WO2021024856A1 - Protein diagnostic biomarkers for interstitial pneumonia - Google Patents
Protein diagnostic biomarkers for interstitial pneumonia Download PDFInfo
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- WO2021024856A1 WO2021024856A1 PCT/JP2020/028866 JP2020028866W WO2021024856A1 WO 2021024856 A1 WO2021024856 A1 WO 2021024856A1 JP 2020028866 W JP2020028866 W JP 2020028866W WO 2021024856 A1 WO2021024856 A1 WO 2021024856A1
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- Prior art keywords
- interstitial pneumonia
- subject
- stratefin
- diffuse alveolar
- pla2
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Definitions
- the present invention relates to a method for assisting in the diagnosis of the onset and pathological condition of interstitial pneumonia.
- Interstitial pneumonia is a general term for inflammatory diseases of the lung interstitium, and is roughly classified into alveolar pneumonia represented by bacterial infection. Interstitial pneumonia is divided into those associated with clear causes such as drug-induced, pneumoconiosis, and collagen disease, and idiopathic diseases of unknown cause, and further classified into various types. Differentiation of these is clinically important because each has different treatments and prognosis.
- Non-Patent Document 1 The type of idiopathic interstitial pneumonia is diagnosed by clinical and imaging findings and histopathological patterns, but drug-induced interstitial pneumonia is also based on idiopathic and similarities with known lung diseases.
- the type of disease is diagnosed (Non-Patent Document 1).
- DAD diffuse alveolar damage
- Non-Patent Document 2 the most serious type is diffuse alveolar damage (DAD).
- DAD is a typical type of disease found in acute exacerbation cases of acute respiratory distress syndrome and idiopathic interstitial pneumonia
- Poor and poor prognosis Non-Patent Documents 1 and 2). Therefore, early detection and diagnosis are of particular importance for DAD.
- Non-Patent Document 3 As pathological types other than DAD, organizing pneumonia (OP), nonspecific interstitial pneumonia (NSIP), etc. can be mentioned, but there are cases and multiple cases that do not fit into any pattern. There are cases where it is difficult to classify by image inspection or pathological examination, such as cases where patterns are mixed, and there is a limit to image pattern classification (Non-Patent Document 3).
- Diagnosis of interstitial pneumonia often takes time to be confirmed, and the use of biomarkers is useful because it imposes a heavy burden on patients economically and physically only by diagnostic imaging and pathological diagnosis.
- KL-6 sialylated sugar chain antigen KL-6
- SP-A pulmonary surfactant protein A
- SP-D pulmonary surfactant protein D
- Non-Patent Documents 4 and 5 KL-6, SP-D, and SP-A have a problem that they show high values for a long period of time even after the condition is improved (Non-Patent Documents 4 and 5), so new biomarkers are required. .. So far, proteins such as UBE2T, HK1, PMSE1, USO1, IFI16, and GLTP (Patent Document 1) and autoantibodies (Patent Document 2) have been proposed and applied for patents as marker candidates.
- An object of the present invention is to develop a biomarker for diagnosing and predicting drug-induced interstitial pneumonia, a disease state, and its type.
- Proteomics analysis was performed on blood samples of drug-induced interstitial pneumonia patients by the SOMAscan method using aptamers (artificial ligands using single-stranded nucleic acids) for 1,310 proteins. Focusing on diffuse alveolar injury (DAD) and organizing pneumonia (OP), based on the relative fluorescence intensity of SOMAscan, a protein that fluctuates significantly in the acute phase of the disease in healthy subjects and convalescent groups is changed. The search was performed using the value and effect size (Hedge's g value) as indicators. While there are many samples in which KL-6 and SP-D levels do not fluctuate, among the 1,310 proteins measured, proteins that increase in the acute phase of DAD but do not change in other types of disease.
- DAD diffuse alveolar injury
- OP organizing pneumonia
- ROC curve Receiveiver Operating Characteristic curve
- DAD and OP A group of patients could be identified as healthy individuals with an AUC of 0.9 or higher. It has been shown that these can be useful biomarkers for interstitial pneumonia including drug-induced.
- the present invention has been completed based on these findings.
- the protein whose expression is to be measured is at least one protein selected from the group consisting of stratephin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T, and the measured value is diffuse.
- the method according to (1) which assists in the diagnosis of alveolar injury.
- the protein whose expression is to be measured is at least one protein selected from the group consisting of stratephine, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T, and the measured value is diffuse.
- the method according to (1) which assists in the specific diagnosis of alveolar injury.
- the method according to (3), wherein the protein whose expression is measured is Stratefin, Cystatin F, NPS-PLA2, IL-1Ra, Netrin-1, Troponin T, or a combination thereof.
- a method for diagnosing diffuse alveolar injury a1. Obtaining a sample from a subject, b1.
- the method comprising determining the presence or absence of the onset of diffuse alveolar injury based on the measurements of b1.
- the method comprising determining the pathology of diffuse alveolar injury based on measurements in b1.
- a method for diagnosing and treating diffuse alveolar injury a1. Obtaining a sample from a subject, b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject. c1. Determine the presence or absence of diffuse alveolar injury based on the measured values of b1 and d1. The method described above comprising treating a subject determined to have a high probability of developing diffuse alveolar injury with treatment for diffuse alveolar injury. (8) A method for diagnosing and treating diffuse alveolar injury.
- a1 Obtaining a sample from a subject, b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject. c1. Determining the pathology of diffuse alveolar injury based on b1 measurements, and d1. The method described above comprising treating a subject determined to be likely to be in the acute phase of diffuse alveolar injury with treatment for diffuse alveolar injury.
- a method for testing interstitial pneumonia in general comprises measuring the expression of at least one protein in a sample derived from a subject.
- Proteins whose expression is to be measured are from stratefine, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6 (CA6).
- a method for diagnosing general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia). a2. Obtaining a sample from the subject, b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6.
- the method comprising determining the presence or absence of the onset of interstitial pneumonia based on the measurements in b2.
- a method for diagnosing general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia).
- a2. Obtaining a sample from the subject, b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6.
- the method comprising determining the pathology of interstitial pneumonia based on the measurements in b2.
- a method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
- a2. Obtaining a sample from the subject, b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6.
- c2. Determine the presence or absence of interstitial pneumonia based on the measured value of b2, and d2. The method described above comprising treating a subject determined to have interstitial pneumonia with a high probability of developing interstitial pneumonia.
- a method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
- a2. Obtaining a sample from the subject, b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6.
- c2. Judging the pathology of interstitial pneumonia based on the measured values of b2, and d2.
- the method comprising treating interstitial pneumonia in a subject determined to be likely to be in the acute phase of interstitial pneumonia.
- a kit for testing for diffuse alveolar injury including.
- a kit for testing interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia), which comprises a reagent capable of measuring the expression of one protein in a sample derived from a subject.
- a protein that exhibits specific fluctuations in diffuse alveolar injury Box plots were used to compare SOMAscan measurements in the acute and convalescent stages of healthy and interstitial pneumonia types for the six proteins with DAD-specific increases. The box shows the quartile deviation (75% and 25%) and median of the SOMAscan measurements.
- HC Healthy
- DAD Diffuse alveolar injury
- OP Organized pneumonia
- NSIP Nonspecific interstitial pneumonia
- A Acute phase
- R Recovery phase
- SFN Stratefin
- CYTF Cystatin F
- NET1 Shows netrin-1.
- C DAD diagnostic performance by a combination assay of typical DAD marker candidate proteins.
- Ac Acute phase
- ALL-R indicates total recovery phase group
- HC indicates healthy subject group
- SFN Stratefin
- CYTF Cystatin F
- NET1 Netrin-1. Distribution of biomarker concentration in healthy subjects, acute phase, convalescent phase, non-symptomatic patients and other lung diseases of drug-induced interstitial pneumonia. The box shows the quartile deviation (75% and 25%) and median of the ELISA measurements.
- HC Healthy
- DILID Drug-induced interstitial pneumonia
- DAD Diffuse alveolar injury
- OP Organic pneumonia
- NSIP Nonspecific interstitial pneumonia
- Ac Acute phase
- All-R Total recovery phase
- No-onset Non-onset
- LuCa Lung cancer
- Infectious Infectious pulmonary disease
- NTM Non-tuberculous mycobacteriosis
- IIPs Idiopathic interstitial pneumonia
- CTD Collagen disease lung
- COPD Chronic obstructive pulmonary disease
- BA Shows bronchial asthma.
- A Arterial oxygen partial pressure / inspired oxygen saturation ratio (PaO 2 / FiO 2 ) and (B) Percutaneous arterial oxygen saturation / inspired oxygen saturation ratio (SpO 2 / FiO 2 ), (C) LDH, (D)
- PaO 2 / FiO 2 Arterial oxygen partial pressure / inspired oxygen saturation ratio
- SpO 2 / FiO 2 Percutaneous arterial oxygen saturation / inspired oxygen saturation ratio
- C LDH
- D For each value of CRP, the value of clinical examination performed in the acute phase of a patient with drug-induced interstitial pneumonia was used. The values of r s and p were calculated from Spearman's correlation coefficient analysis.
- Test method, diagnostic method, treatment method and test kit for diffuse alveolar injury The present invention is at least one selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T.
- a method for testing diffuse alveolar injury which comprises measuring the expression of a protein in a sample derived from a subject.
- Diffuse alveolar injury is a lung lesion that develops due to acute and severe alveolar injury, and CT shows extensive infiltrative and suriglass-like shadows with structural alterations such as traction bronchiectal dilatation and honeycombing.
- CT shows extensive infiltrative and suriglass-like shadows with structural alterations such as traction bronchiectal dilatation and honeycombing.
- the pathological conditions that present with diffuse alveolar injury include acute respiratory distress syndrome (ARDS), acute interstitial pneumonia (AIP), and acute chronic interstitial pneumonia (IPF). Exacerbations can be mentioned.
- the diffuse alveolar injury may be drug-induced or idiopathic.
- Stratefin also known as 14-3-3 sigma, is a 248 amino acid protein encoded by the SFN gene. It is known to be expressed in the epithelium of the esophagus and skin, and in head and neck cancer, lung cancer, cervical cancer and the like. UniProt number P31947.
- Cystatin F is an extracellular protein of 145 amino acids encoded by the gene CST7. Expression is detected in various cancer cells as well as cells in the bone marrow and immune system. UniProt number O76096.
- NPS-PLA2 whose official name is Phospholipase A2 Group IIA, is an extracellularly secreted enzyme of 144 amino acids encoded by the PLA2G2A gene. It requires calcium for activity expression, catalyzes the hydrolysis of the phosphoglyceride sn-2 fatty acid acyl ester bond, and is involved in the production of free fatty acids and lysophospholipids. Strongly expressed in the gastrointestinal tract. UniProt number P14555.
- IL-1Ra whose official name is an interleukin-1 receptor antagonist, is a protein known as an extracellular secretory type of 159 amino acids encoded by the IL1RN gene. It binds to the receptor IL-1R1 and inhibits the activation of interleukin 1 alpha and 1 beta. High expression is observed in cells of the bone marrow, immune system, and digestive tract. UniProt number P18510.
- Netrin-1 is a 604 amino acid protein encoded by the NTN1 gene and is included in the laminin-related secretory protein family. UniProt number O95631. It is known that the expression is increased in breast cancer, kidney cancer, prostate cancer and the like.
- Troponin T is a protein encoded by the TNNT2 gene. Expression is myocardial specific and involved in myocardial contraction. UniProt number P45379.
- the subject is a mammal suspected of developing diffuse alveolar injury, but all mammals at risk of developing may be targeted. It is typically human.
- the sample derived from the subject include cells, tissues, and body fluids obtained from the subject, specifically, the subject's blood (for example, whole blood, serum, plasma, plasma exchange external fluid, etc.), bronchoalveolar lavage fluid, and the like. can do.
- Whole blood, serum or plasma obtained by a normal blood test (clinical test) may be used as a blood sample.
- the expression in a sample derived from a subject may be measured by measuring the abundance of the above protein or a fragment thereof in the sample.
- a known method may be used without particular limitation. It is preferably measured at the protein level, but may be measured at the nucleic acid level.
- the antibody may be either a monoclonal antibody or a polyclonal antibody.
- These antibodies can be produced by known methods, or commercially available ones may be used. Typical methods include immunoassays such as ELISA and immunochromatography. Since the immunoassay method does not require a special device or technique and can easily and quickly detect and quantify the target protein, it can be preferably used for the measurement of the above protein in the present invention as well.
- Antibodies for the above proteins are known, and commercially available products also exist. Further, as described above, since the amino acid sequence of the above protein and the base sequence encoding these are also known, a specific antibody against each protein may be prepared by preparing a general hybridoma.
- the immunoassay itself is well known in this field.
- a sandwich method a competitive method, an agglutination method, a Western blotting method and the like, and based on the label, there are enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, luminescence immunoassay and the like.
- any immunoassay method capable of quantitative detection may be used.
- a sandwich method such as sandwich ELISA can be preferably used.
- the sandwich method the antibody that binds to the target protein is immobilized and reacted with the sample.
- the target protein bound to the immobilized antibody is measured using a detection antibody labeled with an enzyme or the like.
- the detection antibody it is preferable to use an antibody that binds to the target protein at a site different from that of the immobilized antibody.
- the immobilized antibody and the detection antibody may be a polyclonal antibody or a monoclonal antibody, and an antigen-binding fragment of the antibody may be used.
- the target protein bound to the immobilized antibody is reacted with the detection antibody, washed, and then the amount of the bound detection antibody is measured by a signal from the substance labeled on the antibody.
