment of inflammatory bowel disease.
- composition of the present invention is a causative therapy for inflammatory bowel disease, it has excellent preventive and therapeutic effects, and will greatly contribute to the treatment and prevention of recurrence of many patients with inflammatory bowel disease, or the onset of those at risk of inflammatory bowel disease. It is something to do.
- FIG. 4 shows the ROC analysis of the results of FIG. FIG.
- FIG. 5 is a graph showing the results of genome information analysis (metagenomic analysis) of all microorganisms in a fecal sample and showing the Shannon index representing ⁇ -diversity (species diversity) of intestinal microflora.
- FIG. 6 shows the results of genome information analysis (metagenomic analysis) of all microorganisms in fecal samples, showing the results of two-dimensional analysis using the Bray-Curtis index representing ⁇ diversity.
- FIG. 7 is the result of genome information analysis (metagenomic analysis) of all microorganisms in a stool sample, and is a graph showing the FPKM of gene ECSF_3660.
- Figure 8 shows E. coli counts in fecal samples used for the analysis of Figures 1-4. Measured by quantitative PCR.
- FIG. 9 shows lysophospholipid total rank order scores (y-axis) versus E. coli counts (x-axis). Data were analyzed by simple linear regression.
- FIG. 10 shows the full-length ECSF — 3660 band (amplified by PCR) in fecal samples from healthy subjects (HC) and CD patients (CD). *: non-specific band, M: marker
- FIG. 12 shows the full-length ECSF — 3660 band (amplified by PCR) in mouse feces 10 days after transplantation of the gut microbiota of healthy and CD patients into mice. *: non-specific band, M: marker FIG.
- FIG. 13 shows 18:0 LysoPS, 18:1 LysoPS, and total LysoPS concentrations in feces of mice 24 days after transplantation of the gut microbiota of healthy and CD patients.
- Each group n 4; * p ⁇ 0.05, *** p ⁇ 0.005
- FIG. 14 is a graph showing changes in body weight when LysoPS or vehicle was intraperitoneally administered to TNBS-induced colitis model mice for 4 days.
- n 9, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, Student's t-test FIG.
- FIG. 16 shows a histopathological image (H&E staining) (A) and a graph of histological scores (B) of the colon after intraperitoneal administration of LysoPS or vehicle for 4 days to TNBS-induced colitis model mice.
- n 6, ** p ⁇ 0.01, Student's t-test FIG.
- FIG. 17 is a graph showing relative cytokine expression levels (cytokine mRNA expression levels in colonic lamina intestinal lymphocytes) after intraperitoneal administration of LysoPS or vehicle to TNBS-induced colitis model mice for 4 days.
- n 9, n.s.: not significant, * p ⁇ 0.05, Student's t-test
- FIG. 18 is a graph showing changes in body weight when LysoPS or vehicle was intraperitoneally administered to T cell-dependent colitis model mice for 4 days.
- n 4 - 5, * p ⁇ 0.05, Student's t-test FIG.
- FIG. 19 is a photograph (A) and a graph (B) showing colon length after intraperitoneal administration of LysoPS or vehicle to T cell-dependent colitis model mice for 4 days.
- n 4 - 5, ** p ⁇ 0.01, Student's t-test
- FIG. 20 shows a histopathological image (H&E staining) (A) and a graph of histological scores (B) of the colon after intraperitoneal administration of LysoPS or vehicle for 4 days to T cell-dependent colitis model mice.
- n 4 - 5, **** p ⁇ 0.0001, Student's t-test FIG.
- IFN- ⁇ + cells shows IFN- ⁇ + cells, IFN- ⁇ + IL-17A + cells, and IFN- ⁇ + IL-17A + cells in colonic lamina intestinal lymphocytes after intraperitoneal administration of LysoPS or vehicle to T cell-dependent colitis model mice for 4 days.
- Graph showing numbers of IL-10 + cells, IL-17A + cells.
- n 4 - 5, n.s.: not significant, * p ⁇ 0.05, Student's t-test
- the intestinal microorganism involved in lysophosphatidylserine production is not particularly limited as long as it is an intestinal microorganism involved in lysophosphatidylserine production.
- Lysophosphatidylserine (LysoPS) is a phospholipid and has a structure in which an acyl group, a phosphate group, and serine are bound to a glycerol skeleton.
- the fatty acid species of the acyl group is diverse, and there are two types of bonding positions, sn-1 position (1-acyl type) and sn-2 position (2-acyl type) of the glycerol skeleton.
- phosphatidylserine (PS) forming the lipid bilayer of the cell membrane is acted upon by phospholipase A1 (PLA1) or phospholipase A2 (PLA2) to produce LysoPS.
- PKA1 phospholipase A1
- PLA2 phospholipase A2
- LysoPS overactivates Th1 cells to develop or exacerbate symptoms of inflammatory bowel disease.
- the enteric microorganism involved in said LysoPS production is preferably an enteric microorganism expressing phospholipase A.
- intestinal microorganisms expressing phospholipase A include intestinal microorganisms expressing phospholipase A and microorganisms having a phospholipase gene and capable of expressing phospholipase A.
- Gut microbes that express phospholipases disrupt the cell membranes of host intestinal epithelial cells to produce LysoPS.
- the enteric microorganism expressing phospholipase A is preferably a microorganism having the gene ECSF_3660 encoding phospholipase A.
- the enteric microorganism expressing phospholipase A is preferably a bacterium, more preferably a Gram-negative bacterium, such as E. coli, Yersinia and Serratia. Escherichia coli is more preferred, and E. coli having a gene ECSF — 3660 encoding phospholipase A is even more preferred.
- the method for measuring the number of intestinal microorganisms involved in the production of lysophosphatidylserine in the present invention is not particularly limited as long as the amount of the intestinal microorganisms can be measured. Examples thereof include a method of measuring the amount of the intestinal microorganism-derived gene, a method of measuring the phospholipase A expressed by the intestinal microorganism, a method of counting the number of colonies after culturing the intestinal microorganism, and the like.
- a suitable method for measuring the amount of the intestinal microorganism-derived gene includes a method of measuring the amount of the intestinal microorganism-derived gene by PCR, for example, a quantitative real-time PCR method.
- the number of intestinal microorganisms can be determined from the gene content by a known method (for example, the method described in Petr Kralik et al. Front Microbiol 2017 Feb 2;8:108).
- the intestinal microorganism-derived gene is not particularly limited, and may be a gene commonly used for detecting target microorganisms.
- a gene commonly used for detecting target microorganisms For example, in the case of Escherichia coli, quantitative PCR can be performed using primers commonly used for quantification of Escherichia coli.
- a gene encoding phospholipase A is preferable.
- genes encoding phospholipase A include ECSF — 3660.
- ECSF — 3660 is a gene (SEQ ID NO: 1, SEQ ID NO: 2) encoding phospholipase A derived from Escherichia coli and other bacteria (such as Klebsiella pneumoniae).
- Methods for measuring the amount of ECSF_3660 include, for example, a quantitative real-time PCR method.
- One of the preferred embodiments of the method of the present invention is a method of testing for inflammatory bowel disease, comprising the step of measuring the number of E. coli expressing phospholipase A in a sample obtained from a subject, and more preferably, A method for testing inflammatory bowel disease, comprising the step of quantifying the gene ECSF — 3660 by quantitative real-time PCR and measuring the number of E. coli.
- the number of intestinal microorganisms involved in lysophosphatidylserine production can be determined by measuring phospholipase A expressed by the intestinal microorganisms. Examples include methods for measuring phospholipase A present on the surface of microorganisms or within microorganisms. Since phospholipase A is present in the outer membrane of Gram-negative bacteria, a method of measuring phospholipase present in the outer membrane of Gram-negative bacteria can be used. Examples of the intestinal microorganism-derived phospholipase A include phospholipase A encoded by ECSF — 3660.
- One preferred embodiment of the method of the present invention comprises the step of targeting phospholipase A expressed in the outer membrane of Gram-negative bacteria to measure the number of intestinal microorganisms involved in lysophosphatidylserine production. It is a method of examining a disease.
- Phospholipase A preferably includes phospholipase A encoded by ECSF_3660.
- Methods for measuring phospholipase A include a method for measuring the amount of phospholipase A protein and a method for measuring phospholipase A activity.
- the method for measuring the amount of phospholipase A protein is not limited, and for example, a method using an antibody against phospholipase A can be used.
- a preferred measurement method includes an antibody against phospholipase A encoded by ECSF — 3660. Examples of measurement methods using antibodies include ELISA, immunochromatography, latex agglutination, and the like.
- the method for measuring the activity of phospholipase A is not limited. Examples include a colorimetric method for detecting enzyme activity and a method using a fluorogenic lipid as a substrate (David Gonzalez-Bullon et al. Toxins 2018).
- the method of testing for inflammatory bowel disease of the present invention can also be a method comprising the step of measuring the amount of a gene encoding phospholipase A derived from intestinal microorganisms in a sample obtained from a subject.
- the gene encoding the gut microbe-derived phospholipase A is involved in the production of LysoPS.
- Methods for measuring the amount of the gene encoding phospholipase A include, for example, quantitative real-time PCR.
- a preferred gene encoding phospholipase A derived from intestinal microorganisms is ECSF_3660.
- Primers can be designed from the ECSF — 3660 DNA sequence (SEQ ID NO: 1) to perform quantitative real-time PCR. An example of primers is shown below.
- Fw1 5′-ATGCGGACTCTGCAGGGCTGGTTGTTGCCG-3′ (SEQ ID NO: 3)
- Rv1 5′-TCAAAACAGGTCGTTTAGCATAACTCCCAC-3′ (SEQ ID NO: 4)
- One preferred embodiment of the method of the present invention is a method of testing for inflammatory bowel disease comprising measuring the amount of ECSF — 3660 in a sample obtained from a subject.
- the method of testing for inflammatory bowel disease of the present invention can also be a method comprising the step of measuring the concentration of LysoPS in a sample obtained from a subject.
- LysoPS there are 10 or more types of acyl groups constituting LysoPS.
- LysoPS to be measured in the present invention is not limited to the type of acyl group, but is preferably an acyl group having 16 to 22 carbon atoms, more preferably an acyl group having 18 to 20 carbon atoms. and more preferably an acyl group having 18 carbon atoms. Most preferred are 18:0 LysoPS and 18:1 LysoPS. 18:0 indicates stearic acid and 18:1 indicates oleic acid.
- the method of testing for inflammatory bowel disease of the present invention measures at least one LysoPS.
- at least 18:1 LysoPS is measured.
- at least 18:1 LysoPS and 18:0 LysoPS are measured.
- the method for measuring the concentration of LysoPS is not limited. Examples thereof include a measurement method using a combination of HPLC and mass spectrometry, an enzymatic assay method, and the like.
- a preferred measurement method is LC/ESI-MS.
- the method of the invention may comprise measuring at least one other lysophospholipid in addition to measuring LysoPS.
- Other lysophospholipids preferably include lysophosphatidylcholine (hereinafter also referred to as LysoPC).
- LysoPC lysophosphatidylcholine
- Preferred lysophosphatidylcholines include 18:0 LysoPC, 18:1 LysoPC, and 22:1 LysoPC.
- One embodiment includes a mode in which a plurality of lysophospholipids in a sample are measured and the test results are integrated for diagnosis and the like.
- the measurement results of multiple lysophospholipids in a sample can be combined to form a test value.
- fecal concentrations of all or part of 18:0LysoPS, 18:1LysoPS, 18:0LysoPC, 18:1LysoPC, and 22:1LysoPC can be scored and the sum thereof can be used as the test value.
- each concentration of all five lysophospholipids is scored and the total value is used as the test value.
- the method of testing for inflammatory bowel disease of the present invention can also be a method comprising the step of measuring phospholipase A in a sample obtained from a subject.
- the phospholipase A is preferably an intestinal microorganism-derived phospholipase A.
- the phospholipase A expressed by the intestinal microorganism destroys the cell membrane of the host intestinal epithelial cells to produce LysoPS.
- Phospholipase A derived from intestinal microorganisms preferably includes phospholipase A encoded by ECSF_3660.
- ECSF — 3660 is a gene (SEQ ID NO: 1, SEQ ID NO: 2) encoding phospholipase A derived from Escherichia coli and other microorganisms (such as Klebsiella pneumoniae).
- Phospholipase A can be present on the surface of microorganisms, inside microorganisms, and outside microorganisms, and measurement targets include phospholipase A present on the surface of microorganisms, phospholipase A present outside microorganisms, and phospholipase A present inside microorganisms. .
- a method capable of measuring the total amount of expressed phospholipase A, or a method capable of specifying the location thereof may be used. Since phospholipase A is present in the outer membrane of Gram-negative bacteria, a method of measuring phospholipase A present in the outer membrane of Gram-negative bacteria can be used.
- One preferred embodiment of the method of the present invention is a method of testing for inflammatory bowel disease comprising measuring phospholipase A expressed in the outer membrane of Gram-negative bacteria.
- Phospholipase A preferably includes phospholipase A encoded by ECSF_3660.
- Methods for measuring phospholipase A include a method for measuring the amount of phospholipase A protein and a method for measuring phospholipase A activity.
- One of the preferred embodiments of the method of the present invention is a method of testing for inflammatory bowel disease, comprising the step of measuring the protein amount of intestinal microorganism-derived phospholipase A in a sample obtained from a subject. More preferably, it is a method of testing for inflammatory bowel disease, comprising the step of measuring the amount of phospholipase A protein encoded by ECSF — 3660.
- the method for measuring the protein amount of phospholipase A derived from intestinal microorganisms is not limited.
- a measuring method using an antibody against phospholipase A can be mentioned.
- a preferred measurement method includes an antibody against phospholipase A encoded by ECSF — 3660. Examples of the measuring method include ELISA method, immunochromatographic method, latex agglutination method and the like.
- One of the preferred embodiments of the method of the present invention is a method of testing for inflammatory bowel disease comprising the step of measuring the activity of phospholipase A derived from intestinal microorganisms in a sample obtained from a subject. More preferably, it is a method of testing for inflammatory bowel disease, comprising the step of measuring the activity of phospholipase A encoded by gene ECSF_3660.
- the method for measuring the activity of phospholipase A is not limited. Examples include a colorimetric method for detecting enzyme activity and a method using a fluorogenic lipid as a substrate (David Gonzalez-Bullon et al. Toxins 2018).
- Subjects include humans and non-human animals.
- Animals other than humans are preferably mammals, such as pets such as dogs, cats, and hamsters, and domestic animals such as cattle. Humans are preferred.
- the sample obtained from the subject is preferably a sample containing intestinal microflora. More preferably, it is a stool sample.
- Inflammatory bowel disease is a disease that causes inflammation in the intestine, and in the present invention, inflammatory bowel disease includes Crohn's disease and ulcerative colitis, as well as intestinal Behcet's disease, non-specific multiple small intestinal ulcers, familial Mediterranean fever. Associated enteritis and the like are also included. Inflammatory bowel disease is characterized by symptoms such as chronic diarrhea, bloody stools, abdominal pain, and other symptoms such as fever and anal pain. Pathologic examination is characterized by noncaseating epithelioid granulomas in Crohn's disease and crypt abscesses in ulcerative colitis.
- the method of the present invention can be suitably used for examination of inflammatory bowel disease accompanied by excessive activation of Th1 cells.
- Hyperactivation of Th1 cells is characterized by increased secretion of inflammatory cytokines such as IFN- ⁇ from Th1 cells. Hyperactivation of Th1 cells includes increased secretion of inflammatory cytokines per cell, increased production of reactive oxygen species (ROS), increased number of inflammatory cytokine-producing Th1 cells, and differentiation of naive CD4 + T cells into Th1 cells. and promotion of Th1 cell metabolism (particularly promotion of glycolysis).
- ROS reactive oxygen species
- Both Crohn's disease and ulcerative colitis are intractable intestinal inflammatory diseases in which immune cells are involved, and the method of the present invention can be suitably used for examination of Crohn's disease and ulcerative colitis. Particularly preferably, the method of the present invention can be used to test for Crohn's disease.
- Tests in the method of testing for inflammatory bowel disease of the present invention include all tests related to inflammatory bowel disease, tests at the time of diagnosis of inflammatory bowel disease, tests for the risk of developing inflammatory bowel disease, and tests for the risk of developing inflammatory bowel disease. This includes examinations for follow-up after the onset of enteropathy.
- the method of the invention may further comprise the step in which the test value of the sample obtained from the subject and the test value of the control sample are compared.
- the test values measured by the same method for the same test object in the sample and the control sample are compared.
- the control sample is a sample obtained from a healthy subject
- the test value of the sample obtained from the subject is higher than the test value of the control sample, the subject has inflammatory bowel disease or inflammatory bowel It can be determined that the risk of developing the disease is high.
- the test values of samples obtained from healthy subjects include predetermined standard values for healthy subjects.
- test value in the sample obtained from the subject is higher than the test value of the control sample, the subject is worsening or worsening If the test value in the sample obtained from the subject is lower than the test value in the control sample, the subject has improved or may improve can be determined to be high.
- the method for measuring the number of intestinal microorganisms involved in LysoPS production in the screening method for substances or microorganisms for prevention or treatment of inflammatory bowel disease of the present invention is not particularly limited.
- a preferred method includes a method of measuring the number of intestinal microorganisms after contacting the culture solution of the intestinal microorganisms with the candidate substance or candidate microorganisms.
- Another example is a method in which a candidate substance or microorganism is orally ingested by humans or animals other than humans, and the number of intestinal microorganisms in a stool sample is measured. Techniques include quantitative real-time PCR, colony counting, and the like.
- the method of screening for a preventive or therapeutic substance for inflammatory bowel disease of the present invention can be a method comprising the step of measuring phospholipase A activity.
- said phospholipase A is an intestinal microbial phospholipase A.
- a preferred method for measuring the activity of phospholipase A includes a method of contacting phospholipase A with a candidate substance and then measuring phospholipase A activity.
- Said phospholipase A is preferably phospholipase A1.
- a compound library may be known or unknown.
- a compound library for example, PRESTWICK Chemical library (which is a collection of compounds whose patent period has expired), and a compound library which is a collection of compounds that have not yet been approved for food or pharmaceutical use.
- the screening method of the present invention may further comprise a step of selecting substances or microorganisms.
- Substances or microorganisms obtained by the screening method of the present invention can be used for prevention or treatment of inflammatory bowel disease.
- the preventive or therapeutic composition for inflammatory bowel disease of the present invention contains, as an active ingredient, a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production.
- the preventive or therapeutic composition for inflammatory bowel disease of the present invention contains a substance that suppresses the activity of phospholipase A as an active ingredient.
- the prophylactic or therapeutic compositions include drugs, foods, beverages and the like.
- the substance or microorganism that eliminates or reduces intestinal microorganisms involved in the production of lysophosphatidylserine can be the substance or microorganism obtained by the screening method.
- Such substances include, for example, antimicrobial agents and adsorbents.
- the microorganisms may include lactic acid bacteria, bifidobacteria, Xenorhabdus, Photorhabdus, and phages (eg, Enterobacteria Phage P1 and Enterobacteria phage ⁇ ) that infect target microorganisms.
- the substance that suppresses the activity of phospholipase A includes a substance that suppresses expression of phospholipase A and a substance that suppresses biosynthesis.
- a substance that suppresses the activity of phospholipase A can be a substance obtained by the screening method.
- the substance that suppresses the activity of phospholipase A is preferably a substance that suppresses the activity of phospholipase A derived from intestinal microorganisms.
- Examples of the substance that suppresses the activity of phospholipase A include phospholipase A inhibitors, antibodies against phospholipase A, antisense RNA of the phospholipase A gene, and bacteria that inhibit phospholipase A. ⁇ A ⁇ 4-bromophenacyl bromide ⁇ darapladib ⁇ Anagrelide ⁇ OBAA(3-(4-octadecyl)benzoylacrylic acid) ⁇ PACOCF3(palmitoyl trifluoromethyl ketone) ⁇ YM26734 ⁇ LY315920(varespladib methyl ).
- treatment of inflammatory bowel disease includes amelioration, alleviation, suppression of exacerbation, delay of progression, suppression of relapse, and the like of symptoms of inflammatory bowel disease.
- composition of the present invention can be suitably used for prevention and treatment of inflammatory bowel disease accompanied by overactivation of Th1 cells.
- LysoPS causes hyperactivation of Th1 cells via the LysoPS receptor, exacerbating the symptoms of inflammatory bowel disease.
- This hyperactivation of Th1 cells includes increased secretion of inflammatory cytokines per cell, increased number of inflammatory cytokine-producing Th1 cells, enhanced ROS production, enhanced differentiation of naive CD4 + T cells into Th1 cells, and Th1 Includes promotion of cell metabolism (particularly promotion of glycolysis).
- the composition of the present invention can suppress overactivation of Th1 cells, and can suitably prevent or treat inflammatory bowel disease accompanied by overactivation of Th1 cells.
- Both Crohn's disease and ulcerative colitis are intractable intestinal inflammatory diseases in which immune cells are involved, and the composition of the present invention can be suitably used for the prevention and treatment of Crohn's disease and ulcerative colitis. Particularly preferably, the compositions of the invention can be used for the prevention and treatment of Crohn's disease.
- composition of the present invention may contain two or more active ingredients.
- Other inflammatory bowel disease therapeutic agents may be contained, and drugs other than inflammatory bowel disease therapeutic agents may be contained.
- the composition of the present invention may be used in combination with other therapeutic agents for inflammatory bowel disease.
- compositions of the invention can include a pharmaceutically acceptable carrier.
- Such carriers include excipients (e.g., sugar derivatives such as mannitol and sorbitol; starch derivatives such as corn starch and potato starch; or cellulose derivatives such as crystalline cellulose), lubricants (e.g., stearic acid metal stearates such as magnesium; or talc, etc.), binders (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc.), disintegrants (e.g., carboxymethylcellulose, cellulose derivatives such as carboxymethylcellulose calcium, etc.), Water, preservatives (e.g.
- excipients e.g., sugar derivatives such as mannitol and sorbitol; starch derivatives such as corn starch and potato starch; or cellulose derivatives such as crystalline cellulose
- lubricants e.g., stearic acid metal stearates such as magnesium; or talc, etc.
- paraoxybenzoic acid esters such as methylparaben and propylparaben; or alcohols such as chlorobutanol and benzyl alcohol
- pH adjusters e.g. inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid, acetic acid , Organic acids such as succinic acid, fumaric acid or malic acid, or salts thereof
- commonly used carriers for pharmaceutical formulations such as diluents (e.g., water for injection, etc.), alone or in combination of two or more can be blended as
- the composition of the present invention contains a liquid formulation in which the active ingredient is dissolved in water.
- composition of the present invention is a food or beverage
- it may be in the form of being mixed with an existing food or beverage.
- the active ingredient of the present invention can be orally administered in dosage forms such as tablets, granules, capsules, powders, solution formulations, suspension formulations, or emulsified liquid formulations after mixing with the above-mentioned carriers, if necessary. can be done. It can also be administered parenterally in dosage forms such as suppositories, enteral agents, injections, intravenous drips, transdermal agents, transmucosal agents, or inhalants.
- the active ingredient of the present invention is administered to a subject in need thereof, such as a human or an animal, preferably a human, after being formulated into the above dosage form or mixed with food or beverage.
- the dose and frequency of administration of the active ingredient of the present invention can be appropriately changed depending on conditions such as severity of symptoms, patient's age, body weight, sex, type of drug, dosage form, administration route and the like.
- the active ingredient is administered parenterally, for example, subcutaneously, intravenously, intraperitoneally, intramuscularly, or intrarectally, at a dosage of about 0.01 to 10 mg/kg body weight, preferably about 0.01 to 10 mg/kg body weight per administration. about 0.1 to 5 mg/kg body weight, particularly preferably about 0.3 to 3 mg/kg body weight, and orally about 0.01 to 100 mg/kg body weight, preferably about 0.1 to 50 mg/kg body weight. , particularly preferably about 1-30 mg/kg body weight.
- the frequency of administration may be once or multiple times per day, eg, 1-3 times, 1-2 times, or 1 time per day.
- the active ingredient of the present invention can be produced according to known methods.
- C57BL/6J mice were purchased from Clea Japan (Tokyo, Japan).
- C57BL/6J RAG2-deficient mice (Rag2 ⁇ / ⁇ ) were purchased from Taconic Biosciences, Inc. (Hudson, NY, USA).
- Tsl:ICR germ-free mice were purchased from Sankyo Lab Service Co., Ltd. (Tokyo, Japan).
- Male mice aged 8-15 weeks were used and all mice were maintained in an SPF (specific pathogen free) environment.
- TNBS 2,4,6-trinitrobenzenesulfonate
- Anti-mouse CD4-PerCP/Cy5.5 GK1.5
- anti-mouse IL-10-PE JES5-16E3
- anti-mouse IFN- ⁇ -FITC XMG1.2
- 7-AAD anti- human CD4-APC
- anti-human CD45RA-Brilliant Violet 421 HI100
- anti-human CD4-Pacific Blue RPA-T4
- anti-human IFN- ⁇ 4S.B3
- Anti-mouse CD3e-Pe/Cy7 145-2C11
- Anti-mouse IL-17A-Alexa Fluor 647 TC11-18H10 were purchased from BD Biosciences.
- Mouse and human CD4 + T cells were stimulated with 50 ng/ml phorbol myristate acetate (PMA; Sigma-Aldrich) and 5 ⁇ M ionomycin (Sigma-Aldrich) in complete RPMI 1640 for 4 h at 37° C. in the presence of GolgiStop (BD Biosciences). did.
- Cell surface/intracellular staining was performed using the Cytofix/Cytoperm Kit Plus.
- Flow cytometric analysis was performed with a FACSCanto II flow cytometer (BD Biosciences) using FlowJo software (Tree Star, Ashland, OR, USA).
- ⁇ Pathological tissue analysis> Mouse colon and ileum samples were formaldehyde-fixed, paraffin-embedded, sectioned at 4 ⁇ m thickness, and stained with hematoxylin and eosin (H&E). The histological score used for evaluation was according to Iijima, H. et al. J Exp Med 199, 471-482.
- Results are expressed as mean and standard deviation. Mean differences between groups were tested by Student's t-test or One-way ANOVA.
- Example 1 LysoPS concentration measurement (1)> The characteristics of the lipid profile of the intestinal lumen of Crohn's disease patients (hereinafter also referred to as "CD patients") were confirmed. Sample donors were recruited from Crohn's disease patients and healthy subjects. IBD patients were diagnosed according to endoscopic, radiological, histological and clinical diagnostic criteria set by the International Organization for Inflammatory Bowel Disease Research. Crohn's disease patients are defined as in remission if their CDAI score is less than 150. The study was performed in accordance with the Declaration of Helsinki and was approved by the ethics review board of Osaka University Hospital. All participants gave written informed consent before participating in the study. Faecal samples were collected from 43 patients with Crohn's disease (19 active, 24 in remission) and 40 healthy subjects and immediately frozen.
- the fecal lipid composition was clearly different between healthy subjects and CD patients. Similar to plasma phospholipid concentrations (Iwatani et al. 2020), concentrations of total LysoPS, 18:0 LysoPS, and 18:1 LysoPS were higher in CD patients than in healthy subjects. This indicates that LysoPS production is increased in the gut of CD patients.
- Example 2 LysoPS concentration measurement (2)> Furthermore, fecal samples were collected from 11 Crohn's disease patients and 12 healthy subjects, and the lipids in the feces were comprehensively analyzed in the same manner as in Example 1. 529 lipid molecules belonging to 34 classes were identified. Partial least-squares discriminant analysis revealed that the lipid composition in samples from Crohn's disease patients was distinctly different from that in healthy controls.
- lipid molecules were elevated in CD patients compared to healthy subjects.
- concentrations of 18:0LysoPS and 18:1LysoPS in the feces of CD patients were higher than those of healthy subjects (Fig. 1).
- LysoPC levels were also elevated in CD patients (Fig. 2).
- Example 3 Metagenome analysis of fecal samples> DNA was extracted from the fecal samples collected in Example 1. Fecal samples were placed in tubes and RNAlaterTM was added to prepare 10-fold diluted homogenates. Fecal homogenate (200 ⁇ l) was washed twice with PBS, after which 0.3 g glass beads (0.1 mm diameter), 300 ⁇ l Tris-SDS solution, and 500 ⁇ l TE-saturated phenol were added to the suspension. The resulting mixture was vortexed for 30 seconds at power level 5.0 using FastPrep-24 (MP Biomedicals; Kaysersberg, France).
- the sample was centrifuged (20,000 ⁇ g, 4° C., 5 minutes), and 400 ⁇ l of the resulting supernatant was aliquoted and subjected to phenol-chloroform extraction. A 250 ⁇ l aliquot was taken from the supernatant obtained by phenol-chloroform extraction and precipitated with isopropanol. DNA from fecal samples was suspended in 50 ⁇ l of TE buffer.
- FIG. 5 shows the results of ⁇ -diversity represented by the shannon index. Species diversity of the gut microbiota was reduced in the faeces of Crohn's disease patients compared with healthy subjects. As a result of two-dimensional analysis using the Bray-Curtis index representing ⁇ diversity, the microbial community profile in fecal samples clearly differed between Crohn's disease patients and healthy subjects (Fig. 6). Dysbiosis in a patient with Crohn's disease was confirmed.
- Example 4 Measurement of genes encoding phospholipase A (PLA)> From the metagenomic analysis of Example 3, the gene encoding phospholipase A (PLA) was analyzed. Among the 7 genes predicted to encode PLA, ECSF — 3660, mainly derived from Escherichia coli, was identified as a gene significantly increased in Crohn's disease patients compared to samples from healthy subjects (Fig. 7).
- Example 5 Escherichia coli count measurement> The number of E.coli in the CD patient stool sample collected in Example 2 and the E.coli number in the healthy subject stool sample were determined. Quantitative PCR was performed according to a previously reported method (Pareek et al. 2019) for the determination of the E. coli/Shigella group in human and murine fecal samples. That is, 5 ⁇ l of 100-fold diluted DNA as a template and 15 ⁇ l of a master mixed solution were mixed to prepare 20 ⁇ l of each reaction solution.
- the master mix solution contained 4.6 ⁇ l of PCR grade water, 0.2 ⁇ l of forward and reverse primers from 10 ⁇ M stocks, and 10 ⁇ l of GoTaqTM Probe qPCR Master Mix (Promega, Woods Hollow Road Madison, WI, USA). was included. Reactions were carried out in an AB Biosystems StepOnePlusTM system using the following program: 1 cycle of 94° C. 5 min, 40 cycles of 94° C. 15 sec, 60° C. 60 sec, and 72° C. 60 s. Copy number per gram of stool was calculated based on a standard curve obtained for E. coli. The following primer sets were used: 5'-GAGTAAAGTTAATAACCTTTGCTCATTG-3' (SEQ ID NO:6) and 5'-GAGACTCAAGCTKRCCAGTATCAG-3' (SEQ ID NO:7).
- E. coli counts in CD patient samples were significantly higher compared to healthy subjects (Fig. 8). Furthermore, fecal E. coli counts were positively correlated with the relative amounts of fecal lysophospholipids normalized by rank-order scores. In addition, the 6 individuals who were identified as having a high lysophospholipid concentration in Example 2 had a large amount of E. coli (Fig. 9).
- Example 6 LysoPS production by intestinal microorganisms> It has been reported that Gram-negative PLA disrupts host epithelial cell membranes when Gram-negative bacteria invade the host intestinal epithelium (Istivan and Coloe, 2006). On the other hand, both the patient's (host's) own phospholipase A (PLA) and the PLA of intestinal microorganisms are known to hydrolyze cell membrane phospholipids to produce lysophospholipids (Tan et al. 2020). To confirm that the PLA that causes the increase in fecal LysoPS in CD patients is due to microbial PLA that is increased due to intestinal dysbiosis, the following tests were performed.
- PLA phospholipase A
- Amplification conditions were 1 cycle of 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds. The results are shown in FIG. Fecal samples from 4 healthy subjects with healthy microbiota were combined, and stool samples from 2 CD patients harboring ECSF_3660 detectable microbiota were also combined.
- mice Germ-free mice were colonized with human fecal microbiota by a method previously reported (Maeda et al. 2016) with minor modifications.
- Each fecal sample was added to an aerobic buffer (2% Lab-Lemco powder, 0.1% L-cysteine, 0.045% KH 2 PO 4 , 0.09% NaCl, 0.045% (NH 4 ) 2 SO). 4 , 0.045% CaCl 2 , 0.045% MgSO 4 , and 40% glycerin), homogenized by 16-fold dilution (w/v) and stored at -80°C until use.
- an aerobic buffer 2% Lab-Lemco powder, 0.1% L-cysteine, 0.045% KH 2 PO 4 , 0.09% NaCl, 0.045% (NH 4 ) 2 SO). 4 , 0.045% CaCl 2 , 0.045% MgSO 4 , and 40% glycerin
- mice Male germ-free mice (8-9 weeks old) were implanted orally with 250 ⁇ l of fecal suspension from CD patients or healthy subjects. The mice were maintained separately in gnotobiotic isolators for 24 days. At 10 days after transplantation, the number of E. coli in the feces of CD patient microbiota-transplanted mice was clearly higher than that of normal human microbiota-transplanted mice (Fig. 11). In addition, the microbial DNA, ECSF_3660, was detected in the feces of mice harboring the CD patient microbiota. On the other hand, ECSF — 3660 was not detected in the feces of mice carrying the microflora of healthy individuals (Fig. 12).
- Example 7 Methanol is added to the patient's stool sample and homogenized, and the amount of ECSF — 3660 is measured by quantitative real-time PCR method. When the gene dosage is high compared to the amount of ECSF — 3660 in healthy subjects, it is determined that the risk of inflammatory bowel disease is high.
- Example 8 The amount of ECSF_3660 in fecal samples from patients with inflammatory bowel disease is determined as in Example 7. To grasp changes in the patient's condition by comparing with the past examination values of the same patient.
- Example 9 Fecal samples were collected from CD patients and healthy subjects and immediately frozen. Frozen fecal samples were homogenized with the addition of methanol and the concentrations of total LysoPS, 18:0 LysoPS and 18:1 LysoPS were determined by UPLC-ESI-MS/MS. These LysoPS concentrations were higher in Crohn's disease patients than in healthy subjects.
- TNBS-induced colitis model mouse (Crohn's disease model mouse) TNBS-induced colitis model mice were produced by a previously reported method (Iijima, H. et al. J Exp Med 199, 471-482, (2004)) with slight modifications. That is, wild-type C57BL/6J mice (male, 8-10 weeks old) were sensitized on day 1 by applying 150 ⁇ l of a 3.75% TNBS solution in 50% ethanol to the dorsal skin. On day 8, 150 ⁇ l of 2% TNBS solution in 50% ethanol was administered transanally. 18:1 LysoPS was intraperitoneally administered once daily from day 8 to day 11 at a dose of 2.5 mg/kg, and mice were used for analysis on day 12. Control mice received vehicle (70% ethanol).
- T cell-dependent colitis model mouse (Crohn's disease model mouse) A CD4 + T cell-dependent colitis model mouse was generated by slightly modifying the method described in the literature (Ostanin, D. V. et al. Am J Physiol Gastrointest Liver Physiol 296, G135-146, (2009)). Isolated naive CD4 + T cells were diluted with cold phosphate buffered saline (PBS) to a final concentration of 1 ⁇ 10 6 cells/ml and 500 ⁇ l (5 ⁇ 10 5 cells) were injected into Rag2 ⁇ / ⁇ mice (8 males). ⁇ 15 weeks old) were administered intraperitoneally. Seventeen days after administration of naive CD4 + T cells, 18:1 LysoPS (2.5 mg/kg) or vehicle (70% ethanol) was administered intraperitoneally for 4 consecutive days.
- PBS cold phosphate buffered saline
- LysoPS exacerbates colitis by evoking an immunopathological Th1 response in the lamina propria.
- LysoPS exacerbates inflammatory bowel disease by causing hyperactivation of Th1 cells in the intestinal mucosa.
- the present invention contributes to examination of inflammatory bowel disease and prevention and treatment of inflammatory bowel disease.
Abstract
[Problem] Problems addressed are: to provide a new test method for inflammatory bowel disease; to provide a method for screening substances or microorganisms for prevention or treatment of inflammatory bowel disease; and to provide a composition for prevention or treatment of inflammatory bowel disease. [Solution] Provided is a method for testing for inflammatory bowel disease that includes a step for measuring gut microbiota involved in lysophosphatidylserine production, lysophosphatidylserine, phospholipase A, or phospholipase A genes (ECSF_3660, etc.) in a sample. Provided is a screening method for substances or microorganisms for the prevention or treatment of inflammatory bowel disease that includes a step for measuring the gut microbiota count or measuring phospholipase A activity. Provided are a composition for prevention or treatment of inflammatory bowel disease that contains a substance or microorganism for removing or reducing gut microbiota involved in lysophosphatidylserine production and a composition for prevention or treatment of inflammatory bowel disease that contains a phospholipase A activity inhibitor.
Description
本発明は、炎症性腸疾患の検査および治療に関する。
The present invention relates to the testing and treatment of inflammatory bowel disease.
炎症性腸疾患（Ｉｎｆｌａｍｍａｔｏｒｙ ｂｏｗｅｌ ｄｉｓｅａｓｅ：ＩＢＤ）は腸管における免疫の過剰な活性化が関与していると考えられている。腸管に生じる難病であるが、患者数が急速に増加しており問題となっている。ＩＢＤに対する治療薬として抗ＴＮＦ抗体製剤などの免疫抑制機序を持つ新規薬剤が多数登場しており、従来薬と比較して高い治療効果が見られるが、治療薬の継続使用に伴い効果減弱をきたすなど、治療に難渋する患者も少なくない。さまざまな研究が行われているが、未だ疾患の発症、増悪のメカニズムが十分に解明されたとは言えず、根治治療法も開発されていない。
Inflammatory bowel disease (IBD) is thought to involve excessive activation of immunity in the intestinal tract. Although it is an intractable disease that occurs in the intestinal tract, the number of patients is increasing rapidly, and it is becoming a problem. Many new drugs with an immunosuppressive mechanism, such as anti-TNF antibody preparations, have been introduced as therapeutic drugs for IBD, and although they are highly effective compared to conventional drugs, the effect diminishes with continued use of the therapeutic drugs. There are quite a few patients who are difficult to treat, such as getting sick. Although various studies have been conducted, the mechanisms of disease onset and exacerbation have not yet been fully elucidated, and no curative therapy has been developed.
炎症性腸疾患を発症すると、根治的治療法がないため症状が軽減し寛解になっても再燃することが多く、寛解と再燃を繰り返す場合が多い。よって、発症前の発症リスクや発症後の再燃を簡便に検査できることが望まれる。また、再燃の危険性から寛解期であっても定期的な内視鏡検査が必要であるが、内視鏡検査を毎回繰り返し受けることを嫌がる患者も少なくない。よって患者に負担の少ない検査が望まれる。
Once inflammatory bowel disease develops, there is no curative treatment, and even if the symptoms are relieved and go into remission, relapse often occurs, and remission and relapse often occur repeatedly. Therefore, it is desirable to be able to easily examine the onset risk before onset and the relapse after onset. In addition, due to the risk of recurrence, periodic endoscopic examinations are necessary even in the remission period, but many patients are reluctant to undergo repeated endoscopic examinations every time. Therefore, an examination that places less burden on the patient is desired.
腸内微生物叢はそのホストの免疫システムにおいて重要な役割を担っている。ＩＢＤの患者の腸内微生物叢には、微生物叢の多様性の減少と構成の変化によって特徴付けられる異常（Ｄｙｓｂｉｏｓｉｓ）が観察される（非特許文献１）。微生物叢は食物繊維を消化して短鎖脂肪酸（ＳＣＦＡ）を生成する。そのＳＣＦＡは、微生物叢自身やホストの腸上皮細胞のエネルギー源になるだけでなく、Ｔ細胞などの免疫細胞を誘導することによって、ホストの腸のホメオスタシスに寄与している（非特許文献２）。
The gut microbiota plays an important role in its host's immune system. Dysbiosis, characterized by decreased diversity and altered composition of the microbiota, is observed in the intestinal microbiota of IBD patients (Non-Patent Document 1). Microbiota digest dietary fiber to produce short chain fatty acids (SCFA). SCFA not only serve as an energy source for the microbiota itself and host intestinal epithelial cells, but also contribute to host intestinal homeostasis by inducing immune cells such as T cells (Non-Patent Document 2). .
本発明者等は、１６種類のホスファチジルセリン（ＰＳ）と１８：０リゾホスファチジルセリン（ＬｙｓｏＰＳ）の血漿中濃度がクローン病患者では健常者に比較して２倍以上、上昇していることを報告した（非特許文献３）。ＰＳは細胞膜の脂質二重層の中に豊富に存在し、ＬｙｓｏＰＳはホスホリパーゼＡ１／Ａ２（ＰＬＡ１／Ａ２）の酵素の作用によってＰＳから発生して脂質メディエーターとして作用すると考えられている（非特許文献４）。しかし、ＬｙｓｏＰＳの生理学的、病理学的機能は不明である。
The present inventors reported that the plasma concentrations of 16 types of phosphatidylserine (PS) and 18:0 lysophosphatidylserine (LysoPS) were more than doubled in patients with Crohn's disease compared to healthy subjects. (Non-Patent Document 3). PS is abundantly present in the lipid bilayer of the cell membrane, and LysoPS is thought to be generated from PS by the action of the enzyme phospholipase A1/A2 (PLA1/A2) and act as a lipid mediator (Non-Patent Document 4). ). However, the physiological and pathological functions of LysoPS are unknown.