- the substrate of the enzyme may be added into the reaction system, and the amount of color development, fluorescence, and luminescence generated by the enzyme reaction may be measured with a corresponding device.
- Immunity measurement was performed on a standard sample containing a target protein with a known concentration, a calibration curve was created plotting the relationship between the signal of the labeling substance and the concentration, and the same operation was performed on a sample with an unknown target protein concentration. By applying the obtained signal measurement value to the calibration curve, the target protein in the sample can be quantified.
- nucleic acid probe that can specifically hybridize with the mRNA of the above protein (when measuring by Northern blotting).
- at least one pair of nucleic acid primers capable of specifically amplifying the cDNA synthesized using the mRNA of the above protein as a template may be used (when measured by the RT-PCR method).
- Nucleic acid probes and nucleic acid primers can be designed based on the genetic information (described above) of the above proteins.
- nucleic acid probe one having about 15 to 1500 bases is usually suitable.
- the nucleic acid probe may be labeled with a radioactive element, a fluorescent dye, an enzyme or the like.
- nucleic acid primer one having about 15 to 30 bases is usually suitable.
- Nucleic acid primers may be labeled with radioactive elements, fluorescent dyes, enzymes and the like.
- the expression of the protein or gene may be one type or multiple types. More accurate evaluation may be possible by referring to a plurality of gene expression data and a plurality of protein expression data.
- DNA array probe fixed to substrate
- protein chip antibody substrate
- Luminex NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, JUNE 2002, 447-456
- DNA array probe fixed to substrate
- Luminex NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, JUNE 2002, 447-456
- the expression of at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T is measured in a sample derived from the subject, and the expression level is high. In addition, it can be determined that there is a high possibility of developing diffuse alveolar injury, and when the level is low, it can be determined that the possibility of developing diffuse alveolar injury is low.
- the method of the present invention can assist in the diagnosis of diffuse alveolar injury (determination of the presence or absence of the onset of diffuse alveolar injury).
- the present invention is a method for diagnosing diffuse alveolar injury. a1. Obtaining a sample from a subject, b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and c1. Provided above, the method comprising determining the presence or absence of the onset of diffuse alveolar injury based on the measurements of b1.
- the diagnosis of diffuse alveolar injury can be made based on the following criteria. As a result of measuring the expression of at least one of the above proteins in plasma or serum (blood sample) collected from a subject and comparing it with that in a blood sample collected from a healthy person, it is higher than a preset cutoff value or reference value. If a value is obtained, the subject is assessed as developing diffuse alveolar injury. Alternatively, in a patient receiving a drug, if the concentration of at least one of the proteins in a blood sample becomes higher than the cutoff value, the onset of diffuse alveolar injury due to the drug can be suspected. This preset cutoff value can be appropriately set by those skilled in the art.
- the 95% confidence interval of the quantitative value of a healthy person who has not developed diffuse alveolar injury can be used as a reference value, or the cutoff value can be set from the ROC curve.
- the following cutoff values can be set from the ROC curve analysis of Examples described later.
- the cutoff values for differentiating from healthy subjects are Stratefin: 0.2 to 5.0 ng / mL (preferably 0.7 ng / mL) and NPS-PLA2: 4 to 14 ng / mL (preferably 7 ng).
- the cutoff value for detecting the occurrence of diffuse alveolar injury in drug-administered patients was based on the results of comparative analysis with non-symptomatic patients who did not develop interstitial pneumonia. : 0.7 to 7.2 ng / mL (preferably 2.4 ng / mL), NPS-PLA2: 5 to 60 ng / mL (preferably 32 ng / mL), etc. can be set. Alternatively, if at least one of the above proteins follows an upward trend compared to past measurements, the possibility of developing diffuse alveolar injury is suspected.
- the method of the present invention can also be used for specific diagnosis of diffuse alveolar injury.
- specific diagnosis refers to determining whether a subject has interstitial pneumonia with a diffuse alveolar injury pattern. Since diffuse alveolar injury is a particularly serious form of interstitial pneumonia, it is of great clinical significance that it can be used for specific diagnosis of diffuse alveolar injury.
- At least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T is diffusely affected by high expression levels in subject-derived samples. It can be determined that there is a high possibility of sexual alveolar injury.
- the 95% confidence interval of the quantitative value of patients with interstitial pneumonia other than diffuse alveolar injury can be used as the reference value, or the cutoff value can be set from the ROC curve.
- the following cutoff values can be set from the ROC curve analysis of Examples described later. For example, by comparing the measured values of patients with diffuse alveolar injury and other patients with interstitial pneumonia, as a cut-off value for specific diagnosis of diffuse alveolar injury, Stratefin: 1.0 to 5.3 ng / mL ( Preferably 2.4 ng / mL), NPS-PLA2: 10-87 ng / mL (preferably 32 ng / mL).
- the proteins whose expression is measured are stratefine, cystatin F, NPS-PLA2, IL-1Ra, netrin-1, troponin T, or It is good to have a combination of them.
- the combination include a combination of cystatin F and stratefin, a combination of cystatin F and netrin-1, a combination of stratefin and netrin-1, or a combination of netrin-1 and IL-1Ra.
- the method of the present invention can also be used for pathological diagnosis of diffuse alveolar injury.
- disease diagnosis means the determination of the degree and change (severity, therapeutic effect, etc.) of the pathological condition of a subject diagnosed with diffuse alveolar injury.
- Diffuse alveolar at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T at high expression levels in subject-derived samples. It can be determined that there is a high probability of being in the acute phase of the injury.
- the 95% confidence interval of the quantitative value of a patient in the recovery phase of diffuse alveolar injury can be used as a reference value, or the cutoff value can be set from the ROC curve.
- the following cutoff value can be set from the ROC curve analysis of the examples described later.
- Stratefin 0.8-5.0 ng / mL (preferably 2.3 ng / mL)
- NPS-PLA2 8-32 ng / mL (preferably 16 ng / mL).
- the present invention is a method for diagnosing diffuse alveolar injury.
- a1. Obtaining a sample from a subject, b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and c1.
- At least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T is likely to be in the acute phase of diffuse alveolar injury.
- the expression in the sample derived from the subject determined to be was measured once or multiple times at different times, and when the expression level decreased to the cutoff value or the level close to the reference value, the treatment recovered from the diffuse alveolar injury. If the level is high or does not decrease, it can be determined that the treatment has not recovered from the diffuse alveolar injury or the recovery is insufficient.
- the method of the present invention can be used not only for pathological diagnosis of diffuse alveolar injury, but also for prognosis examination and confirmation of therapeutic effect.
- the present invention is a method for diagnosing and treating diffuse alveolar injury.
- a1. Obtaining a sample from a subject, b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
- c1. Determine the presence or absence of diffuse alveolar injury based on the measured values of b1 and d1.
- Provided by the above method which comprises treating a subject who is determined to have a high probability of developing a diffuse alveolar injury with a treatment for the diffuse alveolar injury.
- the present invention is a method for diagnosing and treating diffuse alveolar injury.
- a1. Obtaining a sample from a subject, b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
- c1. Determining the pathology of diffuse alveolar injury based on b1 measurements, and d1.
- Provided by the above method which comprises treating a subject who is determined to be likely to be in the acute phase of diffuse alveolar injury to be treated for diffuse alveolar injury.
- the present invention can measure the expression of at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T in a sample derived from a subject.
- Kits for testing for diffuse alveolar injury, including reagents, are also provided.
- the kit of the present invention contains an antibody capable of specifically recognizing the above protein as a reagent.
- the antibody may be immobilized on a microtiter plate, magnetic beads, a cellulose membrane or a substrate.
- the kit may further include an instrument for collecting a sample derived from a subject, an anticoagulant, a set of reagents for detecting the protein, an instruction manual, and the like.
- the instruction manual should also describe the evaluation and / or differentiation criteria for cases of diffuse alveolar injury or acute exacerbation of acute respiratory distress syndrome or idiopathic interstitial pneumonia. ..
- the kit of the present invention contains a nucleic acid probe that can specifically hybridize with the mRNA of the above protein as a reagent.
- the nucleic acid probe may be fixed to the substrate.
- the kit also includes equipment for collecting biological samples, anticoagulants, reagents for extracting RNA from samples derived from subjects, reagents for detecting RNA, instruction manuals, etc. May be good.
- the instruction manual should also describe the evaluation and / or differentiation criteria for cases of diffuse alveolar injury or acute exacerbation of acute respiratory distress syndrome or idiopathic interstitial pneumonia. ..
- the kit of the present invention contains at least one pair of nucleic acid primers capable of specifically amplifying cDNA synthesized using the above protein mRNA as a reagent.
- the kit also includes equipment for collecting subject-derived samples, anticoagulants, reagents for extracting RNA from subject-derived samples, reagents for detecting RNA, instruction manuals, and the like. It is good to be.
- the instruction manual should also describe the evaluation and / or differentiation criteria for acute exacerbation cases of diffuse alveolar injury, acute respiratory distress syndrome and idiopathic interstitial pneumonia.
- the kit of the present invention may also include a standard protein, a buffer, a substrate (when the antibody is enzyme-labeled), a reaction stop solution, a washing solution, a reaction vessel, and the like.
- the kit of the present invention can be used as a pharmaceutical product for diagnosing a disease.
- test method, diagnostic method, treatment method and test kit for general interstitial pneumonia The present invention also relates to Stratefin, calistatin, NPS-PLA2, PARC (CCL18). , LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6 for at least one protein selected from the group to measure expression in a sample derived from the subject.
- a method for examining interstitial pneumonia in general including.
- Interstitial pneumonia is a condition in which inflammation spreads with the interstitium of the lung as the main lesion, and often causes pulmonary fibrosis. Interstitial pneumonia is divided into those associated with clear causes such as drug-induced, pneumoconiosis, and collagen disease, and idiopathic diseases of unknown cause, and further classified into various types. Typical disease types include diffuse alveolar injury, organizing pneumonia, nonspecific interstitial pneumonia, and eosinophilic pneumonia. In the method for testing interstitial pneumonia of the present invention, either drug-induced interstitial pneumonia or non-drug-induced (idiopathic) interstitial pneumonia may be used regardless of the etiology and type of interstitial pneumonia. , Diffuse alveolar injury or other interstitial pneumonia.
- PARC is a type of chemokine having a CC motif of 89 amino acids encoded by the CCL18 gene, and is classified as homeostatic or plasma chemokine. It is expressed in adipocytes, lungs, lymphocytes, etc. UniProt number P55774.
- LD78-beta is an inflammatory chemokine with a CC motif of 93 amino acids encoded by the CCL3L1 gene and its copy CCL3L3 gene. It is expressed in bone marrow and lymphocytes. It is known to bind to the HIV co-receptor CCR5 and the silent receptor CCBP2 and to be involved in the control of HIV infection. UniProt number P16619.
- PAPP-A paparicin
- PAPP-A is a secretory metalloprotease of 1627 amino acids encoded by the PAPPA gene. It is known to be specifically expressed in the placenta of pregnant women, and its expression in aneurysms has also been reported. UniProt number Q13219.
- Apolipoprotein AI is a secretory protein of 267 amino acids encoded by the APOAI gene. It is strongly expressed in the liver and gastrointestinal tract and is known as the main component of blood HDL. UniProt number P02647.
- Calistatin also called kallikrein inhibitor or serpin A4, is a secretory serine protease inhibitor of 427 amino acids encoded by the SERPINA4 gene. Produced in the liver and abundant in blood, it binds to tissue kallikrein and caridinogenase and inhibits its activity. UniProt number P29622.
- Complement C3b (C3b) is a degradation product of complement C3 encoded by the C3 gene.
- Complement C3 is produced in the liver and decomposed into C3a and C3b in the blood, and C3b is involved in the immunity of the living body by controlling the decomposition of complement C5.
- Carbonic amphidrase 6 is a 308 amino acid metal enzyme encoded by the CA6 gene, which catalyzes the reaction of converting carbon dioxide and water into bicarbonate ions and hydrogen ions. It is strongly expressed in the salivary glands. UniProt number P23280.
- the subject is a mammal suspected of developing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia), but there is a risk of developing it. It may cover all possible mammals. It is typically human.
- the sample derived from the subject and the measurement of expression are as described above.
- the protein or gene whose expression is measured may be one type or a plurality of types.
- the expression of at least one protein selected from the group consisting of stratefin, NPS-PLA2, PARC (CCL18), LD78-beta and PAPP-A (paparicin) and complement C3b was measured in a sample derived from the subject. If the expression level is high, it is determined that there is a high possibility of developing interstitial pneumonia (diffuse alveolar injury or other interstitial pneumonia), and if the level is low, interstitial pneumonia is likely to occur. It can be determined that the possibility of developing is low. On the contrary, when the expression level of at least one protein selected from the group consisting of Apo-AI, calistatin and carbonic anhydrase 6 is measured in a sample derived from a subject and the expression level is low. It is determined that there is a high possibility of developing interstitial pneumonia (diffuse alveolar injury or other interstitial pneumonia), and if the above level is high, it is possible that interstitial pneumonia has developed. It can be determined that the sex is low.
- the method for testing interstitial pneumonia of the present invention can assist in the diagnosis of interstitial pneumonia (determination of the presence or absence of diffuse alveolar injury and other interstitial pneumonia).
- the present invention is a method for diagnosing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
- a2. Obtaining a sample from the subject, b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6.
- Provided above are the methods comprising determining the presence or absence of the onset of interstitial pneumonia based on the measurements of b2.