本発明は、炎症性腸疾患の新たな検査方法を提供すること、炎症性腸疾患の予防または治療用の物質や微生物のスクリーニング方法を提供すること、および炎症性腸疾患の予防用または治療用組成物を提供することを課題とする。
The present invention provides a new test method for inflammatory bowel disease, provides a method for screening substances and microorganisms for preventing or treating inflammatory bowel disease, and provides a method for preventing or treating inflammatory bowel disease. An object is to provide a composition.
本発明者等は、上記課題を解決すべく研究を行った結果、リゾホスファチジルセリンがある種の腸内微生物によって生成されること、及び腸内微生物によって生成されたリゾホスファチジルセリンが、疾患の増悪に直接関与していることを見出し、本発明を完成した。即ち、本発明は以下の態様を含有する。
１．被検体より得られた試料中の、リゾホスファチジルセリン、リゾホスファチジルセリン生成に関与する腸内微生物、腸内微生物由来ホスホリパーゼＡをコードする遺伝子、および、ホスホリパーゼＡからなる群から選択される少なくとも１を測定する工程を含む炎症性腸疾患を検査する方法。
２．被検体より得られた試料中のリゾホスファチジルセリン生成に関与する腸内微生物数を測定する工程を含む、前項１に記載の方法。
３．前記腸内微生物がホスホリパーゼＡを発現する腸内微生物である、前項２に記載の方法。
４．前記ホスホリパーゼＡを発現する腸内微生物が、ホスホリパーゼＡをコードする遺伝子ＥＣＳＦ＿３６６０を有する微生物である、前項３に記載の方法。
５．前記腸内微生物数を測定する工程は、該腸内微生物由来遺伝子の量を測定する工程を含む、前項２～４のいずれか１に記載の方法。
６．前記腸内微生物数を測定する工程は、該腸内微生物発現ホスホリパーゼＡを測定する工程を含む、前項２～４のいずれか１に記載の方法。
７．被検体より得られた試料中の腸内微生物由来ホスホリパーゼＡをコードする遺伝子の量を測定する工程を含む、前項１に記載の方法
８．前記ホスホリパーゼＡをコードする遺伝子が、ＥＣＳＦ＿３６６０である前項７に記載の方法。
９．被検体より得られた試料中のリゾホスファチジルセリンの濃度を測定する工程を含む、前項１に記載の方法。
１０．さらにリゾホスファチジルコリンの濃度を測定する工程を含む前項９に記載の方法。
１１．被検体より得られた試料中のホスホリパーゼＡを測定する工程を含む、前項１に記載の方法。
１２．前記ホスホリパーゼＡが、腸内微生物由来ホスホリパーゼＡである、前項１１に記載の方法。
１３．前記腸内微生物由来ホスホリパーゼＡが、ＥＣＳＦ＿３６６０にコードされるホスホリパーゼＡである、前項１２に記載の方法。
１４．前記ホスホリパーゼＡを測定する工程が、ホスホリパーゼＡのタンパク質量を測定する工程またはホスホリパーゼＡの活性を測定する工程である、前項１１～１３のいずれか１に記載の方法。
１５．前記試料が糞便サンプルである、前項１～１４のいずれか１に記載の方法。
１６．リゾホスファチジルセリン生成に関与する腸内微生物数を測定する工程を含む、炎症性腸疾患の予防用または治療用の物質または微生物のスクリーニング方法。
１７．ホスホリパーゼＡの活性を測定する工程を含む、炎症性腸疾患の予防用または治療用の物質のスクリーニング方法。
１８．リゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させる物質または微生物を含有する炎症性腸疾患の予防用または治療用組成物。
１９．ホスホリパーゼＡの活性を抑制する物質を含有する炎症性腸疾患の予防用または治療用組成物。
２０．被検体より得られた試料の検査値と対照試料の検査値が比較される工程をさらに含む、前項１～１５のいずれか１に記載の方法。
２１．前記対照試料が、健康な被検体から得られた試料または同一被検体から得られた試料である、前項２０に記載の方法。
２２．リゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させる物質または微生物を投与することを含む炎症性腸疾患の予防または治療方法。
２３．腸内微生物由来ホスホリパーゼＡの活性を抑制する物質を投与することを含む炎症性腸疾患の予防または治療方法。
２４．炎症性腸疾患の予防用または治療用組成物の調製のためのリゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させる物質または微生物の使用。
２５．炎症性腸疾患の予防用または治療用組成物の調製のための腸内微生物由来ホスホリパーゼＡの活性を抑制する物質の使用。 As a result of conducting research to solve the above problems, the present inventors found that lysophosphatidylserine is produced by certain intestinal microorganisms, and that lysophosphatidylserine produced by intestinal microorganisms exacerbates the disease. The present invention was completed by discovering that it is directly involved in That is, the present invention includes the following aspects.
1. At least one selected from the group consisting of lysophosphatidylserine, an intestinal microorganism involved in the production of lysophosphatidylserine, a gene encoding phospholipase A derived from intestinal microorganisms, and phospholipase A in a sample obtained from a subject. A method of testing for inflammatory bowel disease comprising the step of measuring.
2. 2. The method according to the precedingitem 1, comprising the step of measuring the number of intestinal microorganisms involved in lysophosphatidylserine production in a sample obtained from a subject.
3. 3. The method according to the precedingitem 2, wherein the intestinal microorganism is an intestinal microorganism that expresses phospholipase A.
4. 4. The method according toitem 3, wherein the intestinal microorganism expressing phospholipase A is a microorganism having a gene ECSF — 3660 encoding phospholipase A.
5. 5. The method according to any one of the precedingitems 2 to 4, wherein the step of measuring the number of intestinal microorganisms includes the step of measuring the amount of the gene derived from the intestinal microorganisms.
6. 5. The method according to any one of the precedingitems 2 to 4, wherein the step of measuring the intestinal microbial count includes the step of measuring the intestinal microbial-expressed phospholipase A.
7. 8. The method according to the precedingitem 1, which comprises the step of measuring the amount of the gene encoding the intestinal microorganism-derived phospholipase A in the sample obtained from the subject. 8. The method according to item 7, wherein the gene encoding phospholipase A is ECSF — 3660.
9. 2. The method according to the precedingitem 1, comprising the step of measuring the concentration of lysophosphatidylserine in a sample obtained from a subject.
10. 10. The method according toitem 9, further comprising the step of measuring the concentration of lysophosphatidylcholine.
11. 2. The method according to the precedingitem 1, comprising the step of measuring phospholipase A in a sample obtained from a subject.
12. 12. The method according toitem 11, wherein the phospholipase A is an intestinal microorganism-derived phospholipase A.
13. 13. The method according toitem 12, wherein the intestinal microorganism-derived phospholipase A is phospholipase A encoded by ECSF_3660.
14. 14. The method according to any one of the precedingitems 11 to 13, wherein the step of measuring phospholipase A is a step of measuring the amount of phospholipase A protein or a step of measuring phospholipase A activity.
15. 15. The method according to any one of the precedingitems 1 to 14, wherein the sample is a stool sample.
16. A method for screening substances or microorganisms for the prevention or treatment of inflammatory bowel disease, comprising the step of measuring the number of intestinal microorganisms involved in lysophosphatidylserine production.
17. A method of screening for a substance for prevention or treatment of inflammatory bowel disease, comprising the step of measuring phospholipase A activity.
18. A composition for preventing or treating inflammatory bowel disease containing a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production.
19. A composition for prevention or treatment of inflammatory bowel disease containing a substance that suppresses the activity of phospholipase A.
20. 16. The method according to any one of the precedingitems 1 to 15, further comprising the step of comparing the test value of the sample obtained from the subject with the test value of the control sample.
21. 21. The method according toitem 20, wherein the control sample is a sample obtained from a healthy subject or a sample obtained from the same subject.
22. A method for preventing or treating inflammatory bowel disease comprising administering a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production.
23. A method for preventing or treating inflammatory bowel disease, comprising administering a substance that suppresses the activity of phospholipase A derived from intestinal microorganisms.
24. Use of a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production for the preparation of a composition for the prevention or treatment of inflammatory bowel disease.
25. Use of a substance that inhibits the activity of intestinal microorganism-derived phospholipase A for the preparation of a composition for the prevention or treatment of inflammatory bowel disease.
１．被検体より得られた試料中の、リゾホスファチジルセリン、リゾホスファチジルセリン生成に関与する腸内微生物、腸内微生物由来ホスホリパーゼＡをコードする遺伝子、および、ホスホリパーゼＡからなる群から選択される少なくとも１を測定する工程を含む炎症性腸疾患を検査する方法。
２．被検体より得られた試料中のリゾホスファチジルセリン生成に関与する腸内微生物数を測定する工程を含む、前項１に記載の方法。
３．前記腸内微生物がホスホリパーゼＡを発現する腸内微生物である、前項２に記載の方法。
４．前記ホスホリパーゼＡを発現する腸内微生物が、ホスホリパーゼＡをコードする遺伝子ＥＣＳＦ＿３６６０を有する微生物である、前項３に記載の方法。
５．前記腸内微生物数を測定する工程は、該腸内微生物由来遺伝子の量を測定する工程を含む、前項２～４のいずれか１に記載の方法。
６．前記腸内微生物数を測定する工程は、該腸内微生物発現ホスホリパーゼＡを測定する工程を含む、前項２～４のいずれか１に記載の方法。
７．被検体より得られた試料中の腸内微生物由来ホスホリパーゼＡをコードする遺伝子の量を測定する工程を含む、前項１に記載の方法
８．前記ホスホリパーゼＡをコードする遺伝子が、ＥＣＳＦ＿３６６０である前項７に記載の方法。
９．被検体より得られた試料中のリゾホスファチジルセリンの濃度を測定する工程を含む、前項１に記載の方法。
１０．さらにリゾホスファチジルコリンの濃度を測定する工程を含む前項９に記載の方法。
１１．被検体より得られた試料中のホスホリパーゼＡを測定する工程を含む、前項１に記載の方法。
１２．前記ホスホリパーゼＡが、腸内微生物由来ホスホリパーゼＡである、前項１１に記載の方法。
１３．前記腸内微生物由来ホスホリパーゼＡが、ＥＣＳＦ＿３６６０にコードされるホスホリパーゼＡである、前項１２に記載の方法。
１４．前記ホスホリパーゼＡを測定する工程が、ホスホリパーゼＡのタンパク質量を測定する工程またはホスホリパーゼＡの活性を測定する工程である、前項１１～１３のいずれか１に記載の方法。
１５．前記試料が糞便サンプルである、前項１～１４のいずれか１に記載の方法。
１６．リゾホスファチジルセリン生成に関与する腸内微生物数を測定する工程を含む、炎症性腸疾患の予防用または治療用の物質または微生物のスクリーニング方法。
１７．ホスホリパーゼＡの活性を測定する工程を含む、炎症性腸疾患の予防用または治療用の物質のスクリーニング方法。
１８．リゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させる物質または微生物を含有する炎症性腸疾患の予防用または治療用組成物。
１９．ホスホリパーゼＡの活性を抑制する物質を含有する炎症性腸疾患の予防用または治療用組成物。
２０．被検体より得られた試料の検査値と対照試料の検査値が比較される工程をさらに含む、前項１～１５のいずれか１に記載の方法。
２１．前記対照試料が、健康な被検体から得られた試料または同一被検体から得られた試料である、前項２０に記載の方法。
２２．リゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させる物質または微生物を投与することを含む炎症性腸疾患の予防または治療方法。
２３．腸内微生物由来ホスホリパーゼＡの活性を抑制する物質を投与することを含む炎症性腸疾患の予防または治療方法。
２４．炎症性腸疾患の予防用または治療用組成物の調製のためのリゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させる物質または微生物の使用。
２５．炎症性腸疾患の予防用または治療用組成物の調製のための腸内微生物由来ホスホリパーゼＡの活性を抑制する物質の使用。 As a result of conducting research to solve the above problems, the present inventors found that lysophosphatidylserine is produced by certain intestinal microorganisms, and that lysophosphatidylserine produced by intestinal microorganisms exacerbates the disease. The present invention was completed by discovering that it is directly involved in That is, the present invention includes the following aspects.
1. At least one selected from the group consisting of lysophosphatidylserine, an intestinal microorganism involved in the production of lysophosphatidylserine, a gene encoding phospholipase A derived from intestinal microorganisms, and phospholipase A in a sample obtained from a subject. A method of testing for inflammatory bowel disease comprising the step of measuring.
2. 2. The method according to the preceding
3. 3. The method according to the preceding
4. 4. The method according to
5. 5. The method according to any one of the preceding
6. 5. The method according to any one of the preceding
7. 8. The method according to the preceding
9. 2. The method according to the preceding
10. 10. The method according to
11. 2. The method according to the preceding
12. 12. The method according to
13. 13. The method according to
14. 14. The method according to any one of the preceding
15. 15. The method according to any one of the preceding
16. A method for screening substances or microorganisms for the prevention or treatment of inflammatory bowel disease, comprising the step of measuring the number of intestinal microorganisms involved in lysophosphatidylserine production.
17. A method of screening for a substance for prevention or treatment of inflammatory bowel disease, comprising the step of measuring phospholipase A activity.
18. A composition for preventing or treating inflammatory bowel disease containing a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production.
19. A composition for prevention or treatment of inflammatory bowel disease containing a substance that suppresses the activity of phospholipase A.
20. 16. The method according to any one of the preceding
21. 21. The method according to
22. A method for preventing or treating inflammatory bowel disease comprising administering a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production.
23. A method for preventing or treating inflammatory bowel disease, comprising administering a substance that suppresses the activity of phospholipase A derived from intestinal microorganisms.
24. Use of a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production for the preparation of a composition for the prevention or treatment of inflammatory bowel disease.
25. Use of a substance that inhibits the activity of intestinal microorganism-derived phospholipase A for the preparation of a composition for the prevention or treatment of inflammatory bowel disease.
本発明の方法は、炎症性腸疾患に関わる腸内微生物の異常を検査できる。腸内微生物の異常（ｄｙｓｂｉｏｓｉｓ）によりホスホリパーゼＡを発現する微生物が増加し、腸内でリゾホスファチジルセリン(以下、ＬｙｓｏＰＳともいう)が増加する。ＬｙｓｏＰＳは炎症性腸疾患の発症や増悪に直接関与している。本発明の方法は、試料中のリゾホスファチジルセリン、リゾホスファチジルセリン生成に関与する腸内微生物、または、リゾホスファチジルセリンを生成する酵素であるホスホリパーゼＡもしくはその遺伝子を測定することにより、炎症性腸疾患を検査することができる。本発明の方法は、炎症性腸疾患の診断のための検査、炎症性腸疾患発症前の発症高リスク者の判定のための検査、炎症性腸疾患患者の経過観察のための検査等、炎症性腸疾患に関わる検査全てに使用できる。本発明の方法は、炎症性腸疾患患者または高リスク者の病状の変化を先んじて捉えることができる。また、本発明の方法は、簡便で患者に負担の少ない検査方法である。
The method of the present invention can examine intestinal microbial abnormalities associated with inflammatory bowel disease. Microorganisms expressing phospholipase A increase due to intestinal microbial dysbiosis, and lysophosphatidylserine (hereinafter also referred to as LysoPS) increases in the intestine. LysoPS is directly involved in the development and exacerbation of inflammatory bowel disease. The method of the present invention measures lysophosphatidylserine in a sample, intestinal microorganisms involved in the production of lysophosphatidylserine, or phospholipase A, which is an enzyme that produces lysophosphatidylserine, or its gene. can be inspected. The method of the present invention can be used for diagnosing inflammatory bowel disease, testing for determining high-risk individuals with pre-onset inflammatory bowel disease, testing for follow-up of inflammatory bowel disease patients, and the like. It can be used for all examinations related to enteropathy. The method of the present invention can anticipate changes in the condition of inflammatory bowel disease patients or high-risk individuals. In addition, the method of the present invention is a simple examination method that imposes little burden on the patient.
本発明のスクリーニング方法は、炎症性腸疾患の原因となるリゾホスファチジルセリン、それを生成するホスホリパーゼＡ、またはリゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させる物質または微生物をスクリーニングすることを可能にし、炎症性腸疾患の治療薬、予防薬、および、治療もしくは予防用食品を提供することを可能にする。
The screening method of the present invention involves screening for substances or microorganisms that eliminate or reduce lysophosphatidylserine that causes inflammatory bowel disease, phospholipase A that produces it, or intestinal microorganisms involved in lysophosphatidylserine production. It is possible to provide a therapeutic drug, a preventive drug, and a therapeutic or preventive food for inflammatory bowel disease.
本発明の組成物は、炎症性腸疾患の原因となるリゾホスファチジルセリン、それを生成するホスホリパーゼＡ、またはリゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させることにより炎症性腸疾患の発症および再燃を予防し、炎症性腸疾患の根治療法を可能にする。本発明の組成物は、炎症性腸疾患の原因となるリゾホスファチジルセリンの生成を抑制し、炎症性腸疾患の発症および再燃を予防し、炎症性腸疾患の根治療法を可能にする。本発明の組成物は炎症性腸疾患の原因療法であることから、予防効果および治療効果に優れ、多くの炎症性腸疾患患者の治療および再燃予防またはその予備軍の発症予防に多大な貢献をするものである。
The composition of the present invention eliminates or reduces lysophosphatidylserine that causes inflammatory bowel disease, phospholipase A that produces it, or intestinal microorganisms involved in lysophosphatidylserine production, thereby reducing the development of inflammatory bowel disease. and prevent relapses, enabling radical treatment of inflammatory bowel disease. The composition of the present invention suppresses the production of lysophosphatidylserine, which causes inflammatory bowel disease, prevents the onset and relapse of inflammatory bowel disease, and enables radical treatment of inflammatory bowel disease. Since the composition of the present invention is a causative therapy for inflammatory bowel disease, it has excellent preventive and therapeutic effects, and will greatly contribute to the treatment and prevention of recurrence of many patients with inflammatory bowel disease, or the onset of those at risk of inflammatory bowel disease. It is something to do.
本発明において、リゾホスファチジルセリン生成に関与する腸内微生物とは、リゾホスファチジルセリン生成に関与している腸内微生物であれば、特に限定されない。リゾホスファチジルセリン（ＬｙｓｏＰＳ）はリン脂質であり、グリセロール骨格にアシル基、リン酸基、セリンが結合している構造を有している。アシル基の脂肪酸種は多様であり、結合位置はグリセロール骨格のｓｎ－１位（１－アシル型）とｓｎ－２位（２－アシル型）の２種類がある。生体内では、細胞膜の脂質二重層を形成しているホスファチジルセリン（ＰＳ）がホスホリパーゼＡ１（ＰＬＡ１）またはホスホリパーゼＡ２（ＰＬＡ２）の作用を受けて、ＬｙｓｏＰＳが生成される。本発明において、ホスホリパーゼＡ１（ＰＬＡ１）および／またはホスホリパーゼＡ２（ＰＬＡ２）をホスホリパーゼＡ（ＰＬＡ）と総称する。ＬｙｓｏＰＳはＴｈ１細胞を過剰活性化させて炎症性腸疾患を発症させ、または症状を増悪させる。
In the present invention, the intestinal microorganism involved in lysophosphatidylserine production is not particularly limited as long as it is an intestinal microorganism involved in lysophosphatidylserine production. Lysophosphatidylserine (LysoPS) is a phospholipid and has a structure in which an acyl group, a phosphate group, and serine are bound to a glycerol skeleton. The fatty acid species of the acyl group is diverse, and there are two types of bonding positions, sn-1 position (1-acyl type) and sn-2 position (2-acyl type) of the glycerol skeleton. In vivo, phosphatidylserine (PS) forming the lipid bilayer of the cell membrane is acted upon by phospholipase A1 (PLA1) or phospholipase A2 (PLA2) to produce LysoPS. In the present invention, phospholipase A1 (PLA1) and/or phospholipase A2 (PLA2) are collectively referred to as phospholipase A (PLA). LysoPS overactivates Th1 cells to develop or exacerbate symptoms of inflammatory bowel disease.
前記ＬｙｓｏＰＳ生成に関与する腸内微生物は、好ましくは、ホスホリパーゼＡを発現する腸内微生物である。本発明において、ホスホリパーゼＡを発現する腸内微生物には、ホスホリパーゼＡを発現している腸内微生物とホスホリパーゼ遺伝子を有する微生物であってホスホリパーゼＡを発現することができる微生物を含む。ホスホリパーゼを発現する腸内微生物は、ホストの腸管上皮細胞の細胞膜を破壊しＬｙｓｏＰＳを生成する。
The enteric microorganism involved in said LysoPS production is preferably an enteric microorganism expressing phospholipase A. In the present invention, intestinal microorganisms expressing phospholipase A include intestinal microorganisms expressing phospholipase A and microorganisms having a phospholipase gene and capable of expressing phospholipase A. Gut microbes that express phospholipases disrupt the cell membranes of host intestinal epithelial cells to produce LysoPS.
前記ホスホリパーゼＡを発現する腸内微生物は、好ましくは、ホスホリパーゼＡをコードする遺伝子ＥＣＳＦ＿３６６０を有する微生物である。
The enteric microorganism expressing phospholipase A is preferably a microorganism having the gene ECSF_3660 encoding phospholipase A.
前記ホスホリパーゼＡを発現する腸内微生物は、好ましくは細菌であり、さらに好ましくはグラム陰性菌であり、例えば大腸菌、エルシニアおよびセラチアが挙げられる。より好ましくは大腸菌（Escherichia coli ）であり、さらに好ましくは、ホスホリパーゼＡをコードする遺伝子ＥＣＳＦ＿３６６０を有する大腸菌である。
The enteric microorganism expressing phospholipase A is preferably a bacterium, more preferably a Gram-negative bacterium, such as E. coli, Yersinia and Serratia. Escherichia coli is more preferred, and E. coli having a gene ECSF — 3660 encoding phospholipase A is even more preferred.
本発明におけるリゾホスファチジルセリン生成に関与する腸内微生物数を測定する方法は、該腸内微生物量を測定できればその手法は特に限定されない。たとえば、該腸内微生物由来遺伝子の量を測定する方法、該腸内微生物発現ホスホリパーゼＡを測定する方法、該腸内微生物を培養した後にコロニー数を計測する方法等が挙げられる。
The method for measuring the number of intestinal microorganisms involved in the production of lysophosphatidylserine in the present invention is not particularly limited as long as the amount of the intestinal microorganisms can be measured. Examples thereof include a method of measuring the amount of the intestinal microorganism-derived gene, a method of measuring the phospholipase A expressed by the intestinal microorganism, a method of counting the number of colonies after culturing the intestinal microorganism, and the like.
前記腸内微生物由来遺伝子の量を測定する好適な方法としては、ＰＣＲによる腸内微生物由来遺伝子の量を測定する方法が挙げられ、例えば定量的リアルタイムＰＣＲ法を挙げることができる。腸内微生物数は遺伝子量から公知の方法（例えば、Petr Kralik et al. Front Microbiol 2017 Feb 2;8:108に記載の方法）により求めることができる。
A suitable method for measuring the amount of the intestinal microorganism-derived gene includes a method of measuring the amount of the intestinal microorganism-derived gene by PCR, for example, a quantitative real-time PCR method. The number of intestinal microorganisms can be determined from the gene content by a known method (for example, the method described in Petr Kralik et al. Front Microbiol 2017 Feb 2;8:108).
前記腸内微生物由来遺伝子は、特に制限されるものではなく、目的の微生物を検出するために通常用いられている遺伝子でよい。例えば、大腸菌であれば通常大腸菌の定量に用いられているプライマーを用いて定量的ＰＣＲを行うことができる。前記腸内微生物由来遺伝子として好ましくはホスホリパーゼＡをコードする遺伝子が挙げられる。
The intestinal microorganism-derived gene is not particularly limited, and may be a gene commonly used for detecting target microorganisms. For example, in the case of Escherichia coli, quantitative PCR can be performed using primers commonly used for quantification of Escherichia coli. As the intestinal microorganism-derived gene, a gene encoding phospholipase A is preferable.
前記ホスホリパーゼＡをコードする遺伝子として、例えば、ＥＣＳＦ＿３６６０が挙げられる。ＥＣＳＦ＿３６６０は大腸菌（Escherichia coli）およびその他の細菌（肺炎桿菌（Klebsiella pneumoniae）等）由来のホスホリパーゼＡをコードする遺伝子（配列番号１、配列番号２）である。ＥＣＳＦ＿３６６０の量を測定する方法として、例えば、定量的リアルタイムＰＣＲ法が挙げられる。
Examples of genes encoding phospholipase A include ECSF — 3660. ECSF — 3660 is a gene (SEQ ID NO: 1, SEQ ID NO: 2) encoding phospholipase A derived from Escherichia coli and other bacteria (such as Klebsiella pneumoniae). Methods for measuring the amount of ECSF_3660 include, for example, a quantitative real-time PCR method.
本発明の方法の好ましい実施形態の１つは、被検体から得られた試料中のホスホリパーゼＡを発現する大腸菌数を測定する工程を含む炎症性腸疾患を検査する方法であり、さらに好ましくは、遺伝子ＥＣＳＦ＿３６６０を定量的リアルタイムＰＣＲ法により定量して該大腸菌数を測定する工程を含む炎症性腸疾患を検査する方法である。
One of the preferred embodiments of the method of the present invention is a method of testing for inflammatory bowel disease, comprising the step of measuring the number of E. coli expressing phospholipase A in a sample obtained from a subject, and more preferably, A method for testing inflammatory bowel disease, comprising the step of quantifying the gene ECSF — 3660 by quantitative real-time PCR and measuring the number of E. coli.
リゾホスファチジルセリン生成に関与する腸内微生物数は、該腸内微生物が発現するホスホリパーゼＡを測定することにより求めることができる。微生物表面または微生物内に存在するホスホリパーゼＡを測定する方法が挙げられる。グラム陰性菌ではホスホリパーゼＡが外膜に存在することより、グラム陰性菌の外膜に存在するホスホリパーゼを測定する方法が挙げられる。前記腸内微生物由来ホスホリパ－ゼＡとしては、ＥＣＳＦ＿３６６０にコードされるホスホリパ－ゼＡが挙げられる。
The number of intestinal microorganisms involved in lysophosphatidylserine production can be determined by measuring phospholipase A expressed by the intestinal microorganisms. Examples include methods for measuring phospholipase A present on the surface of microorganisms or within microorganisms. Since phospholipase A is present in the outer membrane of Gram-negative bacteria, a method of measuring phospholipase present in the outer membrane of Gram-negative bacteria can be used. Examples of the intestinal microorganism-derived phospholipase A include phospholipase A encoded by ECSF — 3660.
本発明の方法の好ましい実施形態の１つは、グラム陰性菌の外膜に発現されているホスホリパーゼＡを標的にしてリゾホスファチジルセリン生成に関与する腸内微生物数を測定する工程を含む炎症性腸疾患を検査する方法である。ホスホリパ－ゼＡとして、好ましくは、ＥＣＳＦ＿３６６０にコードされるホスホリパ－ゼＡが挙げられる。
One preferred embodiment of the method of the present invention comprises the step of targeting phospholipase A expressed in the outer membrane of Gram-negative bacteria to measure the number of intestinal microorganisms involved in lysophosphatidylserine production. It is a method of examining a disease. Phospholipase A preferably includes phospholipase A encoded by ECSF_3660.
ホスホリパ－ゼＡを測定する方法として、ホスホリパ－ゼＡのタンパク質量を測定する方法とホスホリパ－ゼＡの活性を測定する方法が挙げられる。
Methods for measuring phospholipase A include a method for measuring the amount of phospholipase A protein and a method for measuring phospholipase A activity.
ホスホリパ－ゼＡのタンパク質量の測定方法は制限されるものではなく、例えば、ホスホリパーゼＡに対する抗体を用いた測定方法が挙げられる。好ましくは、ＥＣＳＦ＿３６６０にコードされるホスホリパーゼＡに対する抗体を用いた測定方法が挙げられる。抗体を用いた測定方法の例としてＥＬＩＳＡ法、イムノクロマトグラフ法、ラテックス凝集法等が挙げられる。
The method for measuring the amount of phospholipase A protein is not limited, and for example, a method using an antibody against phospholipase A can be used. A preferred measurement method includes an antibody against phospholipase A encoded by ECSF — 3660. Examples of measurement methods using antibodies include ELISA, immunochromatography, latex agglutination, and the like.
ホスホリパーゼＡの活性の測定方法は制限されるものではない。例えば酵素活性検出比色法、蛍光発生脂質を基質とする方法（David Gonzalez-Bullon et al. Toxins 2018）が挙げられる。
The method for measuring the activity of phospholipase A is not limited. Examples include a colorimetric method for detecting enzyme activity and a method using a fluorogenic lipid as a substrate (David Gonzalez-Bullon et al. Toxins 2018).
本発明の炎症性腸疾患を検査する方法は、被検体より得られた試料中の腸内微生物由来ホスホリパーゼＡをコードする遺伝子の量を測定する工程を含む方法でもあり得る。
The method of testing for inflammatory bowel disease of the present invention can also be a method comprising the step of measuring the amount of a gene encoding phospholipase A derived from intestinal microorganisms in a sample obtained from a subject.
前記腸内微生物由来ホスホリパーゼＡをコードする遺伝子は、ＬｙｓｏＰＳの生成に関与している。ホスホリパーゼＡをコードする遺伝子の量を測定する方法として、例えば、定量的リアルタイムＰＣＲ法が挙げられる。腸内微生物由来ホスホリパーゼＡをコードする遺伝子の好ましい遺伝子として、ＥＣＳＦ＿３６６０が挙げられる。ＥＣＳＦ＿３６６０のＤＮＡ配列（配列番号１）よりプライマーをデザインし、定量的リアルタイムＰＣＲをすることができる。プライマーの一例を以下に示す。
The gene encoding the gut microbe-derived phospholipase A is involved in the production of LysoPS. Methods for measuring the amount of the gene encoding phospholipase A include, for example, quantitative real-time PCR. A preferred gene encoding phospholipase A derived from intestinal microorganisms is ECSF_3660. Primers can be designed from the ECSF — 3660 DNA sequence (SEQ ID NO: 1) to perform quantitative real-time PCR. An example of primers is shown below.
Ｆｗ１：５’－ＡＴＧＣＧＧＡＣＴＣＴＧＣＡＧＧＧＣＴＧＧＴＴＧＴＴＧＣＣＧ－３’(配列番号３)
Ｒｖ１：５’－ＴＣＡＡＡＡＣＡＧＧＴＣＧＴＴＴＡＧＣＡＴＡＡＣＴＣＣＣＡＣ－３’（配列番号４） Fw1: 5′-ATGCGGACTCTGCAGGGCTGGTTGTTGCCG-3′ (SEQ ID NO: 3)
Rv1: 5′-TCAAAACAGGTCGTTTAGCATAACTCCCAC-3′ (SEQ ID NO: 4)
Ｒｖ１：５’－ＴＣＡＡＡＡＣＡＧＧＴＣＧＴＴＴＡＧＣＡＴＡＡＣＴＣＣＣＡＣ－３’（配列番号４） Fw1: 5′-ATGCGGACTCTGCAGGGCTGGTTGTTGCCG-3′ (SEQ ID NO: 3)
Rv1: 5′-TCAAAACAGGTCGTTTAGCATAACTCCCAC-3′ (SEQ ID NO: 4)
本発明の方法の好ましい実施形態の１つは、被検体から得られた試料中のＥＣＳＦ＿３６６０の量を測定する工程を含む炎症性腸疾患を検査する方法である。
One preferred embodiment of the method of the present invention is a method of testing for inflammatory bowel disease comprising measuring the amount of ECSF — 3660 in a sample obtained from a subject.
本発明の炎症性腸疾患を検査する方法は、被検体より得られた試料中のＬｙｓｏＰＳの濃度を測定する工程を含む方法でもあり得る。
The method of testing for inflammatory bowel disease of the present invention can also be a method comprising the step of measuring the concentration of LysoPS in a sample obtained from a subject.
ＬｙｓｏＰＳを構成しているアシル基は、１０種類以上存在する。本発明の測定対象のＬｙｓｏＰＳはアシル基の種類に限定されるものではないが、好ましくは、炭素数が１６－２２のアシル基であり、さらに好ましくは、炭素数が１８－２０のアシル基であり、さらに好ましくは、炭素数が１８のアシル基である。最も好ましくは、１８：０ＬｙｓｏＰＳおよび１８：１ＬｙｓｏＰＳである。１８：０はステアリン酸、１８：１はオレイン酸を示す。
There are 10 or more types of acyl groups constituting LysoPS. LysoPS to be measured in the present invention is not limited to the type of acyl group, but is preferably an acyl group having 16 to 22 carbon atoms, more preferably an acyl group having 18 to 20 carbon atoms. and more preferably an acyl group having 18 carbon atoms. Most preferred are 18:0 LysoPS and 18:1 LysoPS. 18:0 indicates stearic acid and 18:1 indicates oleic acid.
本発明の炎症性腸疾患を検査する方法は、少なくとも１つのＬｙｓｏＰＳを測定する。好ましくは、少なくとも１８：１ＬｙｓｏＰＳを測定する。好ましくは、少なくとも１８：１ＬｙｓｏＰＳおよび１８：０ＬｙｓｏＰＳを測定する。
The method of testing for inflammatory bowel disease of the present invention measures at least one LysoPS. Preferably, at least 18:1 LysoPS is measured. Preferably, at least 18:1 LysoPS and 18:0 LysoPS are measured.
ＬｙｓｏＰＳの濃度の測定方法は制限されるものではない。例えば、ＨＰＬＣと質量分析の組み合わせによる測定法、酵素定量法などが挙げられる。好ましくはＬＣ／ＥＳＩ－ＭＳによる測定法が挙げられる。
The method for measuring the concentration of LysoPS is not limited. Examples thereof include a measurement method using a combination of HPLC and mass spectrometry, an enzymatic assay method, and the like. A preferred measurement method is LC/ESI-MS.
本発明の方法は、ＬｙｓｏＰＳの測定に加えて、他のリゾリン脂質の少なくとも１つを測定する工程を含み得る。他のリゾリン脂質として、好ましくはリゾホスファチジルコリン（以下、ＬｙｓｏＰＣともいう）が挙げられる。好ましいリゾホスファチジルコリンとしては、１８：０ＬｙｓｏＰＣ、１８：１ＬｙｓｏＰＣ、および２２：１ＬｙｓｏＰＣが挙げられる。１つの実施形態として、試料中の複数リゾリン脂質を測定し、その検査結果を総合して診断等を行う態様が挙げられる。他の実施形態では、試料中の複数リゾリン脂質の測定結果を総合して検査値とすることができる。例えば、１８：０ＬｙｓｏＰＳ、１８：１ＬｙｓｏＰＳ、１８：０ＬｙｓｏＰＣ、１８：１ＬｙｓｏＰＣ、および２２：１ＬｙｓｏＰＣの全部または一部の糞便中濃度を点数化してその合計を検査値とすることができる。好ましくは、該５つのリゾリン脂質の全部の各濃度を点数化しその合計値を検査値とする。
The method of the invention may comprise measuring at least one other lysophospholipid in addition to measuring LysoPS. Other lysophospholipids preferably include lysophosphatidylcholine (hereinafter also referred to as LysoPC). Preferred lysophosphatidylcholines include 18:0 LysoPC, 18:1 LysoPC, and 22:1 LysoPC. One embodiment includes a mode in which a plurality of lysophospholipids in a sample are measured and the test results are integrated for diagnosis and the like. In another embodiment, the measurement results of multiple lysophospholipids in a sample can be combined to form a test value. For example, fecal concentrations of all or part of 18:0LysoPS, 18:1LysoPS, 18:0LysoPC, 18:1LysoPC, and 22:1LysoPC can be scored and the sum thereof can be used as the test value. Preferably, each concentration of all five lysophospholipids is scored and the total value is used as the test value.
本発明の炎症性腸疾患を検査する方法は、被検体より得られた試料中のホスホリパ－ゼＡを測定する工程を含む方法でもあり得る。
The method of testing for inflammatory bowel disease of the present invention can also be a method comprising the step of measuring phospholipase A in a sample obtained from a subject.
前記ホスホリパ－ゼＡは、好ましくは、腸内微生物由来ホスホリパ－ゼＡである、腸内微生物が発現するホスホリパーゼＡは、ホストの腸管上皮細胞の細胞膜を破壊しＬｙｓｏＰＳを生成する。
The phospholipase A is preferably an intestinal microorganism-derived phospholipase A. The phospholipase A expressed by the intestinal microorganism destroys the cell membrane of the host intestinal epithelial cells to produce LysoPS.
前記腸内微生物由来ホスホリパ－ゼＡとしては、好ましくは、ＥＣＳＦ＿３６６０にコードされるホスホリパ－ゼＡが挙げられる。ＥＣＳＦ＿３６６０は大腸菌（Escherichia coli）およびその他の微生物（肺炎桿菌（Klebsiella pneumoniae）等）由来のホスホリパーゼＡをコードする遺伝子（配列番号１、配列番号２）である。
Phospholipase A derived from intestinal microorganisms preferably includes phospholipase A encoded by ECSF_3660. ECSF — 3660 is a gene (SEQ ID NO: 1, SEQ ID NO: 2) encoding phospholipase A derived from Escherichia coli and other microorganisms (such as Klebsiella pneumoniae).
ホスホリパーゼＡは、微生物表面、微生物内、および微生物外に存在し得、測定対象としては、微生物表面に存在するホスホリパーゼＡ、微生物外に存在するホスホリパーゼＡ、および微生物内に存在するホスホリパーゼＡが挙げられる。発現ホスホリパーゼＡの総量を測定できる方法でもよく、また、存在場所を特定できるような測定方法でもよい。グラム陰性菌ではホスホリパーゼＡが外膜に存在することより、グラム陰性菌の外膜に存在するホスホリパーゼＡを測定する方法が挙げられる。
Phospholipase A can be present on the surface of microorganisms, inside microorganisms, and outside microorganisms, and measurement targets include phospholipase A present on the surface of microorganisms, phospholipase A present outside microorganisms, and phospholipase A present inside microorganisms. . A method capable of measuring the total amount of expressed phospholipase A, or a method capable of specifying the location thereof may be used. Since phospholipase A is present in the outer membrane of Gram-negative bacteria, a method of measuring phospholipase A present in the outer membrane of Gram-negative bacteria can be used.
本発明の方法の好ましい実施形態の１つは、グラム陰性菌の外膜に発現されているホスホリパーゼＡを測定する工程を含む炎症性腸疾患を検査する方法である。ホスホリパ－ゼＡとしては、好ましくは、ＥＣＳＦ＿３６６０にコードされるホスホリパ－ゼＡが挙げられる。
One preferred embodiment of the method of the present invention is a method of testing for inflammatory bowel disease comprising measuring phospholipase A expressed in the outer membrane of Gram-negative bacteria. Phospholipase A preferably includes phospholipase A encoded by ECSF_3660.
ホスホリパ－ゼＡを測定する方法として、ホスホリパ－ゼＡのタンパク質量を測定する方法とホスホリパ－ゼＡの活性を測定する方法が挙げられる。
Methods for measuring phospholipase A include a method for measuring the amount of phospholipase A protein and a method for measuring phospholipase A activity.
本発明の方法の好ましい実施形態の１つは、被検体から得られた試料中の腸内微生物由来ホスホリパーゼＡのタンパク質量を測定する工程を含む炎症性腸疾患を検査する方法である。さらに好ましくは、ＥＣＳＦ＿３６６０にコードされるホスホリパーゼＡのタンパク質量を測定する工程を含む炎症性腸疾患を検査する方法である。
One of the preferred embodiments of the method of the present invention is a method of testing for inflammatory bowel disease, comprising the step of measuring the protein amount of intestinal microorganism-derived phospholipase A in a sample obtained from a subject. More preferably, it is a method of testing for inflammatory bowel disease, comprising the step of measuring the amount of phospholipase A protein encoded by ECSF — 3660.
腸内微生物由来ホスホリパ－ゼＡのタンパク質量の測定方法は制限されるものではない。例えば、ホスホリパーゼＡに対する抗体を用いた測定方法が挙げられる。好ましくは、ＥＣＳＦ＿３６６０にコードされるホスホリパーゼＡに対する抗体を用いた測定方法が挙げられる。その測定方法の例としてＥＬＩＳＡ法、イムノクロマトグラフ法、ラテックス凝集法等が挙げられる。
The method for measuring the protein amount of phospholipase A derived from intestinal microorganisms is not limited. For example, a measuring method using an antibody against phospholipase A can be mentioned. A preferred measurement method includes an antibody against phospholipase A encoded by ECSF — 3660. Examples of the measuring method include ELISA method, immunochromatographic method, latex agglutination method and the like.
本発明の方法の好ましい実施形態の１つは、被検体から得られた試料中の腸内微生物由来ホスホリパーゼＡの活性を測定する工程を含む炎症性腸疾患を検査する方法である。さらに好ましくは、遺伝子ＥＣＳＦ＿３６６０にコードされるホスホリパーゼＡの活性を測定する工程を含む炎症性腸疾患を検査する方法である。ホスホリパーゼＡの活性の測定方法は制限されるものではない。例えば酵素活性検出比色法、蛍光発生脂質を基質とする方法（David Gonzalez-Bullon et al. Toxins 2018）が挙げられる。
One of the preferred embodiments of the method of the present invention is a method of testing for inflammatory bowel disease comprising the step of measuring the activity of phospholipase A derived from intestinal microorganisms in a sample obtained from a subject. More preferably, it is a method of testing for inflammatory bowel disease, comprising the step of measuring the activity of phospholipase A encoded by gene ECSF_3660. The method for measuring the activity of phospholipase A is not limited. Examples include a colorimetric method for detecting enzyme activity and a method using a fluorogenic lipid as a substrate (David Gonzalez-Bullon et al. Toxins 2018).