- the diagnosis of interstitial pneumonia can be made based on the following criteria.
- the expression of at least one of the above proteins in serum or plasma (blood sample) collected from a subject and comparing it with that in a blood sample collected from a healthy person, it is higher than a preset cutoff value or reference value.
- a preset cutoff value or reference value for example, the expression of at least one of the above proteins in serum or plasma (blood sample) collected from a subject and comparing it with that in a blood sample collected from a healthy person.
- the concentration of at least one of the above proteins in a blood sample is higher than the cutoff value in a patient receiving a drug (Stratefin, NPS-PLA2, PARC (CCL18), LD78-beta and PAPP-A). (Paparicin) and complement C3b), or when it becomes low (Apo-AI, calistatin and carbonic anhydrase 6), the occurrence of interstitial pneumonia (drug-induced interstitial pneumonia) due to the drug may be suspected. it can.
- the above-mentioned preset cutoff value can be appropriately set by those skilled in the art.
- the 95% confidence interval of the quantitative value of a healthy person who does not develop interstitial pneumonia can be used as a reference value, or the cutoff value can be set from the ROC curve.
- the following cutoff values can be set from the ROC curve analysis of Examples described later.
- the cut-off values for differentiation from healthy subjects are Stratefin: 0.2 to 1.0 ng / mL (preferably 0.5 ng / mL) and Calistatin: 7.5 to 14.5 ⁇ g / mL (preferably 10 ⁇ g / mL).
- NPS-PLA2 3 to 12 ng / mL (preferably 6 ng / mL)
- PARC CCL18
- the cut-off value for detecting the occurrence of interstitial pneumonia in drug-administered patients was compared with those who did not develop interstitial pneumonia, and Stratefin: 0.2 to 1.7 ng / mL (preferably 0.6 ng).
- calistatin 7.5-14 ⁇ g / mL (preferably 11 ⁇ g / mL)
- NPS-PLA2 7.5-14 ng / mL (preferably 9 ng / mL)
- PARC (CCL18) 20-60 It can be set to ng / mL (preferably 36 ng / mL).
- one of Stratefin, NPS-PLA2, PARC (CCL18), LD78-beta, PAPP-A (Paparicin) and complement C3b follows an upward trend compared to past measurements, or Apo -If AI, calistatin and carbonic anhydrase 6 follow a decreasing trend, suspect the possibility of developing interstitial pneumonia.
- the onset of interstitial pneumonia was suspected by at least one measurement of calistatin, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6. Diffuse if at least one protein selected from the group consisting of stratephin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T is higher than the baseline or cutoff value in the patient. Sexual alveolar injury can be strongly suspected, and if stratephin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T are all within the norm or below the cutoff value, during the rest. You can suspect that you have pneumonia.
- the method for testing interstitial pneumonia of the present invention can also be used for pathological diagnosis of interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia).
- at least one protein selected from the group consisting of Apo-AI, calistatin and carbonic anhydrase 6 is in the acute phase of interstitial pneumonia when the expression level in the sample derived from the subject is low. It can be determined that the possibility is high.
- the 95% confidence interval of the quantitative value of a patient in the recovery phase of interstitial pneumonia can be used as a reference value, or the cutoff value can be set from the ROC curve.
- the following cutoff value can be set from the ROC curve analysis of the examples described later.
- Stratefin 0.2-1.7 ng / mL (preferably 1 ng / mL)
- calistatin 7.5-14.5 ng / mL (preferably 10 ng / mL)
- NPS-PLA2 4-28 (preferably 8 ng) / mL)
- PARC (CCL18) 16-60 ng / mL (preferably 33 ng / mL).
- the present invention is a method for diagnosing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
- a2. Obtaining a sample from the subject, b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6.
- Provided above are methods that include determining the pathology of interstitial pneumonia based on b2 measurements.
- samples from subjects determined to be more likely to be in the acute phase of interstitial pneumonia include Stratefin, calistatin, NPS-PLA2, PARC ( At least one protein selected from the group consisting of CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6, once or multiple times at different times.
- the measurement is within the standard value, it is determined that the patient has recovered from interstitial pneumonia by treatment, and if the level remains outside the standard value, the patient has not recovered from interstitial pneumonia by treatment. Alternatively, it can be determined that the recovery is inadequate.
- the method of the present invention can be used not only for diagnosing the pathology of interstitial pneumonia, but also for prognosis examination and confirmation of therapeutic effect.
- steroid treatment should be considered according to the symptoms and severity.
- the dose of steroid starts from 0.5 to 1.0 mg / kg / day in terms of prednisolone, and gradually decreases and ends in about 2 months.
- immunosuppressive drugs cyclosporine, tacrolimus, etc.
- neutrophil elastase inhibitors selective neutrophil elastase inhibitors
- Mayoclinic offers general treatments for interstitial pneumonia such as drug treatment, oxygen therapy, lung rehabilitation, and lung transplantation (https://www.mayoclinic.org/diseases-conditions/interstitial). -lung-disease / diagnosis-treatment / drc-20353113).
- Pharmacotherapy includes 1) concomitant use of corticosteroids (prednisolone, etc.) and their immunosuppressive drugs: expected to slow or stabilize the progression of interstitial pneumonia, 2) idiopathic lung fibers. Drugs for illness (pirfenidone and nintedanib: expected to slow the progression of interstitial pneumonia).
- gastric acid secretion inhibitors There are gastric acid secretion inhibitors (lansoprazole, omeprazole, etc .: prevention of reflux esophagitis reduces aspiration of gastric juice and suppresses exacerbation of lung damage). Oxygen therapy cannot prevent lung damage, but is expected to ease breathing, prevent and reduce complications due to decreased blood oxygen levels, and reduce blood pressure on the right side of the heart. Lung rehabilitation improves quality of life, including ease of daily living. Lung transplantation is a last resort for patients with severe interstitial pneumonia who have no other treatment options.
- IPF which is a type of interstitial pneumonia
- the number of patients is large and the prognosis is poor. Therefore, treatment guidelines have been published by the American Respiratory Society based on clinical research results (AMERICAN JOURNAL OF RESPIRATORY AND CRINICAL CARE MEDICINE). , JULY 2015. 192 (2): e3-19).
- Nintedanib vascular endothelial cell growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), etc.
- VAGFR vascular endothelial cell growth factor receptor
- FGFR fibroblast growth factor receptor
- PDGFR platelet-derived growth factor receptor
- the present invention is a method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
- a2. Obtaining a sample from the subject, b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6.
- c2. Determine the presence or absence of interstitial pneumonia based on the measured value of b2, and d2.
- Provided by the above method which comprises treating an interstitial pneumonia in a subject determined to have a high possibility of developing interstitial pneumonia.
- the present invention is a method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
- a2. Obtaining a sample from the subject, b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6.
- c2 Judging the pathology of interstitial pneumonia based on the measured values of b2, and d2.
- Provided by the above method which comprises treating an interstitial pneumonia with a subject determined to be likely to be in the acute phase of interstitial pneumonia.
- the present invention is also selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6.
- a kit for testing interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia) containing a reagent capable of measuring the expression of at least one protein in a sample derived from a subject. provide.
- the kit of the present invention contains an antibody capable of specifically recognizing the above protein as a reagent.
- the antibody may be immobilized on a microtiter plate, magnetic beads, a cellulose membrane or a substrate.
- the kit may further include an instrument for collecting a sample derived from a subject, an anticoagulant, a set of reagents for detecting the protein, an instruction manual, and the like.
- the instruction manual should also describe the evaluation and / or discrimination criteria for interstitial pneumonia.
- the kit of the present invention contains a nucleic acid probe that can specifically hybridize with the mRNA of the above protein as a reagent.
- the nucleic acid probe may be fixed to the substrate.
- the kit also includes equipment for collecting biological samples, anticoagulants, reagents for extracting RNA from samples derived from subjects, reagents for detecting RNA, instruction manuals, etc. May be good.
- the instruction manual should also describe the evaluation and / or discrimination criteria for interstitial pneumonia.
- the kit of the present invention contains at least one pair of nucleic acid primers capable of specifically amplifying cDNA synthesized using the above protein mRNA as a reagent.
- the kit also includes equipment for collecting subject-derived samples, anticoagulants, reagents for extracting RNA from subject-derived samples, reagents for detecting RNA, instruction manuals, and the like. It is good to be.
- the instruction manual should also describe the evaluation and / or discrimination criteria for interstitial pneumonia.
- the kit of the present invention may also include a standard protein, a buffer, a substrate (when the antibody is enzyme-labeled), a reaction stop solution, a washing solution, a reaction vessel, and the like.
- the kit of the present invention can be used as a pharmaceutical product for diagnosing a disease.
- Example 1 (1) Regarding human interstitial pneumonia samples used for sample analysis, four base hospitals (Shinshu University, Japan Medical University, Chiba University, Hiroshima University), National Institute of Pharmaceutical and Food Sanitation, Kihara Foundation, Astellas Pharma, And Daiichi Sankyo collected and analyzed with the approval of each research ethics committee.
- blood was collected at the above-mentioned base hospital during the acute phase (near the worst phase) and convalescent phase of drug-induced interstitial pneumonia.
- Blood was collected early in the morning on an empty stomach at the time of admission and at any time during the outpatient department.
- blood is collected using a 7 mL EDTA-2K blood collection tube for plasma collection, and after prompt mixing, centrifugation is performed at 3,000 rpm x 10 minutes (15 ° C to 20 ° C). went.
- the collected plasma was cryopreserved at -80 ° C.
- the patient specimens of drug-induced interstitial pneumonia there are 8 specimens collected in the acute phase and 3 specimens collected in the convalescent phase in the cases showing the diffuse alveolar injury (DAD) pattern in the diagnostic imaging. , Used for analysis. About the cases of organizing pneumonia (OP) pattern, 16 cases of acute phase, 13 cases of convalescent phase, and the cases of nonspecific interstitial pneumonia (NSIP) pattern are 18 cases of acute phase, 16 cases of convalescent phase, and others. For the interstitial pneumonia type, 2 cases in the acute phase and 1 case in the convalescent phase were used. As a healthy person sample, a plasma sample of volunteer healthy adults (24 cases having the same age composition ratio with the group of patients with interstitial pneumonia) collected by Neues Co., Ltd. was used.
- FIG. 1 shows the 6 types of DAD marker candidate proteins (Stratefin, Cystatin F, NPS-PLA2, IL-1Ra, Netrin-1 and Troponin T). The fluctuation pattern in plasma is shown by a box plot. Both proteins showed a marked increase in DAD in the acute phase compared to healthy subjects and decreased to the same extent as in healthy subjects in the recovery phase, but the fluctuations in OP and NSIP were small. These proteins can be useful markers for DAD-specific diagnosis.
- DAD marker candidate proteins Stratefin, Cystatin F, NPS-PLA2, IL-1Ra, Netrin-1 and Troponin T.
- the fluctuation pattern in plasma is shown by a box plot. Both proteins showed a marked increase in DAD in the acute phase compared to healthy subjects and decreased to the same extent as in healthy subjects in the recovery phase, but the fluctuations in OP and NSIP were small. These proteins can be useful markers for DAD-specific diagnosis.
- the AUC value based on the clinical laboratory test value of the existing interstitial pneumonia marker KL-6 was 0.78, which was lower than that of the above protein.
- the AUC values of Stratefin (0.90) and Sistatin F (0.91) are the highest, and other proteins are also AUC.
- the DAD diagnostic performance was 0.8 or higher, which was superior to the existing marker KL-6 (0.67).
- FIGS. 3A and 3B show the results of examining the effect of the combination assay on the DAD diagnostic performance using cystatin F and stratefin, which had low correlation, and netrin-1 and IL-1Ra as examples.
- the detection efficiency of DAD samples was improved, and the AUC value when the combination of cystatin F and stratefin was 0.98, netrin-1 and IL- The AUC value of 1Ra rose to 0.97.
- the combination of cystatin F and netrin-1 and stratefin and netrin-1 was also shown to improve the DAD diagnostic performance to about AUC 0.95.
- the ability to distinguish the acute phase of patients with DAD and OP from the healthy group is that all proteins have an AUC value of 0.9 or higher. It is shown (Table 2). Furthermore, the AUC values of Stratefin and NPS-PLA2, which showed a marked increase in DAD, were 0.87 and 0.95, respectively, showing the ability to distinguish the acute phase of DAD and OP patients from the healthy group. In addition, in patients with acute phase of all interstitial pneumonia including NSIP, although the AUC value decreased slightly, proteins other than complement C3b had an AUC value of 0.89 or more, which differentiated them from healthy subjects.
- the discrimination performance for the convalescent group tended to be inferior to that of healthy subjects in both the acute phase of patients with DAD and OP and the acute phase of patients with total interstitial pneumonia, although it was existing.
- the AUC value was equal to or higher than that of the markers KL-6 and SP-D (Table 2). From the above results, it was shown that the nine proteins shown in Table 2 can be biomarkers for detecting interstitial pneumonia regardless of the disease type.
- Table 1 Diffuse alveolar injury-specific proteins and biomarker performance. Interstitial pneumonia other than DAD acute phase (DAD-Ac), healthy group (HC), total recovery phase group (All-R), and DAD for 6 types of proteins that showed characteristic fluctuations in DAD in SOMAscan analysis.
- the discrimination performance from the group (non-DAD-Ac) is shown by the AUC value calculated from the ROC curve analysis.
- the AUC value of the existing marker KL-6 was calculated from the clinical laboratory test values measured at the time of sample collection.
- Biomarker performance of proteins that detect general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia).
- HC healthy group
- All-Re convalescent group
- ROC curve analysis was performed and the calculated AUC value was shown.
- the AUC values of the existing markers KL-6 and SP-D were calculated from the clinical laboratory test values measured at the time of sample collection. DAD indicates diffuse alveolar injury, OP indicates organizing pneumonia, All indicates interstitial pneumonia in general, and Ac indicates acute phase.
- Lung cancer cases non-onset cases
- lung cancer cases idiopathic interstitial pneumonitis, collagen disease lung, COPD, non-tuberculous mycobacteriosis, bronchial asthma
- a total of 401 cases, including infectious lung diseases (fungal / bacterial infections), or some serum or plasma samples thereof were used for verification using the ELISA kit.
- ELISA Kit Among the proteins shown in Tables 1 and 2, stratefin, calistatin, NPS-PLA2, and PARC were measured by the sandwich ELISA method. Regarding stratefin, an ELISA measurement system was constructed using anti-stratefin mouse monoclonal antibodies (Sigma-Aldrich and Merck), and Escherichia coli recombinant human stratefin (NKMAX) was used as a standard product. Commercially available research ELISA kits were used to verify NPS-PLA2 (Cayman), PARC (R & D), and calistatin (R & D).
- Stratefin did not show high-value samples in infectious lung disease.
- calistatin, NPS-PLA2, and PARC showed fluctuations in drug-induced interstitial pneumonia in general, as well as infectious diseases, idiopathic interstitial pneumonia, and collagen disease lungs. ..
- FIG. 5 and Table 4 show the results of comparing the biomarker performance of each protein for DAD based on the ELISA measurement results. Differentiation performance from healthy subjects for DAD acute phase (confirmation) cases (mixed DAD and DAD type), differentiation performance from total convalescent phase (disease discrimination ability), and differentiation performance from other interstitial pneumonia disease types (DAD diagnosis) Table 4 shows the AUC values calculated from ROC curve analysis (FIGS. 5A to D) for performance) and differentiation performance from the non-symptomatic group.
- Stratefin 0.7 ng / mL
- calistatin 9.2 ⁇ g / mL
- PARC 35 ng / mL
- NPS-PLA2 7.0 ng / mL.
- the cut-off values for determining the pathology of DAD are Stratefin: 2.3 ng / mL, Calistatin: 8.6 ⁇ g / mL, PARC: 32 ng / mL, NPS-PLA2: 16 ng / mL, and DAD diagnosis (others).
- the cut-off values for differentiation from the disease type were Stratefin: 2.4 ng / mL, calistatin: 8.6 ⁇ g / mL, PARC: 57 ng / mL, and NPS-PLA2: 63 ng / mL.
- the cut-off values for differentiation from non-symptomatic cases were Stratefin: 2.4 ng / mL, calistatin: 11 ⁇ g / mL, PARC: 35 ng / mL, and NPS-PLA2: 32 ng / mL.
- differentiation from infectious lung disease is also important for determining treatment policy.
- Verification as a biomarker for interstitial pneumonia in general Table 5 shows the results of comparing the discrimination performance of interstitial pneumonia in general with healthy subjects or in the convalescent period.
- Stratefin, calistatin, NPS-PLA2, and PARC showed high discrimination performance (AUC 0.86 or higher) from healthy subjects (Fig. 6A).
- the ability to discriminate disease in general interstitial pneumonia (compared to the total recovery phase group) is similar to that of SP-D (0.77) for calistatin (AUC 0.76) and PARC (0.74), and KL-6 (same). It exceeded 0.62) (Fig. 6B).
- calistatin AUC 0.81 was superior (Fig. 6C).
- Table 6 sets the cutoff values for stratefin, calistatin, PARC, and NPS-PLA2, and SP-D for each interstitial pneumonia type and positive rate for each disease. This is the result of comparison with KL-6. Clinically used reference values (500 U / mL and 110 ng / mL) were compared for KL-6 and SP-D, and DAD acute (confirmed) cases and non-symptomatic groups were compared for other proteins. From the ROC curve (Fig. 5D), the concentration with the highest sensitivity and specificity was set as the cutoff value.
- Stratefin In Stratefin, no positive cases (> 2.4 ng / mL) were found in the healthy group, while a high positive rate of 92% was shown in DAD acute phase (confirmed) cases (DAD and DAD mixed type). In contrast, other disease types such as OP and NSIP were as low as 20-33%, so by setting an appropriate cutoff value, Stratefin may be useful for DAD-specific serodiagnosis. It has been suggested. In addition, the positive rates (11% and 13%) of stratefin in the total recovery phase group and non-onset group were higher than those of KL-6 (49% and 30%, respectively) and SP-D (42% and 27%, respectively). It was low and was not positive for infectious lung disease or nontuberculous mycobacteriosis.
- NPS-PLA2 also showed a DAD-dominant positive rate (79%), but there were many positive cases of infectious lung disease (62%).
- Calistatin and PARC had high positive rates not only in acute DAD (confirmed) cases, but also in OP and NSIP, and the positive rate in the total recovery group was lower than that in KL-6 and SP-D. This is a result suggesting that calistatin and PARC may be useful for determining the pathological condition of drug-induced interstitial pneumonia in general.
- Table 7 shows the results of examining whether other proteins can be discriminated and detected as positive in drug-induced interstitial lung disease cases in which KL-6 or SP-D was negative.
- KL-6 negative 500 U / mL or less
- SP-D positive 55%
- calistatin was positive ( ⁇ 11 ⁇ g / mL) in 19 cases (86%)
- PARC was also positive in 16 cases (84%) out of 19 cases analyzed.
- calistatin and PARC also had higher positive rates for OP and NSIP.
- calistatin and PARC showed a high positive rate (79-81%). Therefore, it was suggested that the combined use of these proteins may increase the detection efficiency of drug-induced interstitial pneumonia.
- KL-6 and SP-D are cases of acute exacerbation of idiopathic interstitial pneumitis (AE-IIPs, High in idiopathic pulmonary fibrosis (IPF) including acute exacerbation and acute idiopathic pneumonia), but positive in other interstitial pneumonia types such as IPF and NSIP (SP-D> 110 ng / mL, KL-6> 500 U / mL) was the most common.
- Calistatin was also generally positive for idiopathic interstitial pneumonia (cutoff: ⁇ 11 ⁇ g / mL).
- Stratefin tended to be high in AE-IIPs, and was often negative (2.4 ng / mL or less) in other types. Therefore, it was shown that Stratefin is useful as a DAD marker not only for drug-induced but also for idiopathic interstitial pneumonia, and calistatin is also useful for idiopathic interstitial pneumonia in general. ing.
- FIG. 8 shows the results of examining the correlation between the patient's lung function (blood oxygenation ability) and inflammatory parameters and Stratefin.
- Blood stratefin concentration correlates with PaO 2 / FiO 2 ratio and SpO 2 / FiO 2 ratio, which are indicators of blood oxygenation capacity (Figs. 8A and B), and is an indicator of the degree of tissue damage and inflammation. It also showed a correlation with LDH and CRP (Figs. 8C and D). It was suggested that the blood stratefin concentration fluctuates with the decrease of lung function due to tissue damage and inflammation.
- the present invention can be used for in vitro diagnostic agents, clinical tests, and the like.
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Abstract
A development is carried out of biomarkers for the diagnosis and disease state diagnosis of interstitial pneumonia in general or particularly diffuse alveolar damage (DAD), and for the differential diagnosis of other interstitial pneumonias, e.g., organizing pneumonia (OP), and DAD having different treatment responsiveness. Provided is a method for detecting diffuse alveolar damage, comprising measuring the expression in a sample from a subject for at least one protein selected from the group consisting of stratifin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1, and troponin T. Provided is a method for detecting interstitial pneumonia in general (diffuse alveolar damage and other interstitial pneumonias), comprising measuring the expression in a sample from a subject for at least one protein selected from the group consisting of stratifin, kallistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-Al, PAPP-A (pappalysin), complement C3b, and carbonic anhydrase 6.
Description
本発明は、間質性肺炎の発症や病勢の診断を補助するための方法に関する。
The present invention relates to a method for assisting in the diagnosis of the onset and pathological condition of interstitial pneumonia.
間質性肺炎は肺の間質の炎症性疾患の総称であり、細菌感染を代表とする肺胞性肺炎と大別される。間質性肺炎は、薬剤性やじん肺、膠原病などの明確な原因に伴うものと、原因が不明な特発性とに分けられ、さらに様々な病型に分類される。それぞれ治療法や予後が異なるため、これらを鑑別することは臨床上重要である。
Interstitial pneumonia is a general term for inflammatory diseases of the lung interstitium, and is roughly classified into alveolar pneumonia represented by bacterial infection. Interstitial pneumonia is divided into those associated with clear causes such as drug-induced, pneumoconiosis, and collagen disease, and idiopathic diseases of unknown cause, and further classified into various types. Differentiation of these is clinically important because each has different treatments and prognosis.
特発性間質性肺炎の病型は、臨床所見と画像所見、病理組織パターンによって診断されるが、薬剤性間質性肺炎についても、特発性や既知の肺疾患との類似性に基づいて、病型の診断が行われる（非特許文献1）。間質性肺炎の病型の中で、特に重篤な病型は、びまん性肺胞傷害（diffuse alveolar damage、DAD）である。DADは、急性呼吸窮迫症候群や特発性間質性肺炎の急性増悪症例に見られる典型的な病型であり(非特許文献２)、特発性においても薬剤性においても、DADは治療反応性に乏しく予後が悪い（非特許文献１、２）。そのため、DADに対しては、早期の検出と診断が特に重要とされる。また、DAD以外の病型としては、器質化肺炎（organizing pneumonia、OP）、非特異性間質性肺炎（nonspecific interstitial pneumonia、NSIP）などが挙げられるが、どのパターンにも当てはまらない例や複数のパターンが混在する例など、画像検査や病理検査では分類が困難な例も存在し、画像パターン分類には限界がある（非特許文献3）
The type of idiopathic interstitial pneumonia is diagnosed by clinical and imaging findings and histopathological patterns, but drug-induced interstitial pneumonia is also based on idiopathic and similarities with known lung diseases. The type of disease is diagnosed (Non-Patent Document 1). Among the types of interstitial pneumonia, the most serious type is diffuse alveolar damage (DAD). DAD is a typical type of disease found in acute exacerbation cases of acute respiratory distress syndrome and idiopathic interstitial pneumonia (Non-Patent Document 2), and DAD is therapeutically responsive, both idiopathic and drug-induced. Poor and poor prognosis (Non-Patent Documents 1 and 2). Therefore, early detection and diagnosis are of particular importance for DAD. In addition, as pathological types other than DAD, organizing pneumonia (OP), nonspecific interstitial pneumonia (NSIP), etc. can be mentioned, but there are cases and multiple cases that do not fit into any pattern. There are cases where it is difficult to classify by image inspection or pathological examination, such as cases where patterns are mixed, and there is a limit to image pattern classification (Non-Patent Document 3).
間質性肺炎の診断は、確定に時間を要することも多く、画像診断や病理診断だけでは経済的にも体力的にも患者に大きな負担を強いるため、バイオマーカーの利用が有用とされる。
Diagnosis of interstitial pneumonia often takes time to be confirmed, and the use of biomarkers is useful because it imposes a heavy burden on patients economically and physically only by diagnostic imaging and pathological diagnosis.
間質性肺炎のバイオマーカーとしては、現在、シアル化糖鎖抗原KL-6（KL-6）、肺サーファクタントプロテインA（SP-A）、肺サーファクタントプロテインD（SP-D）が、診断の補助に利用されており、KL-6はDADに対する陽性率が高いことが知られている（非特許文献4, 5）。これらは、薬剤性肺障害を反映する指標であるが、喫煙や間質性肺炎以外の疾患でも増加することもあり、肺障害の重症度とは相関しない。KL-6は、薬剤投与前値を基準に経過を追うことが推奨されているものの、既存の間質性肺炎の増悪や日和見感染症でも上昇する。すなわち、これらのマーカーは、DADに特異的ではなく、病型を鑑別するマーカーとしての有用性は低い。また、KL-6、SP-D、およびSP-Aは病状の改善後も長期間高値を示す（非特許文献4, 5）という問題があることから、新たなバイオマーカーが必要とされている。これまでに、UBE2T、HK1、PMSE1、USO1、IFI16、GLTPなどのタンパク質や（特許文献1）、自己抗体（特許文献2）などがマーカー候補として提案、特許申請されている。
Currently, sialylated sugar chain antigen KL-6 (KL-6), pulmonary surfactant protein A (SP-A), and pulmonary surfactant protein D (SP-D) assist in diagnosis as biomarkers for interstitial pneumonia. It is known that KL-6 has a high positive rate for DAD (Non-Patent Documents 4 and 5). Although these are indicators that reflect drug-induced lung injury, they may also increase in diseases other than smoking and interstitial pneumonia and do not correlate with the severity of lung injury. Although it is recommended to follow the course of KL-6 based on the pre-drug administration value, it also increases in the exacerbation of existing interstitial pneumonia and opportunistic infections. That is, these markers are not specific to DAD and are less useful as markers for differentiating the disease type. In addition, KL-6, SP-D, and SP-A have a problem that they show high values for a long period of time even after the condition is improved (Non-Patent Documents 4 and 5), so new biomarkers are required. .. So far, proteins such as UBE2T, HK1, PMSE1, USO1, IFI16, and GLTP (Patent Document 1) and autoantibodies (Patent Document 2) have been proposed and applied for patents as marker candidates.