被検体としては、ヒトおよびヒト以外の動物が挙げられる。ヒト以外の動物としては好ましくは哺乳類であり、例えばイヌ、ネコ、ハムスターなどの愛玩動物、牛等の家畜等が挙げられる。好ましくはヒトである。
Subjects include humans and non-human animals. Animals other than humans are preferably mammals, such as pets such as dogs, cats, and hamsters, and domestic animals such as cattle. Humans are preferred.
被検体から得られた試料は、好適には、腸内微生物叢を含む試料である。さらに好適には糞便サンプルである。
The sample obtained from the subject is preferably a sample containing intestinal microflora. More preferably, it is a stool sample.
炎症性腸疾患は、腸に炎症を起こす疾患であり、本発明において炎症性腸疾患には、クローン病および潰瘍性大腸炎の他に腸管ベーチェット病、非特異性多発小腸潰瘍、家族性地中海熱関連腸炎等も含まれる。炎症性腸疾患は、慢性的な下痢や血便、腹痛などの症状を特徴とし、他にも発熱、肛門痛などの症状が含まれる。病理学的検査では、クローン病では非乾酪性類上皮細胞肉芽腫、潰瘍性大腸炎では陰窩膿瘍が特徴とされる。
Inflammatory bowel disease is a disease that causes inflammation in the intestine, and in the present invention, inflammatory bowel disease includes Crohn's disease and ulcerative colitis, as well as intestinal Behcet's disease, non-specific multiple small intestinal ulcers, familial Mediterranean fever. Associated enteritis and the like are also included. Inflammatory bowel disease is characterized by symptoms such as chronic diarrhea, bloody stools, abdominal pain, and other symptoms such as fever and anal pain. Pathologic examination is characterized by noncaseating epithelioid granulomas in Crohn's disease and crypt abscesses in ulcerative colitis.
本発明の方法は、Ｔｈ１細胞の過剰活性化を伴う炎症性腸疾患の検査に好適に用いられ得る。
INDUSTRIAL APPLICABILITY The method of the present invention can be suitably used for examination of inflammatory bowel disease accompanied by excessive activation of Th1 cells.
Ｔｈ１細胞の過剰活性化は、Ｔｈ１細胞からのＩＦＮ－γ等の炎症性サイトカインの分泌量の上昇を特徴とする。Ｔｈ１細胞の過剰活性化には、細胞あたりの炎症性サイトカインの分泌量上昇、活性酸素種（ＲＯＳ）産生亢進、炎症性サイトカイン産生Ｔｈ１細胞数の増加、ナイーブＣＤ４＋Ｔ細胞のＴｈ１細胞への分化促進、およびＴｈ１細胞の代謝促進（特に解糖系の促進）を含む。
Hyperactivation of Th1 cells is characterized by increased secretion of inflammatory cytokines such as IFN-γ from Th1 cells. Hyperactivation of Th1 cells includes increased secretion of inflammatory cytokines per cell, increased production of reactive oxygen species (ROS), increased number of inflammatory cytokine-producing Th1 cells, and differentiation of naive CD4 + T cells into Th1 cells. and promotion of Th1 cell metabolism (particularly promotion of glycolysis).
クローン病と潰瘍性大腸炎は、ともに免疫細胞が関与する難治性腸管炎症性疾患であり、本発明の方法は、クローン病と潰瘍性大腸炎の検査に好適に用いられ得る。特に好適には、本発明の方法はクローン病の検査に用いられ得る。
Both Crohn's disease and ulcerative colitis are intractable intestinal inflammatory diseases in which immune cells are involved, and the method of the present invention can be suitably used for examination of Crohn's disease and ulcerative colitis. Particularly preferably, the method of the present invention can be used to test for Crohn's disease.
本発明の炎症性腸疾患を検査する方法における検査には、炎症性腸疾患と関連した全ての検査を含み、炎症性腸疾患の診断時の検査、炎症性腸疾患発症リスクの検査、および炎症性腸疾患発症後の経過観察のための検査等を含む。
Tests in the method of testing for inflammatory bowel disease of the present invention include all tests related to inflammatory bowel disease, tests at the time of diagnosis of inflammatory bowel disease, tests for the risk of developing inflammatory bowel disease, and tests for the risk of developing inflammatory bowel disease. This includes examinations for follow-up after the onset of enteropathy.
本発明の方法は、被検体より得られた試料の検査値と対照試料の検査値が比較される工程をさらに含み得る。試料中および対照試料中の同一検査対象を、同一の方法によって測定した検査値を比較する。対照試料が健常者から得られた試料である場合、被検体より得られた試料の検査値が対照試料の検査値より高値の場合は、被検体は炎症性腸疾患である、または炎症性腸疾患を発症するリスクが高いと判定され得る。健常者から得られた試料（対照試料）の検査値は、予め定められた健常者の標準値を含む。対照試料が同一被検体の過去の試料である場合は、被検体より得られた試料中の検査値が対照試料の検査値より高値の場合は、被検体は病状が悪化している、または悪化するリスクが高いと判定され得、逆に被検体より得られた試料中の検査値が対照試料の検査値より低値の場合は、被検体は病状が改善している、または改善する可能性が高いと判定され得る。
The method of the invention may further comprise the step in which the test value of the sample obtained from the subject and the test value of the control sample are compared. The test values measured by the same method for the same test object in the sample and the control sample are compared. When the control sample is a sample obtained from a healthy subject, if the test value of the sample obtained from the subject is higher than the test value of the control sample, the subject has inflammatory bowel disease or inflammatory bowel It can be determined that the risk of developing the disease is high. The test values of samples obtained from healthy subjects (control samples) include predetermined standard values for healthy subjects. When the control sample is a past sample of the same subject, if the test value in the sample obtained from the subject is higher than the test value of the control sample, the subject is worsening or worsening If the test value in the sample obtained from the subject is lower than the test value in the control sample, the subject has improved or may improve can be determined to be high.
本発明の炎症性腸疾患の予防用または治療用の物質または微生物のスクリーニング方法におけるＬｙｓｏＰＳ生成に関与する腸内微生物数の測定方法は、特に限定されるものではない。好ましい方法は、該腸内微生物の培養液と候補物質または候補微生物を接触させた後に該腸内微生物数を測定する方法が挙げられる。また、ヒトまたはヒト以外の動物に候補物質または微生物を経口摂取させて糞便サンプル中の該腸内微生物数を測定する方法が挙げられる。手法としては、定量的リアルタイムＰＣＲ法、コロニー数計測法等が挙げられる。
The method for measuring the number of intestinal microorganisms involved in LysoPS production in the screening method for substances or microorganisms for prevention or treatment of inflammatory bowel disease of the present invention is not particularly limited. A preferred method includes a method of measuring the number of intestinal microorganisms after contacting the culture solution of the intestinal microorganisms with the candidate substance or candidate microorganisms. Another example is a method in which a candidate substance or microorganism is orally ingested by humans or animals other than humans, and the number of intestinal microorganisms in a stool sample is measured. Techniques include quantitative real-time PCR, colony counting, and the like.
本発明の炎症性腸疾患の予防用または治療用の物質のスクリーニング方法は、ホスホリパーゼＡの活性を測定する工程を含む方法であり得る。好適には、該ホスホリパーゼＡは腸内微生物由来ホスホリパーゼＡである。ホスホリパーゼＡの活性の測定方法として好ましい方法は、ホスホリパーゼＡと候補物質を接触させた後にホスホリパーゼＡ活性を測定する方法が挙げられる。または、前記腸内微生物の培養液と候補物質を接触させた後にホスホリパーゼＡ活性を測定する方法が挙げられる。該ホスホリパーゼＡは、好ましくは、ホスホリパーゼＡ１である。
The method of screening for a preventive or therapeutic substance for inflammatory bowel disease of the present invention can be a method comprising the step of measuring phospholipase A activity. Suitably, said phospholipase A is an intestinal microbial phospholipase A. A preferred method for measuring the activity of phospholipase A includes a method of contacting phospholipase A with a candidate substance and then measuring phospholipase A activity. Alternatively, there is a method of measuring the phospholipase A activity after bringing the candidate substance into contact with the culture solution of the intestinal microorganism. Said phospholipase A is preferably phospholipase A1.
前記候補物質は、例えば化合物ライブラリーの化合物を用いることができる。化合物ライブラリーは、公知のものでも公知でないものでもよい。公知の化合物ライブラリーとしては、既に食品（例えば、米国食品医薬品局（ＦＤＡ））または医薬品（例えば、欧州医薬品審査庁（ＥＭＥＡ））の承認を得た化合物を集めた化合物ライブラリー（例えば、ＰＲＥＳＴＷＩＣＫ ＣＨＥＭＩＣＡＬライブラリー）（これは、特許期間が満了した化合物を集めたものである）、および未だそれら食品または医薬品の承認を得ていない化合物を集めた化合物ライブラリー等が挙げられる。
As the candidate substance, for example, compounds in a compound library can be used. A compound library may be known or unknown. As a known compound library, a compound library (for example, PRESTWICK Chemical library) (which is a collection of compounds whose patent period has expired), and a compound library which is a collection of compounds that have not yet been approved for food or pharmaceutical use.
本発明のスクリーニング方法は、物質または微生物を選別する工程をさらに含んでもよい。
The screening method of the present invention may further comprise a step of selecting substances or microorganisms.
本発明のスクリーニング方法で得られた物質または微生物は、炎症性腸疾患の予防または治療に用いることができる。
Substances or microorganisms obtained by the screening method of the present invention can be used for prevention or treatment of inflammatory bowel disease.
本発明の炎症性腸疾患の予防用または治療用組成物は、リゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させる物質または微生物を有効成分として含有する。または、本発明の炎症性腸疾患の予防用または治療用組成物は、ホスホリパーゼＡの活性を抑制する物質を有効成分として含有する。
The preventive or therapeutic composition for inflammatory bowel disease of the present invention contains, as an active ingredient, a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production. Alternatively, the preventive or therapeutic composition for inflammatory bowel disease of the present invention contains a substance that suppresses the activity of phospholipase A as an active ingredient.
前記予防用または治療用組成物には、薬剤、食品、飲料等が含まれる。
The prophylactic or therapeutic compositions include drugs, foods, beverages and the like.
前記リゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させる物質または微生物は、前記スクリーニング方法で得られた物質または微生物であり得る。前記物質としては、例えば抗菌剤および吸着剤が挙げられる。前記微生物としては、乳酸菌、ビフィズス菌、Xenorhabdus、Photorhabdusおよび標的微生物に感染するファージ（例えば、Enterobacteria Phage P1 およびEnterobacteria phage μ）等が含まれ得る。
The substance or microorganism that eliminates or reduces intestinal microorganisms involved in the production of lysophosphatidylserine can be the substance or microorganism obtained by the screening method. Such substances include, for example, antimicrobial agents and adsorbents. The microorganisms may include lactic acid bacteria, bifidobacteria, Xenorhabdus, Photorhabdus, and phages (eg, Enterobacteria Phage P1 and Enterobacteria phage μ) that infect target microorganisms.
前記ホスホリパーゼＡの活性を抑制する物質には、ホスホリパーゼＡの発現を抑制する物質および生合成を抑制する物質等も含まれる。ホスホリパーゼＡの活性を抑制する物質は、前記スクリーニング方法で得られた物質であり得る。前記ホスホリパーゼＡの活性を抑制する物質は、好ましくは腸内微生物由来ホスホリパーゼＡの活性を抑制する物質である。前記ホスホリパーゼＡの活性を抑制する物質としては、例えば、ホスホリパーゼＡ阻害剤、ホスホリパーゼＡに対する抗体、ホスホリパーゼＡ遺伝子のアンチセンスＲＮＡ、ホスホリパーゼＡを阻害する細菌が挙げられる。ホスホリパーゼＡ阻害剤としては、例えば、グリチルリチン酸、グリチルレチン酸、４－ｂｒｏｍｏｐｈｅｎａｃｙｌ ｂｒｏｍｉｄｅ、ｄａｒａｐｌａｄｉｂ、Ａｎａｇｒｅｌｉｄｅ、ＯＢＡＡ（３－（４－ｏｃｔａｄｅｃｙｌ）ｂｅｎｚｏｙｌａｃｒｙｌｉｃ ａｃｉｄ）、ＰＡＣＯＣＦ３（ｐａｌｍｉｔｏｙｌ ｔｒｉｆｌｕｏｒｏｍｅｔｈｙｌ ｋｅｔｏｎｅ）、ＹＭ２６７３４、ＬＹ３１５９２０（ｖａｒｅｓｐｌａｄｉｂ ｍｅｔｈｙｌ）が挙げられる。
The substance that suppresses the activity of phospholipase A includes a substance that suppresses expression of phospholipase A and a substance that suppresses biosynthesis. A substance that suppresses the activity of phospholipase A can be a substance obtained by the screening method. The substance that suppresses the activity of phospholipase A is preferably a substance that suppresses the activity of phospholipase A derived from intestinal microorganisms. Examples of the substance that suppresses the activity of phospholipase A include phospholipase A inhibitors, antibodies against phospholipase A, antisense RNA of the phospholipase A gene, and bacteria that inhibit phospholipase A.ホスホリパーゼＡ阻害剤としては、例えば、グリチルリチン酸、グリチルレチン酸、４－ｂｒｏｍｏｐｈｅｎａｃｙｌ ｂｒｏｍｉｄｅ、ｄａｒａｐｌａｄｉｂ、Ａｎａｇｒｅｌｉｄｅ、ＯＢＡＡ（３－（４－ｏｃｔａｄｅｃｙｌ）ｂｅｎｚｏｙｌａｃｒｙｌｉｃ ａｃｉｄ）、ＰＡＣＯＣＦ３（ｐａｌｍｉｔｏｙｌ ｔｒｉｆｌｕｏｒｏｍｅｔｈｙｌ ｋｅｔｏｎｅ）、ＹＭ２６７３４、ＬＹ３１５９２０（ｖａｒｅｓｐｌａｄｉｂ ｍｅｔｈｙｌ ).
本発明において、炎症性腸疾患の治療には、炎症性腸疾患の症状の改善、軽減、増悪抑制、進行の遅延、および再燃抑制等が含まれる。
In the present invention, treatment of inflammatory bowel disease includes amelioration, alleviation, suppression of exacerbation, delay of progression, suppression of relapse, and the like of symptoms of inflammatory bowel disease.
本発明の組成物は、Ｔｈ１細胞の過剰活性化を伴う炎症性腸疾患の予防および治療に好適に用いられ得る。
The composition of the present invention can be suitably used for prevention and treatment of inflammatory bowel disease accompanied by overactivation of Th1 cells.
ＬｙｓｏＰＳの作用により、ＬｙｓｏＰＳ受容体を介してＴｈ１細胞の過剰活性化が発生し、炎症性腸疾患の症状が悪化する。このＴｈ１細胞の過剰活性化には、細胞あたりの炎症性サイトカインの分泌量上昇、炎症性サイトカイン産生Ｔｈ１細胞数の増加、ＲＯＳ産生亢進、ナイーブＣＤ４＋Ｔ細胞のＴｈ１細胞への分化促進、およびＴｈ１細胞の代謝促進（特に解糖系の促進）を含む。本発明の組成物は、Ｔｈ１細胞の過剰活性化を抑制することができ、Ｔｈ１細胞の過剰活性化を伴う炎症性腸疾患を好適に予防または治療することができる。
The action of LysoPS causes hyperactivation of Th1 cells via the LysoPS receptor, exacerbating the symptoms of inflammatory bowel disease. This hyperactivation of Th1 cells includes increased secretion of inflammatory cytokines per cell, increased number of inflammatory cytokine-producing Th1 cells, enhanced ROS production, enhanced differentiation of naive CD4 + T cells into Th1 cells, and Th1 Includes promotion of cell metabolism (particularly promotion of glycolysis). INDUSTRIAL APPLICABILITY The composition of the present invention can suppress overactivation of Th1 cells, and can suitably prevent or treat inflammatory bowel disease accompanied by overactivation of Th1 cells.
クローン病と潰瘍性大腸炎は、ともに免疫細胞が関与する難治性腸管炎症性疾患であり、本発明の組成物は、クローン病と潰瘍性大腸炎の予防および治療に好適に用いられ得る。特に好適には、本発明の組成物はクローン病の予防および治療に用いられ得る。
Both Crohn's disease and ulcerative colitis are intractable intestinal inflammatory diseases in which immune cells are involved, and the composition of the present invention can be suitably used for the prevention and treatment of Crohn's disease and ulcerative colitis. Particularly preferably, the compositions of the invention can be used for the prevention and treatment of Crohn's disease.
本発明の組成物は、２種以上の有効成分を含有していてもよい。他の炎症性腸疾患治療薬を含有していてもよく、また、炎症性腸疾患治療薬以外の薬物を含有していてもよい。本発明の組成物は他の炎症性腸疾患治療薬と併用されてもよい。
The composition of the present invention may contain two or more active ingredients. Other inflammatory bowel disease therapeutic agents may be contained, and drugs other than inflammatory bowel disease therapeutic agents may be contained. The composition of the present invention may be used in combination with other therapeutic agents for inflammatory bowel disease.
本発明の組成物は医薬的に許容される担体を含むことができる。
Compositions of the invention can include a pharmaceutically acceptable carrier.
その様な担体としては、賦形剤（例えば、マンニトール、ソルビトールの如き糖誘導体；トウモロコシデンプン、バレイショデンプンの如きデンプン誘導体；または、結晶セルロースの如きセルロース誘導体等）、滑沢剤（例えば、ステアリン酸マグネシウムの如きステアリン酸金属塩；またはタルク等）、結合剤（例えば、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、またはポリビニルピロリドン等）、崩壊剤（例えば、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウムの如きセルロース誘導体等）、水、防腐剤（例えば、メチルパラベン、プロピルパラベンの如きパラオキシ安息香酸エステル類；またはクロロブタノール、ベンジルアルコールの如きアルコール類等）、ｐＨ調整剤（例えば、塩酸、硫酸またはリン酸等の無機酸、酢酸、コハク酸、フマル酸またはリンゴ酸等の有機酸、あるいはこれらの塩等）、ならびに希釈剤（例えば、注射用水等）等の通常使用される医薬製剤用担体を、単独または２種以上を混合して配合することができる。本発明の組成物には有効成分を水に溶解させた液剤を含む。
Examples of such carriers include excipients (e.g., sugar derivatives such as mannitol and sorbitol; starch derivatives such as corn starch and potato starch; or cellulose derivatives such as crystalline cellulose), lubricants (e.g., stearic acid metal stearates such as magnesium; or talc, etc.), binders (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc.), disintegrants (e.g., carboxymethylcellulose, cellulose derivatives such as carboxymethylcellulose calcium, etc.), Water, preservatives (e.g. paraoxybenzoic acid esters such as methylparaben and propylparaben; or alcohols such as chlorobutanol and benzyl alcohol), pH adjusters (e.g. inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid, acetic acid , Organic acids such as succinic acid, fumaric acid or malic acid, or salts thereof), and commonly used carriers for pharmaceutical formulations such as diluents (e.g., water for injection, etc.), alone or in combination of two or more can be blended as The composition of the present invention contains a liquid formulation in which the active ingredient is dissolved in water.
本発明の組成物は、食品、飲料である場合には、既存食品または飲料と混合された形態の組成物でもよい。
When the composition of the present invention is a food or beverage, it may be in the form of being mixed with an existing food or beverage.
本発明の有効成分は、必要に応じて上記の担体と混合した後、錠剤、顆粒剤、カプセル剤、粉末剤、溶液製剤、懸濁液製剤、もしくは乳化液剤等の剤形で経口投与することができる。また、坐剤、経腸剤、注射剤、静脈内点滴剤、経皮剤、経粘膜剤、もしくは吸入剤等の剤形で非経口投与することができる。
The active ingredient of the present invention can be orally administered in dosage forms such as tablets, granules, capsules, powders, solution formulations, suspension formulations, or emulsified liquid formulations after mixing with the above-mentioned carriers, if necessary. can be done. It can also be administered parenterally in dosage forms such as suppositories, enteral agents, injections, intravenous drips, transdermal agents, transmucosal agents, or inhalants.
本発明の有効成分は、上記の剤形に製剤化した後、または食品や飲料に混合した後それを必要とする対象、例えばヒトまたは動物、好ましくはヒトに投与される。
The active ingredient of the present invention is administered to a subject in need thereof, such as a human or an animal, preferably a human, after being formulated into the above dosage form or mixed with food or beverage.