本発明は、薬剤性をはじめとする間質性肺炎の診断、病勢、およびその病型を診断、予測するためのバイオマーカーの開発を行うことを目的とする。
An object of the present invention is to develop a biomarker for diagnosing and predicting drug-induced interstitial pneumonia, a disease state, and its type.
1,310種のタンパク質に対するアプタマー（一本鎖核酸による人工リガンド）を用いたSOMAscan法にて、薬剤性間質性肺炎患者の血液試料を対象にプロテオーム解析を行った。びまん性肺胞傷害（DAD）と器質化肺炎（OP）に焦点を当て、SOMAscanの相対蛍光強度から、健常人や回復期群に対して当該疾患の急性期で大きく変動するタンパク質を、Fold change値と効果量（Hedge’s g値）を指標に探索した。KL-6やSP-D値が変動を示していない検体が多数存在する中、測定した1,310種のタンパク質の中から、DADの急性期では増加するが他の病型では変化が認められないタンパク質を6種類（ストラテフィン、シスタチンF、NPS-PLA2、IL-1Ra、ネトリン-1及びトロポニンT）、またDADだけでなく、OPや非特異性間質性肺炎（NSIP）など他の間質性肺炎においても増加もしくは減少するタンパク質を９種類（ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６）見いだした。
Proteomics analysis was performed on blood samples of drug-induced interstitial pneumonia patients by the SOMAscan method using aptamers (artificial ligands using single-stranded nucleic acids) for 1,310 proteins. Focusing on diffuse alveolar injury (DAD) and organizing pneumonia (OP), based on the relative fluorescence intensity of SOMAscan, a protein that fluctuates significantly in the acute phase of the disease in healthy subjects and convalescent groups is changed. The search was performed using the value and effect size (Hedge's g value) as indicators. While there are many samples in which KL-6 and SP-D levels do not fluctuate, among the 1,310 proteins measured, proteins that increase in the acute phase of DAD but do not change in other types of disease. 6 types (Stratefin, Cystatin F, NPS-PLA2, IL-1Ra, Netrin-1 and Troponin T), as well as other interstitial properties such as OP and nonspecific interstitial pneumonia (NSIP) Nine types of proteins that increase or decrease in pneumonia (stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic hydrase 6) I found it.
ROC曲線（Receiver Operatorating Characteristic curve)解析の結果、DADで特異的な変動を示したタンパク質は、DAD患者と健常人を高い精度（area under the curve: AUC 0 .95以上）で鑑別した。さらに、薬剤性間質性肺炎患者の中からDAD患者を判別する能力は、シスタチンF (AUC 0.91)やストラテフィン（0.90）、ネトリン-1（0.85）で比較的高かったが、相関性の低いタンパク質同士を組み合わせることで、AUC値が 0.95以上にまで向上した。また、PARC (CCL18)やLD78-betaなどのケモカインや、Apo-AIやカリスタチン、PAPP-A（パパリシン）等は、DADに限らずOPやNSIPの急性期においても変動が認められ、DADやOP患者の群をAUC 0.9以上で健常人と識別できた。これらは、薬剤性をはじめとする間質性肺炎に対する有用なバイオマーカーとなりうることが示された。
As a result of ROC curve (Receiver Operating Characteristic curve) analysis, proteins showing specific fluctuations in DAD differentiated DAD patients from healthy subjects with high accuracy (area under the curve: AUC 0.95 or higher). Furthermore, the ability to discriminate DAD patients from drug-induced interstitial pneumonia patients was relatively high with cystatin F (AUC 0.91), stratefin (0.90), and netrin-1 (0.85), but the correlation was low. By combining proteins, the AUC value improved to 0.95 or higher. In addition, chemokines such as PARC (CCL18) and LD78-beta, Apo-AI, calistatin, PAPP-A (paparicin), etc. are not limited to DAD, but also fluctuate in the acute phase of OP and NSIP, and DAD and OP A group of patients could be identified as healthy individuals with an AUC of 0.9 or higher. It has been shown that these can be useful biomarkers for interstitial pneumonia including drug-induced.
本発明は、これらの知見に基づいて、完成されたものである。
（１）ストラテフィン（SFN）、NPS-PLA2、シスタチンF（CYTF）、IL-1Ra、ネトリン-1（NET1）及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することを含む、びまん性肺胞傷害の検査方法。
（２）発現を測定するタンパク質が、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質であり、測定値がびまん性肺胞傷害の病勢診断を補助する（１）記載の方法。
（３）発現を測定するタンパク質が、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質であり、測定値がびまん性肺胞傷害の特異的診断を補助する（１）記載の方法。
（４）発現を測定するタンパク質が、ストラテフィン、シスタチンF、NPS-PLA2、IL-1Ra、ネトリン-1、トロポニンT、又はそれらの組み合わせである（３）記載の方法。
（５）びまん性肺胞傷害の診断方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c1. b1の測定値に基づき、びまん性肺胞傷害の発症の有無を判定すること
を含む前記方法。
（６）びまん性肺胞傷害の診断方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c1. b1の測定値に基づき、びまん性肺胞傷害の病勢を判定すること
を含む前記方法。
（７）びまん性肺胞傷害の診断及び治療方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c1. b1の測定値に基づき、びまん性肺胞傷害の発症の有無を判定すること、及び
d1. びまん性肺胞傷害を発症している可能性が高いと判定された被験者にびまん性肺胞傷害の治療を施すこと
を含む前記方法。
（８）びまん性肺胞傷害の診断及び治療方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c1. b1の測定値に基づき、びまん性肺胞傷害の病勢を判定すること、及び
d1. びまん性肺胞傷害の急性期にある可能性が高いと判定された被験者にびまん性肺胞傷害の治療を施すこと
を含む前記方法。
（９）ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６（CA6）からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することを含む、間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の検査方法。
（１０）発現を測定するタンパク質が、ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質であり、測定値が間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の病勢診断を補助する（９）記載の方法。
（１１）間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c2. b2の測定値に基づき、間質性肺炎の発症の有無を判定すること
を含む前記方法。
（１２）間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c2. b2の測定値に基づき、間質性肺炎の病勢を判定すること
を含む前記方法。
（１３）間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断及び治療方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c2. b2の測定値に基づき、間質性肺炎の発症の有無を判定すること、及び
d2. 間質性肺炎を発症している可能性が高いと判定された被験者に間質性肺炎の治療を施すこと
を含む前記方法。
（１４）間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断及び治療方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c2. b2の測定値に基づき、間質性肺炎の病勢を判定すること、及び
d2. 間質性肺炎の急性期にある可能性が高いと判定された被験者に間質性肺炎の治療を施すこと
を含む前記方法。
（１５）ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することができる試薬を含む、びまん性肺胞傷害の検査のためのキット。
（１６）ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することができる試薬を含む、間質性肺炎（びまん性肺胞傷害及びその他の間質性肺炎）の検査のためのキット。 The present invention has been completed based on these findings.
(1) Samples derived from subjects for at least one protein selected from the group consisting of stratefin (SFN), NPS-PLA2, cystatin F (CYTF), IL-1Ra, netrin-1 (NET1) and troponin T. A method of testing for diffuse alveolar injury, including measuring expression in.
(2) The protein whose expression is to be measured is at least one protein selected from the group consisting of stratephin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T, and the measured value is diffuse. The method according to (1), which assists in the diagnosis of alveolar injury.
(3) The protein whose expression is to be measured is at least one protein selected from the group consisting of stratephine, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T, and the measured value is diffuse. The method according to (1), which assists in the specific diagnosis of alveolar injury.
(4) The method according to (3), wherein the protein whose expression is measured is Stratefin, Cystatin F, NPS-PLA2, IL-1Ra, Netrin-1, Troponin T, or a combination thereof.
(5) A method for diagnosing diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and
c1. The method comprising determining the presence or absence of the onset of diffuse alveolar injury based on the measurements of b1.
(6) A method for diagnosing diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and
c1. The method comprising determining the pathology of diffuse alveolar injury based on measurements in b1.
(7) A method for diagnosing and treating diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
c1. Determine the presence or absence of diffuse alveolar injury based on the measured values of b1 and
d1. The method described above comprising treating a subject determined to have a high probability of developing diffuse alveolar injury with treatment for diffuse alveolar injury.
(8) A method for diagnosing and treating diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
c1. Determining the pathology of diffuse alveolar injury based on b1 measurements, and
d1. The method described above comprising treating a subject determined to be likely to be in the acute phase of diffuse alveolar injury with treatment for diffuse alveolar injury.
(9) Select from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6 (CA6). A method for testing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia), which comprises measuring the expression of at least one protein in a sample derived from a subject.
(10) Proteins whose expression is to be measured are from stratefine, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic amphidrase 6. The method according to (9), wherein the protein is at least one protein selected from the group, and the measured value assists the pathological diagnosis of interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
(11) A method for diagnosing general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic amphidrase 6. To measure the expression of a protein in a sample derived from a subject, and
c2. The method comprising determining the presence or absence of the onset of interstitial pneumonia based on the measurements in b2.
(12) A method for diagnosing general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic amphidrase 6. To measure the expression of a protein in a sample derived from a subject, and
c2. The method comprising determining the pathology of interstitial pneumonia based on the measurements in b2.
(13) A method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic amphidrase 6. To measure the expression of a protein in a sample derived from a subject,
c2. Determine the presence or absence of interstitial pneumonia based on the measured value of b2, and
d2. The method described above comprising treating a subject determined to have interstitial pneumonia with a high probability of developing interstitial pneumonia.
(14) A method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic amphidrase 6. To measure the expression of a protein in a sample derived from a subject,
c2. Judging the pathology of interstitial pneumonia based on the measured values of b2, and
d2. The method comprising treating interstitial pneumonia in a subject determined to be likely to be in the acute phase of interstitial pneumonia.
(15) A reagent capable of measuring the expression of at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T in a sample derived from a subject. A kit for testing for diffuse alveolar injury, including.
(16) At least selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic anhydrase 6. A kit for testing interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia), which comprises a reagent capable of measuring the expression of one protein in a sample derived from a subject.
（１）ストラテフィン（SFN）、NPS-PLA2、シスタチンF（CYTF）、IL-1Ra、ネトリン-1（NET1）及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することを含む、びまん性肺胞傷害の検査方法。
（２）発現を測定するタンパク質が、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質であり、測定値がびまん性肺胞傷害の病勢診断を補助する（１）記載の方法。
（３）発現を測定するタンパク質が、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質であり、測定値がびまん性肺胞傷害の特異的診断を補助する（１）記載の方法。
（４）発現を測定するタンパク質が、ストラテフィン、シスタチンF、NPS-PLA2、IL-1Ra、ネトリン-1、トロポニンT、又はそれらの組み合わせである（３）記載の方法。
（５）びまん性肺胞傷害の診断方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c1. b1の測定値に基づき、びまん性肺胞傷害の発症の有無を判定すること
を含む前記方法。
（６）びまん性肺胞傷害の診断方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c1. b1の測定値に基づき、びまん性肺胞傷害の病勢を判定すること
を含む前記方法。
（７）びまん性肺胞傷害の診断及び治療方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c1. b1の測定値に基づき、びまん性肺胞傷害の発症の有無を判定すること、及び
d1. びまん性肺胞傷害を発症している可能性が高いと判定された被験者にびまん性肺胞傷害の治療を施すこと
を含む前記方法。
（８）びまん性肺胞傷害の診断及び治療方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c1. b1の測定値に基づき、びまん性肺胞傷害の病勢を判定すること、及び
d1. びまん性肺胞傷害の急性期にある可能性が高いと判定された被験者にびまん性肺胞傷害の治療を施すこと
を含む前記方法。
（９）ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６（CA6）からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することを含む、間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の検査方法。
（１０）発現を測定するタンパク質が、ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質であり、測定値が間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の病勢診断を補助する（９）記載の方法。
（１１）間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c2. b2の測定値に基づき、間質性肺炎の発症の有無を判定すること
を含む前記方法。
（１２）間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c2. b2の測定値に基づき、間質性肺炎の病勢を判定すること
を含む前記方法。
（１３）間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断及び治療方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c2. b2の測定値に基づき、間質性肺炎の発症の有無を判定すること、及び
d2. 間質性肺炎を発症している可能性が高いと判定された被験者に間質性肺炎の治療を施すこと
を含む前記方法。
（１４）間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断及び治療方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c2. b2の測定値に基づき、間質性肺炎の病勢を判定すること、及び
d2. 間質性肺炎の急性期にある可能性が高いと判定された被験者に間質性肺炎の治療を施すこと
を含む前記方法。
（１５）ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することができる試薬を含む、びまん性肺胞傷害の検査のためのキット。
（１６）ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することができる試薬を含む、間質性肺炎（びまん性肺胞傷害及びその他の間質性肺炎）の検査のためのキット。 The present invention has been completed based on these findings.
(1) Samples derived from subjects for at least one protein selected from the group consisting of stratefin (SFN), NPS-PLA2, cystatin F (CYTF), IL-1Ra, netrin-1 (NET1) and troponin T. A method of testing for diffuse alveolar injury, including measuring expression in.
(2) The protein whose expression is to be measured is at least one protein selected from the group consisting of stratephin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T, and the measured value is diffuse. The method according to (1), which assists in the diagnosis of alveolar injury.
(3) The protein whose expression is to be measured is at least one protein selected from the group consisting of stratephine, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T, and the measured value is diffuse. The method according to (1), which assists in the specific diagnosis of alveolar injury.
(4) The method according to (3), wherein the protein whose expression is measured is Stratefin, Cystatin F, NPS-PLA2, IL-1Ra, Netrin-1, Troponin T, or a combination thereof.
(5) A method for diagnosing diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and
c1. The method comprising determining the presence or absence of the onset of diffuse alveolar injury based on the measurements of b1.
(6) A method for diagnosing diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and
c1. The method comprising determining the pathology of diffuse alveolar injury based on measurements in b1.
(7) A method for diagnosing and treating diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
c1. Determine the presence or absence of diffuse alveolar injury based on the measured values of b1 and
d1. The method described above comprising treating a subject determined to have a high probability of developing diffuse alveolar injury with treatment for diffuse alveolar injury.
(8) A method for diagnosing and treating diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
c1. Determining the pathology of diffuse alveolar injury based on b1 measurements, and
d1. The method described above comprising treating a subject determined to be likely to be in the acute phase of diffuse alveolar injury with treatment for diffuse alveolar injury.
(9) Select from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6 (CA6). A method for testing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia), which comprises measuring the expression of at least one protein in a sample derived from a subject.
(10) Proteins whose expression is to be measured are from stratefine, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
(11) A method for diagnosing general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
c2. The method comprising determining the presence or absence of the onset of interstitial pneumonia based on the measurements in b2.
(12) A method for diagnosing general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
c2. The method comprising determining the pathology of interstitial pneumonia based on the measurements in b2.
(13) A method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
c2. Determine the presence or absence of interstitial pneumonia based on the measured value of b2, and
d2. The method described above comprising treating a subject determined to have interstitial pneumonia with a high probability of developing interstitial pneumonia.
(14) A method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
c2. Judging the pathology of interstitial pneumonia based on the measured values of b2, and
d2. The method comprising treating interstitial pneumonia in a subject determined to be likely to be in the acute phase of interstitial pneumonia.
(15) A reagent capable of measuring the expression of at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T in a sample derived from a subject. A kit for testing for diffuse alveolar injury, including.
(16) At least selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
本発明により見いだされたタンパク質の血液中濃度を、単独、あるいは複数項目を測定することにより、間質性肺炎疑いの患者がびまん性肺胞傷害なのか否か、また患者の病勢や間質性肺炎の活動性について、高い確度で診断することができる。
本明細書は、本願の優先権の基礎である日本国特許出願、特願2019‐142706の明細書および／または図面に記載される内容を包含する。 By measuring the blood concentration of the protein found by the present invention, alone or in multiple items, whether or not the patient suspected of having interstitial pneumonia has diffuse alveolar injury, and the patient's pathology and interstitial nature. The activity of pneumonia can be diagnosed with high accuracy.
This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2019-142706 and / or drawings which are the basis of the priority of the present application.
本明細書は、本願の優先権の基礎である日本国特許出願、特願2019‐142706の明細書および／または図面に記載される内容を包含する。 By measuring the blood concentration of the protein found by the present invention, alone or in multiple items, whether or not the patient suspected of having interstitial pneumonia has diffuse alveolar injury, and the patient's pathology and interstitial nature. The activity of pneumonia can be diagnosed with high accuracy.
This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2019-142706 and / or drawings which are the basis of the priority of the present application.
以下、本発明の実施の形態を詳細に説明する。
１．びまん性肺胞傷害の検査方法、診断方法、治療方法及び検査キット
本発明は、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することを含む、びまん性肺胞傷害の検査方法を提供する。 Hereinafter, embodiments of the present invention will be described in detail.
1. 1. Test method, diagnostic method, treatment method and test kit for diffuse alveolar injury The present invention is at least one selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T. Provided is a method for testing diffuse alveolar injury, which comprises measuring the expression of a protein in a sample derived from a subject.
１．びまん性肺胞傷害の検査方法、診断方法、治療方法及び検査キット
本発明は、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することを含む、びまん性肺胞傷害の検査方法を提供する。 Hereinafter, embodiments of the present invention will be described in detail.
1. 1. Test method, diagnostic method, treatment method and test kit for diffuse alveolar injury The present invention is at least one selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T. Provided is a method for testing diffuse alveolar injury, which comprises measuring the expression of a protein in a sample derived from a subject.
びまん性肺胞傷害は、急性で高度の肺胞傷害により出現する肺病変で、CTでは広範な浸潤影やスリガラス様陰影に、牽引性気管支拡張や蜂巣肺などの構造改変所見が認められるのが特徴で、びまん性肺胞傷害を呈する病態としては、急性呼吸窮迫症候群（ARDS）や急性間質性肺炎（AIP）、特発性肺線維症（IPF）をはじめとする慢性間質性肺炎の急性増悪などが挙げられる。本発明の方法において、びまん性肺胞傷害は、薬剤性であっても、特発性であってもよい。
Diffuse alveolar injury is a lung lesion that develops due to acute and severe alveolar injury, and CT shows extensive infiltrative and suriglass-like shadows with structural alterations such as traction bronchiectal dilatation and honeycombing. Characteristically, the pathological conditions that present with diffuse alveolar injury include acute respiratory distress syndrome (ARDS), acute interstitial pneumonia (AIP), and acute chronic interstitial pneumonia (IPF). Exacerbations can be mentioned. In the method of the invention, the diffuse alveolar injury may be drug-induced or idiopathic.
ストラテフィンは、別名14-3-3シグマと呼ばれ、SFN遺伝子にコードされる248アミノ酸のタンパク質である。食道や皮膚の上皮に発現し、頭頸部がん、肺がん、子宮頸がんなどで発現することが知られている。UniProt番号P31947。
Stratefin, also known as 14-3-3 sigma, is a 248 amino acid protein encoded by the SFN gene. It is known to be expressed in the epithelium of the esophagus and skin, and in head and neck cancer, lung cancer, cervical cancer and the like. UniProt number P31947.
シスタチンFは、遺伝子CST7にコードされている145アミノ酸の細胞外分泌タンパク質である。骨髄や免疫系の細胞のほか、様々ながん細胞からも発現が検出される。UniProt番号O76096。
Cystatin F is an extracellular protein of 145 amino acids encoded by the gene CST7. Expression is detected in various cancer cells as well as cells in the bone marrow and immune system. UniProt number O76096.
NPS-PLA2は、正式名はホスホリパーゼA2グループIIAであり、PLA2G2A遺伝子にコードされる144アミノ酸の細胞外分泌型の酵素である。活性発現にはカルシウムを必要とし、ホスホグリセリドのsn-2脂肪酸アシルエステル結合の加水分解を触媒し、遊離脂肪酸とリゾリン脂質の生成に関与する。消化管で強く発現する。UniProt番号P14555。
NPS-PLA2, whose official name is Phospholipase A2 Group IIA, is an extracellularly secreted enzyme of 144 amino acids encoded by the PLA2G2A gene. It requires calcium for activity expression, catalyzes the hydrolysis of the phosphoglyceride sn-2 fatty acid acyl ester bond, and is involved in the production of free fatty acids and lysophospholipids. Strongly expressed in the gastrointestinal tract. UniProt number P14555.
IL-1Raは、正式名はインターロイキン－１レセプターアンタゴニストであり、IL1RN遺伝子にコードされる159アミノ酸の細胞外分泌型として知られるタンパク質である。受容体IL-1R1に結合し、インターロイキン１アルファや１ベータの活性化を阻害する。骨髄や免疫系の細胞、消化管の細胞で高い発現が認められる。UniProt番号P18510。
IL-1Ra, whose official name is an interleukin-1 receptor antagonist, is a protein known as an extracellular secretory type of 159 amino acids encoded by the IL1RN gene. It binds to the receptor IL-1R1 and inhibits the activation of interleukin 1 alpha and 1 beta. High expression is observed in cells of the bone marrow, immune system, and digestive tract. UniProt number P18510.
ネトリン-1は、NTN1遺伝子にコードされる604アミノ酸のタンパク質であり、ラミニン関連分泌タンパク質ファミリーに含まれる。UniProt番号O95631。乳がん、腎がん、前立腺がんなどで発現上昇することが知られている。
Netrin-1 is a 604 amino acid protein encoded by the NTN1 gene and is included in the laminin-related secretory protein family. UniProt number O95631. It is known that the expression is increased in breast cancer, kidney cancer, prostate cancer and the like.
トロポニンTは、TNNT2遺伝子にコードされるタンパク質である。発現は心筋特異的であり、心筋の収縮に関与する。UniProt番号P45379。
Troponin T is a protein encoded by the TNNT2 gene. Expression is myocardial specific and involved in myocardial contraction. UniProt number P45379.
本発明において、被験者は、びまん性肺胞傷害の発症が疑われる哺乳動物であるが、発症の危険性が考えられるすべての哺乳動物を対象としてもよい。典型的にはヒトである。被験者由来の試料としては、被験者から得た細胞、組織、体液など、具体的には、被験者の血液（例えば、全血、血清、血漿、血漿交換外液など）や気管支肺胞洗浄液などを例示することができる。通常の血液検査（臨床検査）で得られる全血、血清あるいは血漿を血液サンプルとして使用するとよい。
In the present invention, the subject is a mammal suspected of developing diffuse alveolar injury, but all mammals at risk of developing may be targeted. It is typically human. Examples of the sample derived from the subject include cells, tissues, and body fluids obtained from the subject, specifically, the subject's blood (for example, whole blood, serum, plasma, plasma exchange external fluid, etc.), bronchoalveolar lavage fluid, and the like. can do. Whole blood, serum or plasma obtained by a normal blood test (clinical test) may be used as a blood sample.
本発明の方法において、被験者由来の試料における発現の測定は、試料中の上記タンパク質またはその断片の存在量を測定すればよい。測定する手段としては、特に限定されることなく公知の方法を用いても良い。タンパク質レベルで測定するのが好ましいが、核酸レベルで測定してもよい。
In the method of the present invention, the expression in a sample derived from a subject may be measured by measuring the abundance of the above protein or a fragment thereof in the sample. As the means for measuring, a known method may be used without particular limitation. It is preferably measured at the protein level, but may be measured at the nucleic acid level.
上記タンパク質の発現をタンパク質レベルで測定するためには、上記タンパク質を特異的に認識する抗体を用いるとよい。抗体は、モノクローナル抗体、ポリクローナル抗体のいずれであってもよい。これらの抗体は公知の方法で製造することができるし、また市販のものを使用してもよい。典型的な方法としては、ELISA法やイムノクロマトグラフィー法などの免疫測定法が挙げられる。免疫測定法は、特殊な装置や技術を必要とせず、簡便迅速に標的タンパク質を検出、定量することが可能であるため、本発明においても上記タンパク質の測定に好ましく用いることができる。上記タンパク質の抗体は公知であり、市販品も存在する。また、上述したように、上記タンパク質のアミノ酸配列およびこれらをコードする塩基配列も公知であるので、一般的なハイブリドーマを作成することにより、各タンパク質に対する特異抗体を作製してもよい。
In order to measure the expression of the protein at the protein level, it is advisable to use an antibody that specifically recognizes the protein. The antibody may be either a monoclonal antibody or a polyclonal antibody. These antibodies can be produced by known methods, or commercially available ones may be used. Typical methods include immunoassays such as ELISA and immunochromatography. Since the immunoassay method does not require a special device or technique and can easily and quickly detect and quantify the target protein, it can be preferably used for the measurement of the above protein in the present invention as well. Antibodies for the above proteins are known, and commercially available products also exist. Further, as described above, since the amino acid sequence of the above protein and the base sequence encoding these are also known, a specific antibody against each protein may be prepared by preparing a general hybridoma.
免疫測定法自体はこの分野で周知されている。反応形式により、サンドイッチ法、競合法、凝集法、ウェスタンブロット法等があり、また、標識に基づいて、酵素免疫分析、放射免疫分析、蛍光免疫分析、発光免疫分析等がある。本発明においては、定量的検出が可能な免疫測定法のいずれを用いてもよい。特に限定されないが、例えば、サンドイッチELISA等のサンドイッチ法を好ましく用いることができる。サンドイッチ法では、標的タンパク質に結合する抗体を固相化し、試料と反応させる。固相化抗体に結合した標的タンパク質は、酵素などで標識した検出抗体を用いて測定する。検出抗体は、固相化抗体とは異なる部位で標的タンパク質に結合する抗体を用いるのが好ましい。固相化抗体と検出抗体はポリクローナル抗体でもモノクローナル抗体でもよく、該抗体の抗原結合性の断片を用いることもできる。固相化抗体と結合した標的タンパク質を検出抗体と反応させ、洗浄を行った後、結合した検出抗体の量を、抗体に標識した物質からのシグナルで測定する。例えば、アルカリホスファターゼで標識した抗体を検出抗体として用いた場合、該酵素の基質を反応系内に添加し、酵素反応により生じる発色や蛍光、発光の量を対応する装置で測定すればよい。標的タンパク質を含む濃度既知の標準試料について免疫測定を行ない、標識物質のシグナルと濃度との関係をプロットした検量線を作成しておき、標的タンパク質が濃度未知の試料についても、同じ操作を行ない、得られるシグナル測定値を当該検量線に当てはめることにより、試料中の標的タンパク質を定量することができる。
The immunoassay itself is well known in this field. Depending on the reaction format, there are a sandwich method, a competitive method, an agglutination method, a Western blotting method and the like, and based on the label, there are enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, luminescence immunoassay and the like. In the present invention, any immunoassay method capable of quantitative detection may be used. Although not particularly limited, for example, a sandwich method such as sandwich ELISA can be preferably used. In the sandwich method, the antibody that binds to the target protein is immobilized and reacted with the sample. The target protein bound to the immobilized antibody is measured using a detection antibody labeled with an enzyme or the like. As the detection antibody, it is preferable to use an antibody that binds to the target protein at a site different from that of the immobilized antibody. The immobilized antibody and the detection antibody may be a polyclonal antibody or a monoclonal antibody, and an antigen-binding fragment of the antibody may be used. The target protein bound to the immobilized antibody is reacted with the detection antibody, washed, and then the amount of the bound detection antibody is measured by a signal from the substance labeled on the antibody. For example, when an antibody labeled with alkaline phosphatase is used as a detection antibody, the substrate of the enzyme may be added into the reaction system, and the amount of color development, fluorescence, and luminescence generated by the enzyme reaction may be measured with a corresponding device. Immunity measurement was performed on a standard sample containing a target protein with a known concentration, a calibration curve was created plotting the relationship between the signal of the labeling substance and the concentration, and the same operation was performed on a sample with an unknown target protein concentration. By applying the obtained signal measurement value to the calibration curve, the target protein in the sample can be quantified.