本発明の有効成分の投与量および投与回数は、症状の重篤度、患者の年齢、体重、性別、薬物の種類、剤形、投与経路等の条件によって適宜変化しうる。ヒトに投与する場合、有効成分は、例えば非経口的には皮下、静脈内、腹腔内、筋肉内、または直腸内等に、１回の投与当たり、約０．０１～１０ｍｇ／ｋｇ体重、好ましくは約０．１～５ｍｇ／ｋｇ体重、特に好ましくは約０．３～３ｍｇ／ｋｇ体重、また経口的には約０．０１～１００ｍｇ／ｋｇ体重、好ましくは約０．１～５０ｍｇ／ｋｇ体重、特に好ましくは約１～３０ｍｇ／ｋｇ体重投与される。また、投与回数は、１日当たり１回または複数回、例えば１日当たり１～３回、１～２回、または１回であってよい。
The dose and frequency of administration of the active ingredient of the present invention can be appropriately changed depending on conditions such as severity of symptoms, patient's age, body weight, sex, type of drug, dosage form, administration route and the like. When administered to humans, the active ingredient is administered parenterally, for example, subcutaneously, intravenously, intraperitoneally, intramuscularly, or intrarectally, at a dosage of about 0.01 to 10 mg/kg body weight, preferably about 0.01 to 10 mg/kg body weight per administration. about 0.1 to 5 mg/kg body weight, particularly preferably about 0.3 to 3 mg/kg body weight, and orally about 0.01 to 100 mg/kg body weight, preferably about 0.1 to 50 mg/kg body weight. , particularly preferably about 1-30 mg/kg body weight. Also, the frequency of administration may be once or multiple times per day, eg, 1-3 times, 1-2 times, or 1 time per day.
本発明の有効成分は、公知の方法に従って製造できる。
The active ingredient of the present invention can be produced according to known methods.
以下に、実施例を挙げて本発明をさらに具体的に説明するが、本発明はこれらに限定されるものではない。
EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these.
＜材料と試験方法＞
<Materials and test methods>
＜マウス＞
Ｃ５７ＢＬ／６Ｊのマウスは日本クレア社（東京、日本）から購入した。Ｃ５７ＢＬ／６Ｊ ＲＡＧ２欠損マウス（Ｒａｇ２－／－）はTaconic Biosciences, Inc. (Hudson, NY, USA)から購入した。Ｔｓｌ：ＩＣＲ無菌マウスは三協ラボサービス株式会社（東京、日本）から購入した。８-１５週齢の雄のマウスを使用し、すべてのマウスはＳＰＦ（specific pathogen free）環境下で維持した。 <Mouse>
C57BL/6J mice were purchased from Clea Japan (Tokyo, Japan). C57BL/6J RAG2-deficient mice (Rag2 −/− ) were purchased from Taconic Biosciences, Inc. (Hudson, NY, USA). Tsl:ICR germ-free mice were purchased from Sankyo Lab Service Co., Ltd. (Tokyo, Japan). Male mice aged 8-15 weeks were used and all mice were maintained in an SPF (specific pathogen free) environment.
Ｃ５７ＢＬ／６Ｊのマウスは日本クレア社（東京、日本）から購入した。Ｃ５７ＢＬ／６Ｊ ＲＡＧ２欠損マウス（Ｒａｇ２－／－）はTaconic Biosciences, Inc. (Hudson, NY, USA)から購入した。Ｔｓｌ：ＩＣＲ無菌マウスは三協ラボサービス株式会社（東京、日本）から購入した。８-１５週齢の雄のマウスを使用し、すべてのマウスはＳＰＦ（specific pathogen free）環境下で維持した。 <Mouse>
C57BL/6J mice were purchased from Clea Japan (Tokyo, Japan). C57BL/6J RAG2-deficient mice (Rag2 −/− ) were purchased from Taconic Biosciences, Inc. (Hudson, NY, USA). Tsl:ICR germ-free mice were purchased from Sankyo Lab Service Co., Ltd. (Tokyo, Japan). Male mice aged 8-15 weeks were used and all mice were maintained in an SPF (specific pathogen free) environment.
＜試薬＞
2、4、6‐トリニトロベンゼンスルホン酸ナトリウム（ＴＮＢＳ）はSigma‐Aldrich (St. Louis, MO, USA) から購入した。１８：０ ＬｙｓｏＰＳ（1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt)）、および１８：１ ＬｙｓｏＰＳ（1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt)）はAvanti Polar Lipids, Inc (Alabaster, AL, USA) から購入し、７０％エタノールに溶解して試験に用いた。 <Reagent>
Sodium 2,4,6-trinitrobenzenesulfonate (TNBS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 18:0 LysoPS (1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt)), and 18:1 LysoPS (1-oleoyl-2-hydroxy-sn-glycero-3- phospho-L-serine (sodium salt)) was purchased from Avanti Polar Lipids, Inc (Alabaster, AL, USA) and dissolved in 70% ethanol for use in the test.
2、4、6‐トリニトロベンゼンスルホン酸ナトリウム（ＴＮＢＳ）はSigma‐Aldrich (St. Louis, MO, USA) から購入した。１８：０ ＬｙｓｏＰＳ（1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt)）、および１８：１ ＬｙｓｏＰＳ（1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt)）はAvanti Polar Lipids, Inc (Alabaster, AL, USA) から購入し、７０％エタノールに溶解して試験に用いた。 <Reagent>
＜フローサイトメトリー＞
Anti-mouse CD4-PerCP/Cy5.5 (GK1.5), anti-mouse IL-10-PE (JES5-16E3), anti-mouse IFN-γ -FITC (XMG1.2), 7-AAD, anti-human CD4-APC (clone SK3), anti-human CD45RA-Brilliant Violet 421 (HI100), anti-human CD4-Pacific Blue (RPA-T4), anti-human IFN-γ (4S.B3)は、BioLegend (San Diego, CA, USA)から購入した。Anti-mouse CD3e-Pe/Cy7 (145-2C11), anti-mouse IL-17A-Alexa Fluor 647 (TC11-18H10) はBD Biosciences から購入した。マウスおよびヒトのＣＤ４＋Ｔ細胞を、GolgiStop (BD Biosciences)存在下、４ｈ３７℃で、完全ＲＰＭＩ１６４０中５０ｎｇ／ｍｌホルボールミリステートアセテート（ＰＭＡ； Sigma-Aldrich）と５μＭイオノマイシン（Sigma-Aldrich）によって刺激した。細胞表面／細胞内を、Cytofix/Cytoperm Kit Plusを使って染色した。フローサイトメトリー分析はFlowJo software (Tree Star, Ashland, OR, USA)を使用したFACSCanto II flow cytometer (BD Biosciences)によって実行した。 <Flow cytometry>
Anti-mouse CD4-PerCP/Cy5.5 (GK1.5), anti-mouse IL-10-PE (JES5-16E3), anti-mouse IFN-γ -FITC (XMG1.2), 7-AAD, anti- human CD4-APC (clone SK3), anti-human CD45RA-Brilliant Violet 421 (HI100), anti-human CD4-Pacific Blue (RPA-T4), anti-human IFN-γ (4S.B3) are BioLegend (San Diego, Calif., USA). Anti-mouse CD3e-Pe/Cy7 (145-2C11), anti-mouse IL-17A-Alexa Fluor 647 (TC11-18H10) were purchased from BD Biosciences. Mouse and human CD4 + T cells were stimulated with 50 ng/ml phorbol myristate acetate (PMA; Sigma-Aldrich) and 5 μM ionomycin (Sigma-Aldrich) in complete RPMI 1640 for 4 h at 37° C. in the presence of GolgiStop (BD Biosciences). did. Cell surface/intracellular staining was performed using the Cytofix/Cytoperm Kit Plus. Flow cytometric analysis was performed with a FACSCanto II flow cytometer (BD Biosciences) using FlowJo software (Tree Star, Ashland, OR, USA).
Anti-mouse CD4-PerCP/Cy5.5 (GK1.5), anti-mouse IL-10-PE (JES5-16E3), anti-mouse IFN-γ -FITC (XMG1.2), 7-AAD, anti-human CD4-APC (clone SK3), anti-human CD45RA-Brilliant Violet 421 (HI100), anti-human CD4-Pacific Blue (RPA-T4), anti-human IFN-γ (4S.B3)は、BioLegend (San Diego, CA, USA)から購入した。Anti-mouse CD3e-Pe/Cy7 (145-2C11), anti-mouse IL-17A-Alexa Fluor 647 (TC11-18H10) はBD Biosciences から購入した。マウスおよびヒトのＣＤ４＋Ｔ細胞を、GolgiStop (BD Biosciences)存在下、４ｈ３７℃で、完全ＲＰＭＩ１６４０中５０ｎｇ／ｍｌホルボールミリステートアセテート（ＰＭＡ； Sigma-Aldrich）と５μＭイオノマイシン（Sigma-Aldrich）によって刺激した。細胞表面／細胞内を、Cytofix/Cytoperm Kit Plusを使って染色した。フローサイトメトリー分析はFlowJo software (Tree Star, Ashland, OR, USA)を使用したFACSCanto II flow cytometer (BD Biosciences)によって実行した。 <Flow cytometry>
Anti-mouse CD4-PerCP/Cy5.5 (GK1.5), anti-mouse IL-10-PE (JES5-16E3), anti-mouse IFN-γ -FITC (XMG1.2), 7-AAD, anti- human CD4-APC (clone SK3), anti-human CD45RA-Brilliant Violet 421 (HI100), anti-human CD4-Pacific Blue (RPA-T4), anti-human IFN-γ (4S.B3) are BioLegend (San Diego, Calif., USA). Anti-mouse CD3e-Pe/Cy7 (145-2C11), anti-mouse IL-17A-Alexa Fluor 647 (TC11-18H10) were purchased from BD Biosciences. Mouse and human CD4 + T cells were stimulated with 50 ng/ml phorbol myristate acetate (PMA; Sigma-Aldrich) and 5 μM ionomycin (Sigma-Aldrich) in complete RPMI 1640 for 4 h at 37° C. in the presence of GolgiStop (BD Biosciences). did. Cell surface/intracellular staining was performed using the Cytofix/Cytoperm Kit Plus. Flow cytometric analysis was performed with a FACSCanto II flow cytometer (BD Biosciences) using FlowJo software (Tree Star, Ashland, OR, USA).
＜病理組織解析＞
マウスの結腸と回腸のサンプルはホルムアルデヒドで固定し、パラフィン包埋し、４μｍの厚さの切片を作製し、ヘマトキシリンとエオジン（Ｈ＆Ｅ）によって染色した。評価に用いた組織学的スコアは、Iijima, H. et al. J Exp Med 199, 471-482に準じた。 <Pathological tissue analysis>
Mouse colon and ileum samples were formaldehyde-fixed, paraffin-embedded, sectioned at 4 μm thickness, and stained with hematoxylin and eosin (H&E). The histological score used for evaluation was according to Iijima, H. et al. J Exp Med 199, 471-482.
マウスの結腸と回腸のサンプルはホルムアルデヒドで固定し、パラフィン包埋し、４μｍの厚さの切片を作製し、ヘマトキシリンとエオジン（Ｈ＆Ｅ）によって染色した。評価に用いた組織学的スコアは、Iijima, H. et al. J Exp Med 199, 471-482に準じた。 <Pathological tissue analysis>
Mouse colon and ileum samples were formaldehyde-fixed, paraffin-embedded, sectioned at 4 μm thickness, and stained with hematoxylin and eosin (H&E). The histological score used for evaluation was according to Iijima, H. et al. J Exp Med 199, 471-482.
＜統計分析＞
結果は平均と標準偏差で表される。グループ間の平均値の差は、Student's t-test、またはOne-way ANOVAによって検定した。 <Statistical analysis>
Results are expressed as mean and standard deviation. Mean differences between groups were tested by Student's t-test or One-way ANOVA.
結果は平均と標準偏差で表される。グループ間の平均値の差は、Student's t-test、またはOne-way ANOVAによって検定した。 <Statistical analysis>
Results are expressed as mean and standard deviation. Mean differences between groups were tested by Student's t-test or One-way ANOVA.
＜実施例１：ＬｙｓｏＰＳ濃度測定（１）＞
クローン病患者（以下、「ＣＤ患者」ともいう）の腸管腔の脂質プロファイルの特徴を確認した。クローン病患者と健常者からサンプル提供者を募集した。炎症性腸疾患研究の国際機関によって定められた内視鏡的、放射線学的、組織学的、臨床診断基準に従ってＩＢＤ患者を診断した。クローン病患者は、ＣＤＡＩスコアが１５０より小さい場合に寛解期と定義される。研究はヘルシンキ宣言に従って実行され、大阪大学医学部附属病院の倫理審査委員会によって承認された。研究に参加する前に、すべての参加者に書面によるインフォームド・コンセントが行われた。クローン病患者４３人（活動期１９人、寛解期２４人）と健常者４０人から糞便サンプルを収集し、直ちに凍結した。 <Example 1: LysoPS concentration measurement (1)>
The characteristics of the lipid profile of the intestinal lumen of Crohn's disease patients (hereinafter also referred to as "CD patients") were confirmed. Sample donors were recruited from Crohn's disease patients and healthy subjects. IBD patients were diagnosed according to endoscopic, radiological, histological and clinical diagnostic criteria set by the International Organization for Inflammatory Bowel Disease Research. Crohn's disease patients are defined as in remission if their CDAI score is less than 150. The study was performed in accordance with the Declaration of Helsinki and was approved by the ethics review board of Osaka University Hospital. All participants gave written informed consent before participating in the study. Faecal samples were collected from 43 patients with Crohn's disease (19 active, 24 in remission) and 40 healthy subjects and immediately frozen.
クローン病患者（以下、「ＣＤ患者」ともいう）の腸管腔の脂質プロファイルの特徴を確認した。クローン病患者と健常者からサンプル提供者を募集した。炎症性腸疾患研究の国際機関によって定められた内視鏡的、放射線学的、組織学的、臨床診断基準に従ってＩＢＤ患者を診断した。クローン病患者は、ＣＤＡＩスコアが１５０より小さい場合に寛解期と定義される。研究はヘルシンキ宣言に従って実行され、大阪大学医学部附属病院の倫理審査委員会によって承認された。研究に参加する前に、すべての参加者に書面によるインフォームド・コンセントが行われた。クローン病患者４３人（活動期１９人、寛解期２４人）と健常者４０人から糞便サンプルを収集し、直ちに凍結した。 <Example 1: LysoPS concentration measurement (1)>
The characteristics of the lipid profile of the intestinal lumen of Crohn's disease patients (hereinafter also referred to as "CD patients") were confirmed. Sample donors were recruited from Crohn's disease patients and healthy subjects. IBD patients were diagnosed according to endoscopic, radiological, histological and clinical diagnostic criteria set by the International Organization for Inflammatory Bowel Disease Research. Crohn's disease patients are defined as in remission if their CDAI score is less than 150. The study was performed in accordance with the Declaration of Helsinki and was approved by the ethics review board of Osaka University Hospital. All participants gave written informed consent before participating in the study. Faecal samples were collected from 43 patients with Crohn's disease (19 active, 24 in remission) and 40 healthy subjects and immediately frozen.
報告されている方法（Muranaka et al. 2017）に少し修正を加えて、糞便サンプル中の脂質分子の濃度を測定した。すなわち、凍結された糞便サンプルを押しつぶし、いくつかの断片を１％酢酸を含むメタノールの中で均質化した（サンプル１０ｍｇあたり１００μｌ）。その後６０秒氷上で超音波処理した。得られたホモジネート１００μｌ、内部標準を含むクロロホルム、メタノール、およびエタノール（１：２：２比率）混合液２ｍｌ、並びにクロロホルム、メタノール、エタノール、および酢酸（２：１：１：１の比率）混合液１００μｌを混合し、４℃で１０分間遠心分離した。有機層を収集し、ＥＺ－２ Ｐｌｕｓ Ｇｅｎｅｖａｃ ｃｅｎｔｒｉｆｕｇａｌ ｅｖａｐｏｒａｔｏｒ (SP Scientific)で濃縮した。得られた乾燥体を、クロロホルム、メタノール、およびエタノール（１：２：２比率）混合液２８０μｌに溶解した。糞便中の脂質分子種を分析するために、得られた溶液の４μｌをＵＰＬＣ－ＥＳＩ－ＭＳ／ＭＳに使用した。各人の糞便サンプルあたり２回脂質濃度を測定し、これらの濃度の平均がその後の分析のために使われた。データは、ＭｅｔａｂｏＡｎａｌｙｓｔ ５．０（https://www.metaboanalyst.ca/MetaboAnalyst/ModuleView.xhtml）を使って分析された。
We measured the concentration of lipid molecules in fecal samples using a reported method (Muranaka et al. 2017) with minor modifications. Briefly, frozen fecal samples were crushed and some pieces were homogenized in methanol containing 1% acetic acid (100 μl per 10 mg of sample). It was then sonicated on ice for 60 seconds. 100 μl of the resulting homogenate, 2 ml of a mixture of chloroform, methanol and ethanol (1:2:2 ratio) containing the internal standard and a mixture of chloroform, methanol, ethanol and acetic acid (2:1:1:1 ratio). 100 μl were mixed and centrifuged at 4° C. for 10 minutes. The organic layer was collected and concentrated with an EZ-2 Plus Genevac centrifugal evaporator (SP Scientific). The resulting dried product was dissolved in 280 μl of a mixture of chloroform, methanol and ethanol (1:2:2 ratio). 4 μl of the resulting solution was used for UPLC-ESI-MS/MS to analyze lipid molecular species in feces. Lipid concentrations were measured twice per individual fecal sample and the average of these concentrations was used for subsequent analyses. Data were analyzed using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/MetaboAnalyst/ModuleView.xhtml).
糞便中の脂質組成は健常者とＣＤ患者の間で明確に相違していた。血漿中リン脂質濃度(Iwatani et al. 2020)に類似して、全ＬｙｓｏＰＳ、１８：０ＬｙｓｏＰＳ、および１８：１ＬｙｓｏＰＳの濃度は、健常者よりＣＤ患者で高かった。これは、ＬｙｓｏＰＳ産生がＣＤ患者の腸の中で増加していることを示す。
The fecal lipid composition was clearly different between healthy subjects and CD patients. Similar to plasma phospholipid concentrations (Iwatani et al. 2020), concentrations of total LysoPS, 18:0 LysoPS, and 18:1 LysoPS were higher in CD patients than in healthy subjects. This indicates that LysoPS production is increased in the gut of CD patients.
＜実施例２：ＬｙｓｏＰＳ濃度測定（２）＞
さらにクローン病患者１１人と健常者１２人から糞便サンプル収集し、実施例１と同様の方法で糞便中の脂質を網羅的に解析した。３４クラスに属する５２９の脂質分子を特定した。部分的最小二乗判別分析により、クローン病患者のサンプル中の脂質組成が、健常者の脂質組成と明確に異なることが明らかになった。 <Example 2: LysoPS concentration measurement (2)>
Furthermore, fecal samples were collected from 11 Crohn's disease patients and 12 healthy subjects, and the lipids in the feces were comprehensively analyzed in the same manner as in Example 1. 529 lipid molecules belonging to 34 classes were identified. Partial least-squares discriminant analysis revealed that the lipid composition in samples from Crohn's disease patients was distinctly different from that in healthy controls.
さらにクローン病患者１１人と健常者１２人から糞便サンプル収集し、実施例１と同様の方法で糞便中の脂質を網羅的に解析した。３４クラスに属する５２９の脂質分子を特定した。部分的最小二乗判別分析により、クローン病患者のサンプル中の脂質組成が、健常者の脂質組成と明確に異なることが明らかになった。 <Example 2: LysoPS concentration measurement (2)>
Furthermore, fecal samples were collected from 11 Crohn's disease patients and 12 healthy subjects, and the lipids in the feces were comprehensively analyzed in the same manner as in Example 1. 529 lipid molecules belonging to 34 classes were identified. Partial least-squares discriminant analysis revealed that the lipid composition in samples from Crohn's disease patients was distinctly different from that in healthy controls.
１５の脂質分子が健常者に比較してＣＤ患者で増加していた。ＣＤ患者の糞便中の１８：０ＬｙｓｏＰＳおよび１８：１ＬｙｓｏＰＳの濃度は、健常者より高値を示した（図１）。ＬｙｓｏＰＳに加えて、ＬｙｓｏＰＣの濃度も、ＣＤ患者において高値を示した(図２)。
Fifteen lipid molecules were elevated in CD patients compared to healthy subjects. The concentrations of 18:0LysoPS and 18:1LysoPS in the feces of CD patients were higher than those of healthy subjects (Fig. 1). In addition to LysoPS, LysoPC levels were also elevated in CD patients (Fig. 2).
ＣＤ患者の糞便中の１８：０／１８：１ＬｙｓｏＰＳ、そして、１８：０／１８：１／２２：１ＬｙｓｏＰＣの増加についてさらに検討するために、我々は、ランク-オーダースコアモデル（ｒａｎｋ－ｏｒｄｅｒ ｓｃｏｒｉｎｇ ｍｏｄｅｌ）を導入した。すなわち、各リゾリン脂質の糞便の濃度に、１（最も低い）から２３（最も高い）の正規化された値を割り当てた。各スコアの合計値（トータルランク-オーダースコア）は、ＣＤ患者において明確な増加を示した（図３）。ＲＯＣ解析（ＡＵＣ＝０．７９９２、ｐ＝０．００３６、図４）によりカットオフ値が７８ポイント（特異度が１００％になるトータルランク-オーダースコアの最低値）であることが判明した。トータルランク-オーダースコア７８ポイント以上のＣＤ患者６人（ＣＤ０３，ＣＤ０５，ＣＤ０６，ＣＤ０７，ＣＤ０９およびＣＤ１１）を、リゾリン脂質高濃度保持者と認定した(図３)。
To further investigate the increase in 18:0/18:1 LysoPS and 18:0/18:1/22:1 LysoPC in the feces of CD patients, we used a rank-order scoring model. ) was introduced. That is, the fecal concentration of each lysophospholipid was assigned a normalized value from 1 (lowest) to 23 (highest). The sum of each score (total rank-order score) showed a clear increase in CD patients (Fig. 3). ROC analysis (AUC=0.7992, p=0.0036, FIG. 4) revealed a cut-off value of 78 points (lowest total rank-order score with 100% specificity). Six CD patients (CD03, CD05, CD06, CD07, CD09 and CD11) with a total rank-order score of 78 points or more were identified as high lysophospholipid concentration carriers (Fig. 3).
＜実施例３：糞便サンプルのメタゲノム解析＞
実施例１で収集した糞便サンプルからＤＮＡを抽出した。糞便のサンプルをチューブに入れ、ＲＮＡｌａｔｅｒ（商標）を１０倍希釈ホモジネートを調製するために添加した。糞便ホモジネート（２００μｌ）をＰＢＳで２回洗い、その後、ガラスビーズ（直径０．１ｍｍ）０．３ｇ、トリスＳＤＳ溶液３００μｌ、およびＴＥ飽和フェノール５００μｌを懸濁液に追加した。得られた混合液を、ＦａｓｔＰｒｅｐ－２４（MP Biomedicals; Kaysersberg, France）を使って、パワーレベル５．０で３０秒ボルテックスした。サンプルを遠心分離（２０，０００×ｇ、４℃、５分間）し、得られた上清から４００μｌを分取してフェノールクロロホルム抽出を行った。フェノールクロロホルム抽出により得られた上清から２５０μｌを分取し、イソプロパノールによる沈殿を得た。糞便のサンプルからのＤＮＡをＴＥ緩衝液５０μｌに懸濁した。 <Example 3: Metagenome analysis of fecal samples>
DNA was extracted from the fecal samples collected in Example 1. Fecal samples were placed in tubes and RNAlater™ was added to prepare 10-fold diluted homogenates. Fecal homogenate (200 μl) was washed twice with PBS, after which 0.3 g glass beads (0.1 mm diameter), 300 μl Tris-SDS solution, and 500 μl TE-saturated phenol were added to the suspension. The resulting mixture was vortexed for 30 seconds at power level 5.0 using FastPrep-24 (MP Biomedicals; Kaysersberg, France). The sample was centrifuged (20,000×g, 4° C., 5 minutes), and 400 μl of the resulting supernatant was aliquoted and subjected to phenol-chloroform extraction. A 250 μl aliquot was taken from the supernatant obtained by phenol-chloroform extraction and precipitated with isopropanol. DNA from fecal samples was suspended in 50 μl of TE buffer.
実施例１で収集した糞便サンプルからＤＮＡを抽出した。糞便のサンプルをチューブに入れ、ＲＮＡｌａｔｅｒ（商標）を１０倍希釈ホモジネートを調製するために添加した。糞便ホモジネート（２００μｌ）をＰＢＳで２回洗い、その後、ガラスビーズ（直径０．１ｍｍ）０．３ｇ、トリスＳＤＳ溶液３００μｌ、およびＴＥ飽和フェノール５００μｌを懸濁液に追加した。得られた混合液を、ＦａｓｔＰｒｅｐ－２４（MP Biomedicals; Kaysersberg, France）を使って、パワーレベル５．０で３０秒ボルテックスした。サンプルを遠心分離（２０，０００×ｇ、４℃、５分間）し、得られた上清から４００μｌを分取してフェノールクロロホルム抽出を行った。フェノールクロロホルム抽出により得られた上清から２５０μｌを分取し、イソプロパノールによる沈殿を得た。糞便のサンプルからのＤＮＡをＴＥ緩衝液５０μｌに懸濁した。 <Example 3: Metagenome analysis of fecal samples>
DNA was extracted from the fecal samples collected in Example 1. Fecal samples were placed in tubes and RNAlater™ was added to prepare 10-fold diluted homogenates. Fecal homogenate (200 μl) was washed twice with PBS, after which 0.3 g glass beads (0.1 mm diameter), 300 μl Tris-SDS solution, and 500 μl TE-saturated phenol were added to the suspension. The resulting mixture was vortexed for 30 seconds at power level 5.0 using FastPrep-24 (MP Biomedicals; Kaysersberg, France). The sample was centrifuged (20,000×g, 4° C., 5 minutes), and 400 μl of the resulting supernatant was aliquoted and subjected to phenol-chloroform extraction. A 250 μl aliquot was taken from the supernatant obtained by phenol-chloroform extraction and precipitated with isopropanol. DNA from fecal samples was suspended in 50 μl of TE buffer.
実施例１で収集した糞便サンプル中の全微生物のゲノム情報を得るために、次世代シーケンサーを用いたメタゲノムのショットガンシーケンス（メタゲノム解析）を行った。α多様性をｓｈａｎｎｏｎインデックスで表した結果を図５に示す。健常者に比較してクローン病患者の糞便中では、腸内微生物叢の種多様性が減少していた。β多様性を表すＢｒａｙ－Ｃｕｒｔｉｓインデックスを用いて二次元解析を行った結果、クローン病患者と健常者では糞便サンプル中の微生物群プロファイルが明らかに相違した（図６）。クローン病患者のｄｙｓｂｉｏｓｉｓが確認された。
In order to obtain the genomic information of all microorganisms in the fecal samples collected in Example 1, shotgun sequencing of metagenomics (metagenomic analysis) was performed using a next-generation sequencer. FIG. 5 shows the results of α-diversity represented by the shannon index. Species diversity of the gut microbiota was reduced in the faeces of Crohn's disease patients compared with healthy subjects. As a result of two-dimensional analysis using the Bray-Curtis index representing β diversity, the microbial community profile in fecal samples clearly differed between Crohn's disease patients and healthy subjects (Fig. 6). Dysbiosis in a patient with Crohn's disease was confirmed.
<実施例４：ホスホリパーゼＡ（ＰＬＡ）をコードする遺伝子測定＞
実施例３のメタゲノム解析から、ホスホリパーゼＡ（ＰＬＡ）をコードしている遺伝子を解析した。ＰＬＡをコードすると予想される７つの遺伝子の中で、健常者のサンプルと比較してクローン病患者で顕著に増加してる遺伝子として、主として大腸菌（Escherichia coli）に由来するＥＣＳＦ＿３６６０ が同定された（図７）。 <Example 4: Measurement of genes encoding phospholipase A (PLA)>
From the metagenomic analysis of Example 3, the gene encoding phospholipase A (PLA) was analyzed. Among the 7 genes predicted to encode PLA, ECSF — 3660, mainly derived from Escherichia coli, was identified as a gene significantly increased in Crohn's disease patients compared to samples from healthy subjects (Fig. 7).
実施例３のメタゲノム解析から、ホスホリパーゼＡ（ＰＬＡ）をコードしている遺伝子を解析した。ＰＬＡをコードすると予想される７つの遺伝子の中で、健常者のサンプルと比較してクローン病患者で顕著に増加してる遺伝子として、主として大腸菌（Escherichia coli）に由来するＥＣＳＦ＿３６６０ が同定された（図７）。 <Example 4: Measurement of genes encoding phospholipase A (PLA)>
From the metagenomic analysis of Example 3, the gene encoding phospholipase A (PLA) was analyzed. Among the 7 genes predicted to encode PLA, ECSF — 3660, mainly derived from Escherichia coli, was identified as a gene significantly increased in Crohn's disease patients compared to samples from healthy subjects (Fig. 7).
＜実施例５：大腸菌数測定＞
実施例２で収集したＣＤ患者糞便サンプル中の大腸菌数と健常者糞便サンプルに中の大腸菌数を測定した。ヒトおよびネズミの糞便サンプル中のＥ. coli/Shigella groupの測定について以前報告されている方法（Pareek et al. 2019）に従って、定量的ＰＣＲを行った。すなわち、テンプレートとして１００倍希釈されたＤＮＡ５μｌおよびマスター混合溶液１５μｌを混合して各２０μｌの反応溶液を調製した。そのマスター混合溶液には、ＰＣＲグレードの水４．６μｌ、１０μＭストックからのフォワードおよびリバースプライマー０．２μｌ、およびＧｏＴａｑ(商標) Ｐｒｏｂｅ ｑＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ（Promega, Woods Hollow Road Madison, WI, USA）１０μｌが含まれていた。反応は、ＡＢ Ｂｉｏｓｙｓｔｅｍｓ ＳｔｅｐＯｎｅＰｌｕｓ(商標)システムにおいて、以下のプログラムを使って実行された：９４℃５分の１サイクル、９４℃１５秒、６０℃６０秒、および７２℃６０ｓの４０サイクル。糞便１グラムあたりのコピー数は、大腸菌用に得られた標準曲線に基づいて、計算された。次のプライマーセットを使用した：５’－ＧＡＧＴＡＡＡＧＴＴＡＡＴＡＣＣＴＴＴＧＣＴＣＡＴＴＧ －３’(配列番号６)、および、５’－ＧＡＧＡＣＴＣＡＡＧＣＴＫＲＣＣＡＧＴＡＴＣＡＧ－３’(配列番号７)。 <Example 5: Escherichia coli count measurement>
The number of E.coli in the CD patient stool sample collected in Example 2 and the E.coli number in the healthy subject stool sample were determined. Quantitative PCR was performed according to a previously reported method (Pareek et al. 2019) for the determination of the E. coli/Shigella group in human and murine fecal samples. That is, 5 μl of 100-fold diluted DNA as a template and 15 μl of a master mixed solution were mixed to prepare 20 μl of each reaction solution. The master mix solution contained 4.6 μl of PCR grade water, 0.2 μl of forward and reverse primers from 10 μM stocks, and 10 μl of GoTaq™ Probe qPCR Master Mix (Promega, Woods Hollow Road Madison, WI, USA). was included. Reactions were carried out in an AB Biosystems StepOnePlus™ system using the following program: 1 cycle of 94° C. 5 min, 40 cycles of 94° C. 15 sec, 60° C. 60 sec, and 72° C. 60 s. Copy number per gram of stool was calculated based on a standard curve obtained for E. coli. The following primer sets were used: 5'-GAGTAAAGTTAATAACCTTTGCTCATTG-3' (SEQ ID NO:6) and 5'-GAGACTCAAGCTKRCCAGTATCAG-3' (SEQ ID NO:7).
実施例２で収集したＣＤ患者糞便サンプル中の大腸菌数と健常者糞便サンプルに中の大腸菌数を測定した。ヒトおよびネズミの糞便サンプル中のＥ. coli/Shigella groupの測定について以前報告されている方法（Pareek et al. 2019）に従って、定量的ＰＣＲを行った。すなわち、テンプレートとして１００倍希釈されたＤＮＡ５μｌおよびマスター混合溶液１５μｌを混合して各２０μｌの反応溶液を調製した。そのマスター混合溶液には、ＰＣＲグレードの水４．６μｌ、１０μＭストックからのフォワードおよびリバースプライマー０．２μｌ、およびＧｏＴａｑ(商標) Ｐｒｏｂｅ ｑＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ（Promega, Woods Hollow Road Madison, WI, USA）１０μｌが含まれていた。反応は、ＡＢ Ｂｉｏｓｙｓｔｅｍｓ ＳｔｅｐＯｎｅＰｌｕｓ(商標)システムにおいて、以下のプログラムを使って実行された：９４℃５分の１サイクル、９４℃１５秒、６０℃６０秒、および７２℃６０ｓの４０サイクル。糞便１グラムあたりのコピー数は、大腸菌用に得られた標準曲線に基づいて、計算された。次のプライマーセットを使用した：５’－ＧＡＧＴＡＡＡＧＴＴＡＡＴＡＣＣＴＴＴＧＣＴＣＡＴＴＧ －３’(配列番号６)、および、５’－ＧＡＧＡＣＴＣＡＡＧＣＴＫＲＣＣＡＧＴＡＴＣＡＧ－３’(配列番号７)。 <Example 5: Escherichia coli count measurement>
The number of E.coli in the CD patient stool sample collected in Example 2 and the E.coli number in the healthy subject stool sample were determined. Quantitative PCR was performed according to a previously reported method (Pareek et al. 2019) for the determination of the E. coli/Shigella group in human and murine fecal samples. That is, 5 μl of 100-fold diluted DNA as a template and 15 μl of a master mixed solution were mixed to prepare 20 μl of each reaction solution. The master mix solution contained 4.6 μl of PCR grade water, 0.2 μl of forward and reverse primers from 10 μM stocks, and 10 μl of GoTaq™ Probe qPCR Master Mix (Promega, Woods Hollow Road Madison, WI, USA). was included. Reactions were carried out in an AB Biosystems StepOnePlus™ system using the following program: 1 cycle of 94° C. 5 min, 40 cycles of 94° C. 15 sec, 60° C. 60 sec, and 72° C. 60 s. Copy number per gram of stool was calculated based on a standard curve obtained for E. coli. The following primer sets were used: 5'-GAGTAAAGTTAATAACCTTTGCTCATTG-3' (SEQ ID NO:6) and 5'-GAGACTCAAGCTKRCCAGTATCAG-3' (SEQ ID NO:7).
ＣＤ患者サンプル中の大腸菌数は健常者と比較して有意に高かった(図８)。さらに、糞便中の大腸菌数はランクオーダースコアにより正規化された糞便リゾリン脂質の相対量と正に相関していた。また、実施例２でリゾリン脂質高濃度保持者に認定された６人は、多くの大腸菌を保持していた（図９）。
E. coli counts in CD patient samples were significantly higher compared to healthy subjects (Fig. 8). Furthermore, fecal E. coli counts were positively correlated with the relative amounts of fecal lysophospholipids normalized by rank-order scores. In addition, the 6 individuals who were identified as having a high lysophospholipid concentration in Example 2 had a large amount of E. coli (Fig. 9).
＜実施例６：腸内微生物によるＬｙｓｏＰＳ生成＞
グラム陰性菌がホストの腸管上皮に侵襲する際に、グラム陰性菌のＰＬＡがホストの上皮細胞膜を破壊することが報告されている(Istivan and Coloe, 2006)。一方、患者(ホスト)自身のホスホリパーゼＡ（ＰＬＡ）と腸内微生物のＰＬＡはいずれも、細胞膜リン脂質を加水分解し、リゾリン脂質を生成することが知られている(Tan et al. 2020)。ＣＤ患者の糞便中ＬｙｓｏＰＳの増加を引き起こすＰＬＡが、腸内微生物叢の異常（ｄｙｓｂｉｏｓｉｓ）により増加した微生物由来のＰＬＡによるものであることを確認するために、以下の試験を行った。 <Example 6: LysoPS production by intestinal microorganisms>
It has been reported that Gram-negative PLA disrupts host epithelial cell membranes when Gram-negative bacteria invade the host intestinal epithelium (Istivan and Coloe, 2006). On the other hand, both the patient's (host's) own phospholipase A (PLA) and the PLA of intestinal microorganisms are known to hydrolyze cell membrane phospholipids to produce lysophospholipids (Tan et al. 2020). To confirm that the PLA that causes the increase in fecal LysoPS in CD patients is due to microbial PLA that is increased due to intestinal dysbiosis, the following tests were performed.
グラム陰性菌がホストの腸管上皮に侵襲する際に、グラム陰性菌のＰＬＡがホストの上皮細胞膜を破壊することが報告されている(Istivan and Coloe, 2006)。一方、患者(ホスト)自身のホスホリパーゼＡ（ＰＬＡ）と腸内微生物のＰＬＡはいずれも、細胞膜リン脂質を加水分解し、リゾリン脂質を生成することが知られている(Tan et al. 2020)。ＣＤ患者の糞便中ＬｙｓｏＰＳの増加を引き起こすＰＬＡが、腸内微生物叢の異常（ｄｙｓｂｉｏｓｉｓ）により増加した微生物由来のＰＬＡによるものであることを確認するために、以下の試験を行った。 <Example 6: LysoPS production by intestinal microorganisms>
It has been reported that Gram-negative PLA disrupts host epithelial cell membranes when Gram-negative bacteria invade the host intestinal epithelium (Istivan and Coloe, 2006). On the other hand, both the patient's (host's) own phospholipase A (PLA) and the PLA of intestinal microorganisms are known to hydrolyze cell membrane phospholipids to produce lysophospholipids (Tan et al. 2020). To confirm that the PLA that causes the increase in fecal LysoPS in CD patients is due to microbial PLA that is increased due to intestinal dysbiosis, the following tests were performed.
健常者とＣＤ患者から新鮮な糞便サンプルを収集し、糞便サンプル中の、ＣＳＦ＿３６６０（全長８７０ｂｐ）を、定量的ＰＣＲによって検出した。プライマーとして、５’－ＡＴＧＣＧＧＡＣＴＣＴＧＣＡＧＧＧＣＴＧＧＴＴＧＴＴＧＣＣＧ－３’(配列番号３)、および、５’－ＴＣＡＡＡＡＣＡＧＧＴＣＧＴＴＴＡＧＣＡＴＡＡＣＴＣＣＣＡＣ－３’（配列番号４）を用いた。増幅条件は、９４℃５分の１サイクル、９４℃３０秒、６０℃３０秒、および７２℃６０秒の３５のサイクルとした。結果を図１０に示す。健全な微生物叢を持つ４人の健常者からの糞便サンプルを混合し、また、ＥＣＳＦ＿３６６０検出可能な微生物叢を保菌する２人のＣＤ患者からの糞便サンプルを混合した。
Fresh stool samples were collected from healthy subjects and CD patients, and CSF — 3660 (full length 870 bp) was detected in the stool samples by quantitative PCR. As primers, 5'-ATGCGGACTCTGCAGGGCTGGTTGTTGCCG-3' (SEQ ID NO: 3) and 5'-TCAAACAGGTCGTTTAGCATAACTCCCAC-3' (SEQ ID NO: 4) were used. Amplification conditions were 1 cycle of 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds. The results are shown in FIG. Fecal samples from 4 healthy subjects with healthy microbiota were combined, and stool samples from 2 CD patients harboring ECSF_3660 detectable microbiota were also combined.
既報 (Maeda et al. 2016)にわずかな修正を加えた方法により、無菌マウスにヒトの糞便の微生物叢を定着させた。
Germ-free mice were colonized with human fecal microbiota by a method previously reported (Maeda et al. 2016) with minor modifications.
各糞便のサンプルを好気性緩衝液（２％ Ｌａｂ－Ｌｅｍｃｏ ｐｏｗｄｅｒ、０．１％ Ｌ－ｃｙｓｔｅｉｎｅ、０．０４５％ ＫＨ２ＰＯ４、０．０９％ ＮａＣｌ、０．０４５％ （ＮＨ４）２ＳＯ４、０．０４５％ ＣａＣｌ２、０．０４５％のＭｇＳＯ４、および、４０％ グリセリン）を用いて１６倍希釈（ｗ／ｖ）し均質化し、使用するまで－８０℃で保存した。
Each fecal sample was added to an aerobic buffer (2% Lab-Lemco powder, 0.1% L-cysteine, 0.045% KH 2 PO 4 , 0.09% NaCl, 0.045% (NH 4 ) 2 SO). 4 , 0.045% CaCl 2 , 0.045% MgSO 4 , and 40% glycerin), homogenized by 16-fold dilution (w/v) and stored at -80°C until use.
雄の無菌マウス（８－９週齢）にＣＤ患者または健常者からの糞便の懸濁液２５０μｌを経口で移植した。これらマウスを２４日の間ノトバイオートのアイソレータ中に別々に維持した。移植後１０日において、ＣＤ患者微生物叢移植マウスの糞便中の大腸菌数は、健常者微生物叢移植マウスの糞便中の大腸菌数に比較して、明らかに高値を示した（図１１）。加えて、ＣＤ患者微生物叢を保菌しているマウスの糞便から、微生物ＤＮＡであるＥＣＳＦ＿３６６０を検出した。一方、健常者微生物叢を保菌しているマウスの糞便からはＥＣＳＦ＿３６６０は検出されなかった（図１２）。我々は、移植後２４日に更に１２１の脂質種の糞便中濃度を測定した。ＣＤ患者微生物叢保菌マウスの糞便中濃度と健常者微生物叢保菌マウスの糞便中濃度を比較すると、７つのクラスの３７の脂質種の濃度が、ＣＤ患者微生物叢保菌マウスで高かった。１８：０、１８：１および総ＬｙｓｏＰＳの糞便中濃度は、健常者微生物叢保菌マウスと比較してＣＤ患者微生物叢保菌マウスにおいて高値であった(図１３)。これらの結果は、ＣＤ患者における異常な腸内微生物叢の保菌（ｄｙｓｂｉｏｔｉｃ）が腸内のＬｙｓｏＰＳの生成に関連することを示唆する。
Male germ-free mice (8-9 weeks old) were implanted orally with 250 μl of fecal suspension from CD patients or healthy subjects. The mice were maintained separately in gnotobiotic isolators for 24 days. At 10 days after transplantation, the number of E. coli in the feces of CD patient microbiota-transplanted mice was clearly higher than that of normal human microbiota-transplanted mice (Fig. 11). In addition, the microbial DNA, ECSF_3660, was detected in the feces of mice harboring the CD patient microbiota. On the other hand, ECSF — 3660 was not detected in the feces of mice carrying the microflora of healthy individuals (Fig. 12). We measured fecal concentrations of an additional 121 lipid species 24 days after transplantation. Comparing fecal concentrations in CD microbiota-bearing mice with those in normal microbiota-bearing mice, concentrations of 37 lipid species from seven classes were higher in CD microbiota-bearing mice. Fecal concentrations of 18:0, 18:1 and total LysoPS were higher in CD patient microbiota-colonized mice compared to healthy control microbiota-colonized mice (FIG. 13). These results suggest that abnormal gut microbiota dysbiotics in CD patients are associated with the generation of intestinal LysoPS.