上記タンパク質の発現を核酸レベルで測定するためには、上記タンパク質のmRNAと特異的にハイブリダイズできる核酸プローブを用いるとよい（ノーザンブロット法で測定する場合）。あるいはまた、上記タンパク質のmRNAを鋳型として合成されるcDNAを特異的に増幅できる少なくとも１対の核酸プライマーを用いてもよい（RT-PCR法で測定する場合）。核酸プローブ及び核酸プライマーは、上記タンパク質の遺伝子情報（上述）に基づいて設計することができる。核酸プローブは、通常、約１５～１５００塩基のものが適当である。核酸プローブは、放射性元素、蛍光色素、酵素などで標識するとよい。核酸プライマーは、通常、約１５～３０塩基のものが適当である。核酸プライマーを放射性元素、蛍光色素、酵素などで標識してもよい。
In order to measure the expression of the above protein at the nucleic acid level, it is preferable to use a nucleic acid probe that can specifically hybridize with the mRNA of the above protein (when measuring by Northern blotting). Alternatively, at least one pair of nucleic acid primers capable of specifically amplifying the cDNA synthesized using the mRNA of the above protein as a template may be used (when measured by the RT-PCR method). Nucleic acid probes and nucleic acid primers can be designed based on the genetic information (described above) of the above proteins. As the nucleic acid probe, one having about 15 to 1500 bases is usually suitable. The nucleic acid probe may be labeled with a radioactive element, a fluorescent dye, an enzyme or the like. As the nucleic acid primer, one having about 15 to 30 bases is usually suitable. Nucleic acid primers may be labeled with radioactive elements, fluorescent dyes, enzymes and the like.
発現を測定するタンパク質又は遺伝子は１種類でもよいし、複数種類であってもよい。複数の遺伝子発現や複数のタンパク発現データを参照することにより、より正確な評価が可能となりうる。複数の遺伝子発現や複数のタンパク発現を同時に検出するためには、ＤＮＡアレイ（プローブを基板に固定）（NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, DECEMBER 2002, 951-960）、プロテインチップ（抗体を基板に固定）（NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, SEPTEMBER 2002, 683-695）、ルミネックス（NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, JUNE 2002, 447-456）、等の検出法を用いてもよい。
The expression of the protein or gene may be one type or multiple types. More accurate evaluation may be possible by referring to a plurality of gene expression data and a plurality of protein expression data. In order to detect multiple gene expression and multiple protein expression at the same time, DNA array (probe fixed to substrate) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, DECEMBER 2002, 951-960), protein chip (antibody substrate) (Fixed to) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, SEPTEMBER 2002, 683-695), Luminex (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, JUNE 2002, 447-456), etc. may be used.
ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定し、その発現レベルが高い場合に、びまん性肺胞傷害を発症している可能性が高いと判定し、前記レベルが低い場合に、びまん性肺胞傷害を発症している可能性が低いと判定することができる。
The expression of at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T is measured in a sample derived from the subject, and the expression level is high. In addition, it can be determined that there is a high possibility of developing diffuse alveolar injury, and when the level is low, it can be determined that the possibility of developing diffuse alveolar injury is low.
よって、本発明の方法は、びまん性肺胞傷害の診断（びまん性肺胞傷害の発症の有無の判定）を補助することができる。本発明は、びまん性肺胞傷害の診断方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c1. b1の測定値に基づき、びまん性肺胞傷害の発症の有無を判定すること
を含む前記方法を提供する。 Therefore, the method of the present invention can assist in the diagnosis of diffuse alveolar injury (determination of the presence or absence of the onset of diffuse alveolar injury). The present invention is a method for diagnosing diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and
c1. Provided above, the method comprising determining the presence or absence of the onset of diffuse alveolar injury based on the measurements of b1.
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c1. b1の測定値に基づき、びまん性肺胞傷害の発症の有無を判定すること
を含む前記方法を提供する。 Therefore, the method of the present invention can assist in the diagnosis of diffuse alveolar injury (determination of the presence or absence of the onset of diffuse alveolar injury). The present invention is a method for diagnosing diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and
c1. Provided above, the method comprising determining the presence or absence of the onset of diffuse alveolar injury based on the measurements of b1.
本発明の一つの例として、びまん性肺胞傷害の診断は、以下のような基準で行うことができる。被験者から採取した血漿または血清（血液試料）における上記タンパク質の少なくとも１個の発現を測定し、健常人から採取した血液試料におけるそれと比較した結果、予め設定されたカットオフ値や基準値よりも高い値が得られた場合、被験者はびまん性肺胞傷害を発症していると評価する。あるいは、医薬品投与中の患者において、上記タンパク質の少なくとも１個の血液試料中濃度がカットオフ値よりも高くなった場合、当該医薬品によるびまん性肺胞傷害の発症を疑うことができる。この予め設定するカットオフ値は、当業者が適宜設定することができる。例えば、びまん性肺胞傷害を発症していない健常者の定量値の95％信頼区間を基準値としたり、ROC曲線からカットオフ値を設定したりすることができる。一実施態様として、後述の実施例のROC曲線解析から、以下のカットオフ値を設定することができる。例えば健常人との鑑別のためのカットオフ値としては、ストラテフィン：0.2～5.0 ng/mL（好ましくは、0.7 ng/mL）、NPS-PLA2：4～14 ng/mL（好ましくは、7 ng/mL）が挙げられ、医薬品投与患者におけるびまん性肺胞傷害の発生を検出するためのカットオフ値は、間質性肺炎を発症しなかった非発症例との比較解析の結果より、ストラテフィン：0.7～7.2 ng/mL（好ましくは、2.4 ng/mL）、NPS-PLA2：5～60 ng/mL（好ましくは、32 ng/mL）などと設定できる。あるいは、過去の測定値と比較して、上記タンパク質の少なくとも一つが上昇の傾向を辿った場合、びまん性肺胞傷害の発症の可能性を疑う。
As an example of the present invention, the diagnosis of diffuse alveolar injury can be made based on the following criteria. As a result of measuring the expression of at least one of the above proteins in plasma or serum (blood sample) collected from a subject and comparing it with that in a blood sample collected from a healthy person, it is higher than a preset cutoff value or reference value. If a value is obtained, the subject is assessed as developing diffuse alveolar injury. Alternatively, in a patient receiving a drug, if the concentration of at least one of the proteins in a blood sample becomes higher than the cutoff value, the onset of diffuse alveolar injury due to the drug can be suspected. This preset cutoff value can be appropriately set by those skilled in the art. For example, the 95% confidence interval of the quantitative value of a healthy person who has not developed diffuse alveolar injury can be used as a reference value, or the cutoff value can be set from the ROC curve. As one embodiment, the following cutoff values can be set from the ROC curve analysis of Examples described later. For example, the cutoff values for differentiating from healthy subjects are Stratefin: 0.2 to 5.0 ng / mL (preferably 0.7 ng / mL) and NPS-PLA2: 4 to 14 ng / mL (preferably 7 ng). / mL), and the cutoff value for detecting the occurrence of diffuse alveolar injury in drug-administered patients was based on the results of comparative analysis with non-symptomatic patients who did not develop interstitial pneumonia. : 0.7 to 7.2 ng / mL (preferably 2.4 ng / mL), NPS-PLA2: 5 to 60 ng / mL (preferably 32 ng / mL), etc. can be set. Alternatively, if at least one of the above proteins follows an upward trend compared to past measurements, the possibility of developing diffuse alveolar injury is suspected.
本発明の方法は、びまん性肺胞傷害の特異的診断にも利用できる。本明細書において、「特異的診断」とは、被験者が、びまん性肺胞傷害パターンの間質性肺炎なのか否かを判定することを指す。びまん性肺胞傷害は、間質性肺炎の病型の中でも特に重篤な病型であるので、びまん性肺胞傷害の特異的診断に利用できることは、臨床上の意義が大きい。ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現レベルが高い場合に、病型がびまん性肺胞傷害である可能性が高いと判定することができる。例えば、びまん性肺胞傷害以外の間質性肺炎患者の定量値の95％信頼区間を基準値としたり、ROC曲線からカットオフ値を設定したりすることができる。一実施態様として、後述の実施例のROC曲線解析から、以下のカットオフ値を設定することができる。例えばびまん性肺胞傷害とそれ以外の間質性肺炎の患者の測定値を比較することで、びまん性肺胞傷害の特異的診断のカットオフ値として、ストラテフィン： 1.0～5.3 ng/mL（好ましくは、2.4 ng/mL）、NPS-PLA2：10～87 ng/mL（好ましくは、32 ng/mL）。
The method of the present invention can also be used for specific diagnosis of diffuse alveolar injury. As used herein, "specific diagnosis" refers to determining whether a subject has interstitial pneumonia with a diffuse alveolar injury pattern. Since diffuse alveolar injury is a particularly serious form of interstitial pneumonia, it is of great clinical significance that it can be used for specific diagnosis of diffuse alveolar injury. At least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T is diffusely affected by high expression levels in subject-derived samples. It can be determined that there is a high possibility of sexual alveolar injury. For example, the 95% confidence interval of the quantitative value of patients with interstitial pneumonia other than diffuse alveolar injury can be used as the reference value, or the cutoff value can be set from the ROC curve. As one embodiment, the following cutoff values can be set from the ROC curve analysis of Examples described later. For example, by comparing the measured values of patients with diffuse alveolar injury and other patients with interstitial pneumonia, as a cut-off value for specific diagnosis of diffuse alveolar injury, Stratefin: 1.0 to 5.3 ng / mL ( Preferably 2.4 ng / mL), NPS-PLA2: 10-87 ng / mL (preferably 32 ng / mL).
本発明の方法をびまん性肺胞傷害の特異的診断に利用する場合には、発現を測定するタンパク質が、ストラテフィン、シスタチンF、NPS-PLA2、IL-1Ra、ネトリン-1、トロポニンT、又はそれらの組み合わせであるとよい。組み合わせとしては、シスタチンFとストラテフィンの組み合わせ、シスタチンFとネトリン-1の組み合わせ、ストラテフィンとネトリン-1の組み合わせ又はネトリン-1とIL-1Raの組み合わせを例示することができる。
When the method of the present invention is used for the specific diagnosis of diffuse alveolar injury, the proteins whose expression is measured are stratefine, cystatin F, NPS-PLA2, IL-1Ra, netrin-1, troponin T, or It is good to have a combination of them. Examples of the combination include a combination of cystatin F and stratefin, a combination of cystatin F and netrin-1, a combination of stratefin and netrin-1, or a combination of netrin-1 and IL-1Ra.
また、本発明の方法は、びまん性肺胞傷害の病勢診断にも利用できる。本明細書において、「病勢診断」とは、びまん性肺胞傷害と診断された被験者の、病態の程度や変化（重症度、治療効果等）の判断を意味する。ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現レベルが高い場合に、びまん性肺胞傷害の急性期にある可能性が高いと判定することができる。例えば、びまん性肺胞傷害の回復期にある患者の定量値の95％信頼区間を基準値としたり、ROC曲線からカットオフ値を設定したりすることができる。一実施態様として、後述の実施例のROC曲線解析から、例えば以下のカットオフ値を設定することができる。ストラテフィン：0.8～5.0 ng/mL（好ましくは、2.3 ng/mL）、NPS-PLA2：8～32 ng/mL（好ましくは、16 ng/mL）。
The method of the present invention can also be used for pathological diagnosis of diffuse alveolar injury. As used herein, the term "disease diagnosis" means the determination of the degree and change (severity, therapeutic effect, etc.) of the pathological condition of a subject diagnosed with diffuse alveolar injury. Diffuse alveolar at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T at high expression levels in subject-derived samples. It can be determined that there is a high probability of being in the acute phase of the injury. For example, the 95% confidence interval of the quantitative value of a patient in the recovery phase of diffuse alveolar injury can be used as a reference value, or the cutoff value can be set from the ROC curve. As one embodiment, for example, the following cutoff value can be set from the ROC curve analysis of the examples described later. Stratefin: 0.8-5.0 ng / mL (preferably 2.3 ng / mL), NPS-PLA2: 8-32 ng / mL (preferably 16 ng / mL).
本発明は、びまん性肺胞傷害の診断方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c1. b1の測定値に基づき、びまん性肺胞傷害の病勢を判定すること
を含む前記方法を提供する。 The present invention is a method for diagnosing diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and
c1. Provided above are methods that include determining the pathology of diffuse alveolar injury based on measurements in b1.