以上の結果は、ＣＤ患者の腸内では、ｄｙｓｂｉｏｓｉｓによりＰＬＡを産生する大腸菌等が増加し、ＬｙｓｏＰＳ濃度の上昇を引き起こしていることを示す。
The above results indicate that dysbiosis increases PLA-producing Escherichia coli and the like in the intestines of CD patients, causing an increase in LysoPS concentration.
実施例７
患者の糞便サンプルにメタノール添加のうえホモジナイズし、定量的リアルタイムＰＣＲ法によりＥＣＳＦ＿３６６０の量を測定する。健常者のＥＣＳＦ＿３６６０の量と比較して遺伝子量が多い場合は、炎症性腸疾患のリスクが高いと判定する。 Example 7
Methanol is added to the patient's stool sample and homogenized, and the amount of ECSF — 3660 is measured by quantitative real-time PCR method. When the gene dosage is high compared to the amount of ECSF — 3660 in healthy subjects, it is determined that the risk of inflammatory bowel disease is high.
患者の糞便サンプルにメタノール添加のうえホモジナイズし、定量的リアルタイムＰＣＲ法によりＥＣＳＦ＿３６６０の量を測定する。健常者のＥＣＳＦ＿３６６０の量と比較して遺伝子量が多い場合は、炎症性腸疾患のリスクが高いと判定する。 Example 7
Methanol is added to the patient's stool sample and homogenized, and the amount of ECSF — 3660 is measured by quantitative real-time PCR method. When the gene dosage is high compared to the amount of ECSF — 3660 in healthy subjects, it is determined that the risk of inflammatory bowel disease is high.
実施例８
実施例７と同様に炎症性腸疾患患者の糞便サンプル中のＥＣＳＦ＿３６６０の量を測定する。同一患者の過去の検査値と比較して患者の病状の変化を把握する。 Example 8
The amount of ECSF_3660 in fecal samples from patients with inflammatory bowel disease is determined as in Example 7. To grasp changes in the patient's condition by comparing with the past examination values of the same patient.
実施例７と同様に炎症性腸疾患患者の糞便サンプル中のＥＣＳＦ＿３６６０の量を測定する。同一患者の過去の検査値と比較して患者の病状の変化を把握する。 Example 8
The amount of ECSF_3660 in fecal samples from patients with inflammatory bowel disease is determined as in Example 7. To grasp changes in the patient's condition by comparing with the past examination values of the same patient.
実施例９
ＣＤ患者と健常者から糞便サンプルを収集し、直ちに凍結した。凍結した糞便サンプルをメタノール添加のうえホモジナイズし、ＵＰＬＣ－ＥＳＩ－ＭＳ／ＭＳによって全ＬｙｓｏＰＳ、１８：０ ＬｙｓｏＰＳ、および１８：１ ＬｙｓｏＰＳの濃度を測定した。これらＬｙｓｏＰＳの濃度は健常者よりクローン病患者の方が高かった。 Example 9
Fecal samples were collected from CD patients and healthy subjects and immediately frozen. Frozen fecal samples were homogenized with the addition of methanol and the concentrations of total LysoPS, 18:0 LysoPS and 18:1 LysoPS were determined by UPLC-ESI-MS/MS. These LysoPS concentrations were higher in Crohn's disease patients than in healthy subjects.
ＣＤ患者と健常者から糞便サンプルを収集し、直ちに凍結した。凍結した糞便サンプルをメタノール添加のうえホモジナイズし、ＵＰＬＣ－ＥＳＩ－ＭＳ／ＭＳによって全ＬｙｓｏＰＳ、１８：０ ＬｙｓｏＰＳ、および１８：１ ＬｙｓｏＰＳの濃度を測定した。これらＬｙｓｏＰＳの濃度は健常者よりクローン病患者の方が高かった。 Example 9
Fecal samples were collected from CD patients and healthy subjects and immediately frozen. Frozen fecal samples were homogenized with the addition of methanol and the concentrations of total LysoPS, 18:0 LysoPS and 18:1 LysoPS were determined by UPLC-ESI-MS/MS. These LysoPS concentrations were higher in Crohn's disease patients than in healthy subjects.
＜試験例１：ＬｙｓｏＰＳによる大腸炎の増悪＞
ＬｙｓｏＰＳレベル上昇が腸炎の重症度に与える影響を２種類の大腸炎モデルマウスを用いて解析した。 <Test Example 1: Exacerbation of colitis by LysoPS>
The effect of elevated LysoPS levels on the severity of enteritis was analyzed using two types of colitis model mice.
ＬｙｓｏＰＳレベル上昇が腸炎の重症度に与える影響を２種類の大腸炎モデルマウスを用いて解析した。 <Test Example 1: Exacerbation of colitis by LysoPS>
The effect of elevated LysoPS levels on the severity of enteritis was analyzed using two types of colitis model mice.
Ａ．ＴＮＢＳ誘導大腸炎モデルマウス（クローン病モデルマウス）
ＴＮＢＳ誘導大腸炎モデルマウスを以前報告した方法（Iijima, H. et al. J Exp Med 199, 471-482, (2004)） にわずかな修正を加えて作製した。すなわち、野生型Ｃ５７ＢＬ／６Ｊマウス（雄、８－１０週齢）をｄａｙ１に５０％のエタノール中３．７５％ＴＮＢＳ溶液１５０μｌを背皮膚へ塗布することにより感作した。ｄａｙ８に、５０％エタノール中２％ＴＮＢＳ溶液１５０μｌを経肛門投与した。ｄａｙ８からｄａｙ１１まで毎日１回１８:１ ＬｙｓｏＰＳを２．５ｍｇ／ｋｇの投与量で腹腔内投与し、ｄａｙ１２にマウスを解析に用いた。コントロールマウスにはビークル（７０％エタノール）を投与した。 A. TNBS-induced colitis model mouse (Crohn's disease model mouse)
TNBS-induced colitis model mice were produced by a previously reported method (Iijima, H. et al. J Exp Med 199, 471-482, (2004)) with slight modifications. That is, wild-type C57BL/6J mice (male, 8-10 weeks old) were sensitized onday 1 by applying 150 μl of a 3.75% TNBS solution in 50% ethanol to the dorsal skin. On day 8, 150 μl of 2% TNBS solution in 50% ethanol was administered transanally. 18:1 LysoPS was intraperitoneally administered once daily from day 8 to day 11 at a dose of 2.5 mg/kg, and mice were used for analysis on day 12. Control mice received vehicle (70% ethanol).
ＴＮＢＳ誘導大腸炎モデルマウスを以前報告した方法（Iijima, H. et al. J Exp Med 199, 471-482, (2004)） にわずかな修正を加えて作製した。すなわち、野生型Ｃ５７ＢＬ／６Ｊマウス（雄、８－１０週齢）をｄａｙ１に５０％のエタノール中３．７５％ＴＮＢＳ溶液１５０μｌを背皮膚へ塗布することにより感作した。ｄａｙ８に、５０％エタノール中２％ＴＮＢＳ溶液１５０μｌを経肛門投与した。ｄａｙ８からｄａｙ１１まで毎日１回１８:１ ＬｙｓｏＰＳを２．５ｍｇ／ｋｇの投与量で腹腔内投与し、ｄａｙ１２にマウスを解析に用いた。コントロールマウスにはビークル（７０％エタノール）を投与した。 A. TNBS-induced colitis model mouse (Crohn's disease model mouse)
TNBS-induced colitis model mice were produced by a previously reported method (Iijima, H. et al. J Exp Med 199, 471-482, (2004)) with slight modifications. That is, wild-type C57BL/6J mice (male, 8-10 weeks old) were sensitized on
ＬｙｓｏＰＳ投与マウスでは、コントロールマウスと比較して、体重減少と結腸の短縮が観察され（図１４、図１５）、病理組織解析においても悪化は明らかであった（図１６）。結腸の粘膜固有層リンパ球中のサイトカインｍＲＮＡ発現量をｑＲＴ－ＰＣＲを用いて測定したところ、ＬｙｓｏＰＳ投与マウスでは、コントロールマウスに比較して、ＩｆｎｇとＩｌ１７ａの発現量が高かったが、Ｉｌ１０、Ｉｌ１２ｂ、およびＩｌ２３ａの発現量に有意差はなかった（図１７）。
Weight loss and colon shortening were observed in LysoPS-administered mice compared with control mice (FIGS. 14 and 15), and the deterioration was also evident in histopathological analysis (FIG. 16). Cytokine mRNA expression levels in colonic lamina propria lymphocytes were measured using qRT-PCR. Compared to control mice, LysoPS-administered mice had higher expression levels of Ifng and Il17a, but Il10 and Il12b. , and Il23a expression levels were not significantly different (FIG. 17).
Ｂ．Ｔ細胞依存的大腸炎モデルマウス（クローン病モデルマウス）
ＣＤ４＋Ｔ細胞依存的大腸炎モデルマウスを文献（Ostanin, D. V. et al. Am J Physiol Gastrointest Liver Physiol 296, G135-146, (2009)）に記載の方法にわずかな修正を加えて作製した。単離したナイーブＣＤ４＋Ｔ細胞を冷リン酸緩衝液（ＰＢＳ）で終濃度１×１０６ｃｅｌｌｓ／ｍｌに希釈して、５００μｌ（５×１０５ｃｅｌｌｓ）をＲａｇ２－/－マウス（雄、８－１５週齢）に腹腔内投与した。ナイーブＣＤ４＋Ｔ細胞を投与した１７日後、連続４日間１８：１ ＬｙｓｏＰＳ（２．５ｍｇ／ｋｇ）またはビークル（７０％エタノール）を腹腔内に投与した。 B. T cell-dependent colitis model mouse (Crohn's disease model mouse)
A CD4 + T cell-dependent colitis model mouse was generated by slightly modifying the method described in the literature (Ostanin, D. V. et al. Am J Physiol Gastrointest Liver Physiol 296, G135-146, (2009)). Isolated naive CD4 + T cells were diluted with cold phosphate buffered saline (PBS) to a final concentration of 1×10 6 cells/ml and 500 μl (5×10 5 cells) were injected into Rag2 −/− mice (8 males). −15 weeks old) were administered intraperitoneally. Seventeen days after administration of naive CD4 + T cells, 18:1 LysoPS (2.5 mg/kg) or vehicle (70% ethanol) was administered intraperitoneally for 4 consecutive days.
ＣＤ４＋Ｔ細胞依存的大腸炎モデルマウスを文献（Ostanin, D. V. et al. Am J Physiol Gastrointest Liver Physiol 296, G135-146, (2009)）に記載の方法にわずかな修正を加えて作製した。単離したナイーブＣＤ４＋Ｔ細胞を冷リン酸緩衝液（ＰＢＳ）で終濃度１×１０６ｃｅｌｌｓ／ｍｌに希釈して、５００μｌ（５×１０５ｃｅｌｌｓ）をＲａｇ２－/－マウス（雄、８－１５週齢）に腹腔内投与した。ナイーブＣＤ４＋Ｔ細胞を投与した１７日後、連続４日間１８：１ ＬｙｓｏＰＳ（２．５ｍｇ／ｋｇ）またはビークル（７０％エタノール）を腹腔内に投与した。 B. T cell-dependent colitis model mouse (Crohn's disease model mouse)
A CD4 + T cell-dependent colitis model mouse was generated by slightly modifying the method described in the literature (Ostanin, D. V. et al. Am J Physiol Gastrointest Liver Physiol 296, G135-146, (2009)). Isolated naive CD4 + T cells were diluted with cold phosphate buffered saline (PBS) to a final concentration of 1×10 6 cells/ml and 500 μl (5×10 5 cells) were injected into Rag2 −/− mice (8 males). −15 weeks old) were administered intraperitoneally. Seventeen days after administration of naive CD4 + T cells, 18:1 LysoPS (2.5 mg/kg) or vehicle (70% ethanol) was administered intraperitoneally for 4 consecutive days.
ＬｙｓｏＰＳ投与マウスで、結腸の短縮、体重減少が観察された（図１８、図１９）。さらに、病理組織解析ではＬｙｓｏＰＳによる大腸組織病変の顕著な増悪が観察された（図２０）。結腸の病理組織による重症度の評価は、Liu, Z. et al. J Immunol 164, 6005-6014に記載の方法に準じて行った。大腸の粘膜固有層のＣＤ４＋Ｔ細胞中のサイトカイン産生細胞数をフローサイトメトリー法で解析したところ、コントロールマウスと比較して、ＬｙｓｏＰＳ投与マウスでは大腸炎の増悪に伴いＩＦＮ－γ＋細胞およびＩＦＮ－γ＋ＩＬ－１７Ａ＋細胞が増加したが、ＩＬ－１７Ａ＋細胞およびＩＬ－１０＋細胞は増加しなかった（図２１）。
Colon shortening and weight loss were observed in LysoPS-administered mice (FIGS. 18 and 19). Furthermore, in histopathological analysis, significant exacerbation of colonic tissue lesions by LysoPS was observed (Fig. 20). Evaluation of severity by colon histopathology was performed according to the method described in Liu, Z. et al. J Immunol 164, 6005-6014. Analysis of the number of cytokine-producing cells among CD4 + T cells in the lamina propria of the large intestine by flow cytometry revealed that compared with control mice, LysoPS-treated mice increased IFN-γ + cells and IFN-γ + cells with exacerbation of colitis. -γ + IL-17A + cells increased, but IL-17A + and IL-10 + cells did not (FIG. 21).
これら結果は、ＬｙｓｏＰＳが粘膜固有層において免疫病理学的なＴｈ１応答を呼び起すことによって大腸炎を悪化させることを示す。すなわち、ＬｙｓｏＰＳは腸粘膜においてＴｈ１細胞の過剰活性化を引き起こして炎症性腸疾患を悪化させる。
These results indicate that LysoPS exacerbates colitis by evoking an immunopathological Th1 response in the lamina propria. Thus, LysoPS exacerbates inflammatory bowel disease by causing hyperactivation of Th1 cells in the intestinal mucosa.
本発明は、炎症性腸疾患の検査並びに炎症性腸疾患の予防および治療に貢献する。
INDUSTRIAL APPLICABILITY The present invention contributes to examination of inflammatory bowel disease and prevention and treatment of inflammatory bowel disease.
Claims (19)
- 被検体より得られた試料中の、リゾホスファチジルセリン、リゾホスファチジルセリン生成に関与する腸内微生物、腸内微生物由来ホスホリパーゼＡをコードする遺伝子、および、ホスホリパーゼＡからなる群から選択される少なくとも１を測定する工程を含む炎症性腸疾患を検査する方法。 At least one selected from the group consisting of lysophosphatidylserine, an intestinal microorganism involved in the production of lysophosphatidylserine, a gene encoding phospholipase A derived from intestinal microorganisms, and phospholipase A in a sample obtained from a subject. A method of testing for inflammatory bowel disease comprising the step of measuring.
- 被検体より得られた試料中のリゾホスファチジルセリン生成に関与する腸内微生物数を測定する工程を含む、請求項１に記載の方法。 2. The method of claim 1, comprising measuring the number of intestinal microorganisms involved in lysophosphatidylserine production in a sample obtained from a subject.
- 前記腸内微生物がホスホリパーゼＡを発現する腸内微生物である、請求項２に記載の方法。 3. The method of claim 2, wherein said gut microbe is a phospholipase A-expressing gut microbe.
- 前記ホスホリパーゼＡを発現する腸内微生物が、ホスホリパーゼＡをコードする遺伝子ＥＣＳＦ＿３６６０を有する微生物である、請求項３に記載の方法。 4. The method of claim 3, wherein the phospholipase A-expressing intestinal microorganism is a microorganism having the phospholipase A-encoding gene ECSF_3660.
- 前記腸内微生物数を測定する工程は、該腸内微生物由来遺伝子の量を測定する工程を含む、請求項２～４のいずれか１に記載の方法。 The method according to any one of claims 2 to 4, wherein the step of measuring the intestinal microbial count includes the step of measuring the amount of the intestinal microbial-derived gene.
- 前記腸内微生物数を測定する工程は、該腸内微生物発現ホスホリパーゼＡを測定する工程を含む、請求項２～４のいずれか１に記載の方法。 The method according to any one of claims 2 to 4, wherein the step of measuring the intestinal microbial count comprises measuring the intestinal microbial-expressed phospholipase A.
- 被検体より得られた試料中の腸内微生物由来ホスホリパーゼＡをコードする遺伝子の量を測定する工程を含む、請求項１に記載の方法 2. The method according to claim 1, comprising the step of measuring the amount of the gene encoding the intestinal microbial phospholipase A in the sample obtained from the subject.
- 前記ホスホリパーゼＡをコードする遺伝子が、ＥＣＳＦ＿３６６０である請求項７に記載の方法。 8. The method of claim 7, wherein the gene encoding phospholipase A is ECSF_3660.
- 被検体より得られた試料中のリゾホスファチジルセリンの濃度を測定する工程を含む、請求項１に記載の方法。 2. The method of claim 1, comprising measuring the concentration of lysophosphatidylserine in a sample obtained from the subject.
- さらにリゾホスファチジルコリンの濃度を測定する工程を含む請求項９に記載の方法。 10. The method of claim 9, further comprising measuring the concentration of lysophosphatidylcholine.
- 被検体より得られた試料中のホスホリパーゼＡを測定する工程を含む、請求項１に記載の方法。 2. The method of claim 1, comprising measuring phospholipase A in a sample obtained from the subject.
- 前記ホスホリパーゼＡが、腸内微生物由来ホスホリパーゼＡである、請求項１１に記載の方法。 12. The method of claim 11, wherein the phospholipase A is an intestinal microbial phospholipase A.
- 前記腸内微生物由来ホスホリパーゼＡが、ＥＣＳＦ＿３６６０にコードされるホスホリパーゼＡである、請求項１２に記載の方法。 13. The method of claim 12, wherein the enteric microbial phospholipase A is phospholipase A encoded by ECSF_3660.
- 前記ホスホリパーゼＡを測定する工程が、ホスホリパーゼＡのタンパク質量を測定する工程またはホスホリパーゼＡの活性を測定する工程である、請求項１１～１３のいずれか１に記載の方法。 The method according to any one of claims 11 to 13, wherein the step of measuring phospholipase A is a step of measuring the protein amount of phospholipase A or a step of measuring phospholipase A activity.
- 前記試料が糞便サンプルである、請求項１～１４のいずれか１に記載の方法。 15. The method of any one of claims 1-14, wherein the sample is a fecal sample.
- リゾホスファチジルセリン生成に関与する腸内微生物数を測定する工程を含む、炎症性腸疾患の予防用または治療用の物質または微生物のスクリーニング方法。 A method for screening substances or microorganisms for the prevention or treatment of inflammatory bowel disease, comprising the step of measuring the number of intestinal microorganisms involved in lysophosphatidylserine production.
- ホスホリパーゼＡの活性を測定する工程を含む、炎症性腸疾患の予防用または治療用の物質のスクリーニング方法。 A method of screening for a substance for prevention or treatment of inflammatory bowel disease, comprising the step of measuring phospholipase A activity.
- リゾホスファチジルセリン生成に関与する腸内微生物を除去または減少させる物質または微生物を含有する炎症性腸疾患の予防用または治療用組成物。 A composition for preventing or treating inflammatory bowel disease containing a substance or microorganism that eliminates or reduces intestinal microorganisms involved in lysophosphatidylserine production.
- ホスホリパーゼＡの活性を抑制する物質を含有する炎症性腸疾患の予防用または治療用組成物。
A composition for prevention or treatment of inflammatory bowel disease containing a substance that suppresses the activity of phospholipase A.
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| IWATANI SHUKO, HIDEKI IIJIMA,YURIKO OTAKE,TAKAHIRO AMANO,MIZUKI TANI,TAKEO YOSHIHARA,TAKU TASHIRO,YOSHIKI TSUJII,TAKAHIRO INOUE,YO: "Novel mass spectrometry-based comprehensive lipidomic analysis of plasma from patients with inflammatory bowel disease", J. GASTROENTEROL. HEPATOL., vol. 35, no. 8, 14 April 2020 (2020-04-14), pages 1355 - 1364, XP055969443, DOI: 10.1111/jgh.15067 * |
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