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c1. b1の測定値に基づき、びまん性肺胞傷害の病勢を判定すること
を含む前記方法を提供する。 The present invention is a method for diagnosing diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and
c1. Provided above are methods that include determining the pathology of diffuse alveolar injury based on measurements in b1.
さらに、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、びまん性肺胞傷害の急性期にある可能性が高いと判定された被験者由来の試料における発現を１回又は異なる時期に複数回測定し、発現レベルがカットオフ値もしくは基準値に近いレベルにまで低下した場合に、治療によりびまん性肺胞傷害から回復したと判定し、前記レベルが高いあるいは低下しない場合に、治療によりびまん性肺胞傷害から回復していない、あるいは回復が不十分であると判定することができる。本発明の方法は、びまん性肺胞傷害の病勢診断の他、予後の検査、治療効果の確認にも利用できる。
In addition, at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T is likely to be in the acute phase of diffuse alveolar injury. The expression in the sample derived from the subject determined to be was measured once or multiple times at different times, and when the expression level decreased to the cutoff value or the level close to the reference value, the treatment recovered from the diffuse alveolar injury. If the level is high or does not decrease, it can be determined that the treatment has not recovered from the diffuse alveolar injury or the recovery is insufficient. The method of the present invention can be used not only for pathological diagnosis of diffuse alveolar injury, but also for prognosis examination and confirmation of therapeutic effect.
被験者がびまん性肺胞傷害を発症している可能性が高いと判断された場合には、薬剤性が疑われる場合は、被疑薬はすみやかに中止する。次いで、喀痰や血清の感染症検査、画像検査、気管支肺胞洗浄検査や病理検査などの各種検査を組み合わせて行い、びまん性肺胞傷害の診断を確定させる。びまん性肺胞傷害の診断が下された時は、すみやかに副腎皮質ステロイドの投与を開始する。日本呼吸器学会の治療指針では、メチルプレドニゾロン500～1000 mg/日を3日間投与するパルス療法を行い、プレドニゾロン換算で0.5～1.0 mg/kg/日で継続し、漸減することが推奨されている。漸減の速度に一定の基準はなく、治療反応性をみながら減らしていく。ステロイド治療に抵抗性、あるいは難治性の肺障害では、免疫抑制薬（シクロスポリン、タクロリムスなど）や好中球エラスターゼ阻害薬（シベレスタット）の投与、ポリミキシンB固定化線維カラム（PMX）療法などを組み合わせた集学的治療を行う。これらの治療は薬剤性肺障害に対するエビデンスに乏しく保険適用もないため、実際にはIPFの急性増悪や急性呼吸窮迫症候群に準じて用いられる。呼吸不全には対しては、高流量酸素投与、非侵襲的陽圧換気療法、あるいは気管内挿管下での人工呼吸管理など、重症度に応じた対処を行う。
If it is judged that the subject is likely to have diffuse alveolar injury, and if drug-induced is suspected, the suspected drug should be discontinued immediately. Next, various tests such as sputum and serum infectious disease tests, imaging tests, bronchoalveolar lavage tests, and pathological tests are performed in combination to confirm the diagnosis of diffuse alveolar injury. When a diagnosis of diffuse alveolar injury is made, administration of corticosteroids should be started immediately. According to the treatment guidelines of the Japanese Respiratory Society, it is recommended to perform pulse therapy in which methylprednisolone 500 to 1000 mg / day is administered for 3 days, continue at 0.5 to 1.0 mg / kg / day in terms of prednisolone, and gradually decrease. .. There is no fixed standard for the rate of tapering, and it is reduced while observing treatment responsiveness. For pulmonary disorders that are refractory or refractory to steroid treatment, a combination of immunosuppressive drugs (cyclosporine, tacrolimus, etc.), neutrophil elastase inhibitor (sivelestat), polymyxin B-immobilized fiber column (PMX) therapy, etc. Perform multidisciplinary treatment. Since these treatments have little evidence for drug-induced lung injury and are not covered by insurance, they are actually used according to the acute exacerbation of IPF and acute respiratory distress syndrome. Respiratory failure should be treated according to severity, such as high flow oxygen administration, non-invasive positive pressure ventilation therapy, or mechanical ventilation management under endotracheal intubation.
米国においても、IPFの急性増悪例（IPF患者の5-10%）と診断した場合は、コルチコステロイド（プレドニゾロン等）の投与が推奨されているが、投与量、投与経路および期間は、各医療施設の方針に委ねられる（AMERICAN JOURNAL OF RESPIRATORY AND CRINICAL CARE MEDICINE, MARCH 2011. 15;183(6):788-824）。
In the United States as well, if a patient is diagnosed with an acute exacerbation of IPF (5-10% of IPF patients), administration of corticosteroids (prednisolone, etc.) is recommended, but the dose, route of administration, and duration are different. It is left to the policy of the medical facility (AMERICAN JOURNAL OF RESPIRATORY AND CRINICAL CARE MEDICINE, MARCH 2011. 15; 183 (6): 788-824).
本発明は、びまん性肺胞傷害の診断及び治療方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c1. b1の測定値に基づき、びまん性肺胞傷害の発症の有無を判定すること、及び
d1. びまん性肺胞傷害を発症している可能性が高いと判定された被験者にびまん性肺胞傷害の治療を施すこと
を含む前記方法を提供する。 The present invention is a method for diagnosing and treating diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
c1. Determine the presence or absence of diffuse alveolar injury based on the measured values of b1 and
d1. Provided by the above method, which comprises treating a subject who is determined to have a high probability of developing a diffuse alveolar injury with a treatment for the diffuse alveolar injury.
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c1. b1の測定値に基づき、びまん性肺胞傷害の発症の有無を判定すること、及び
d1. びまん性肺胞傷害を発症している可能性が高いと判定された被験者にびまん性肺胞傷害の治療を施すこと
を含む前記方法を提供する。 The present invention is a method for diagnosing and treating diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
c1. Determine the presence or absence of diffuse alveolar injury based on the measured values of b1 and
d1. Provided by the above method, which comprises treating a subject who is determined to have a high probability of developing a diffuse alveolar injury with a treatment for the diffuse alveolar injury.
また、本発明は、びまん性肺胞傷害の診断及び治療方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c1. b1の測定値に基づき、びまん性肺胞傷害の病勢を判定すること、及び
d1. びまん性肺胞傷害の急性期にある可能性が高いと判定された被験者にびまん性肺胞傷害の治療を施すこと
を含む前記方法を提供する。 The present invention is a method for diagnosing and treating diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
c1. Determining the pathology of diffuse alveolar injury based on b1 measurements, and
d1. Provided by the above method, which comprises treating a subject who is determined to be likely to be in the acute phase of diffuse alveolar injury to be treated for diffuse alveolar injury.
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c1. b1の測定値に基づき、びまん性肺胞傷害の病勢を判定すること、及び
d1. びまん性肺胞傷害の急性期にある可能性が高いと判定された被験者にびまん性肺胞傷害の治療を施すこと
を含む前記方法を提供する。 The present invention is a method for diagnosing and treating diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
c1. Determining the pathology of diffuse alveolar injury based on b1 measurements, and
d1. Provided by the above method, which comprises treating a subject who is determined to be likely to be in the acute phase of diffuse alveolar injury to be treated for diffuse alveolar injury.
本発明は、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することができる試薬を含む、びまん性肺胞傷害の検査のためのキットも提供する。
The present invention can measure the expression of at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T in a sample derived from a subject. Kits for testing for diffuse alveolar injury, including reagents, are also provided.
一つの例として、本発明のキットは、上記タンパク質を特異的に認識できる抗体を試薬として含む。抗体はマイクロタイタープレートや磁気ビーズ、セルロース膜や基板に固定されていてもよい。キットには、さらに、被験者由来の試料を採取するための器具、抗凝固剤、上記タンパク質を検出するための試薬一式、取扱説明書などが含まれてもよい。取扱説明書には、キットの使用方法の他、びまん性肺胞傷害、あるいは急性呼吸窮迫症候群や特発性間質性肺炎の急性増悪症例の評価及び／又は鑑別基準なども記載しておくとよい。
As an example, the kit of the present invention contains an antibody capable of specifically recognizing the above protein as a reagent. The antibody may be immobilized on a microtiter plate, magnetic beads, a cellulose membrane or a substrate. The kit may further include an instrument for collecting a sample derived from a subject, an anticoagulant, a set of reagents for detecting the protein, an instruction manual, and the like. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or differentiation criteria for cases of diffuse alveolar injury or acute exacerbation of acute respiratory distress syndrome or idiopathic interstitial pneumonia. ..
別の一例として、本発明のキットは、上記タンパク質のmRNAと特異的にハイブリダイズできる核酸プローブを試薬として含む。核酸プローブは基板に固定されていてもよい。キットには、さらに、生体試料を採取するための器具、抗凝固剤、被験者由来の試料からRNAを抽出するための試薬類、RNAを検出するための試薬類、取扱説明書などが含まれてもよい。取扱説明書には、キットの使用方法の他、びまん性肺胞傷害、あるいは急性呼吸窮迫症候群や特発性間質性肺炎の急性増悪症例の評価及び／又は鑑別基準なども記載しておくとよい。
As another example, the kit of the present invention contains a nucleic acid probe that can specifically hybridize with the mRNA of the above protein as a reagent. The nucleic acid probe may be fixed to the substrate. The kit also includes equipment for collecting biological samples, anticoagulants, reagents for extracting RNA from samples derived from subjects, reagents for detecting RNA, instruction manuals, etc. May be good. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or differentiation criteria for cases of diffuse alveolar injury or acute exacerbation of acute respiratory distress syndrome or idiopathic interstitial pneumonia. ..
さらに別の一例として、本発明のキットは上記タンパク質のmRNAを鋳型として合成されるcDNAを特異的に増幅できる少なくとも１対の核酸プライマーを試薬として含む。キットには、さらに、被験者由来の試料を採取するための器具、抗凝固剤、被験者由来の試料からRNAを抽出するための試薬類、RNAを検出するための試薬類、取扱説明書などが含まれるとよい。取扱説明書には、キットの使用方法の他、びまん性肺胞傷害、急性呼吸窮迫症候群や特発性間質性肺炎の急性増悪症例の評価及び／又は鑑別基準なども記載しておくとよい。
As yet another example, the kit of the present invention contains at least one pair of nucleic acid primers capable of specifically amplifying cDNA synthesized using the above protein mRNA as a reagent. The kit also includes equipment for collecting subject-derived samples, anticoagulants, reagents for extracting RNA from subject-derived samples, reagents for detecting RNA, instruction manuals, and the like. It is good to be. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or differentiation criteria for acute exacerbation cases of diffuse alveolar injury, acute respiratory distress syndrome and idiopathic interstitial pneumonia.
本発明のキットには、この他、標準タンパク質、バッファー、基質（抗体が酵素標識されている場合）、反応停止液、洗浄液、反応容器などを含めてもよい。
The kit of the present invention may also include a standard protein, a buffer, a substrate (when the antibody is enzyme-labeled), a reaction stop solution, a washing solution, a reaction vessel, and the like.
本発明のキットは、疾病を診断するための医薬品として用いることができる。
The kit of the present invention can be used as a pharmaceutical product for diagnosing a disease.
２．間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の検査方法、診断方法、治療方法及び検査キット
また、本発明は、ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することを含む間質性肺炎全般（びまん性肺胞傷害やその他の病型の間質性肺炎）の検査方法を提供する。 2. 2. Test method, diagnostic method, treatment method and test kit for general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia) The present invention also relates to Stratefin, calistatin, NPS-PLA2, PARC (CCL18). , LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic amphidrase 6 for at least one protein selected from the group to measure expression in a sample derived from the subject. Provided is a method for examining interstitial pneumonia in general (diffuse alveolar injury and other types of interstitial pneumonia) including.
また、本発明は、ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することを含む間質性肺炎全般（びまん性肺胞傷害やその他の病型の間質性肺炎）の検査方法を提供する。 2. 2. Test method, diagnostic method, treatment method and test kit for general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia) The present invention also relates to Stratefin, calistatin, NPS-PLA2, PARC (CCL18). , LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
間質性肺炎は、肺の間質を病変の主座とした炎症が広がる病態をいい、しばしば肺線維症を起こす。間質性肺炎は、薬剤性やじん肺、膠原病などの明確な原因に伴うものと、原因が不明な特発性とに分けられ、さらに様々な病型に分類される。代表的な病型として、びまん性肺胞傷害の他、器質化肺炎や非特異性間質性肺炎、好酸球性肺炎などがある。本発明の間質性肺炎の検査方法において、間質性肺炎の病因、病型は問わず、薬剤性間質性肺炎又は非薬剤性（特発性）間質性肺炎のいずれであってもよく、びまん性肺胞傷害や他の間質性肺炎であってもよい。
Interstitial pneumonia is a condition in which inflammation spreads with the interstitium of the lung as the main lesion, and often causes pulmonary fibrosis. Interstitial pneumonia is divided into those associated with clear causes such as drug-induced, pneumoconiosis, and collagen disease, and idiopathic diseases of unknown cause, and further classified into various types. Typical disease types include diffuse alveolar injury, organizing pneumonia, nonspecific interstitial pneumonia, and eosinophilic pneumonia. In the method for testing interstitial pneumonia of the present invention, either drug-induced interstitial pneumonia or non-drug-induced (idiopathic) interstitial pneumonia may be used regardless of the etiology and type of interstitial pneumonia. , Diffuse alveolar injury or other interstitial pneumonia.
PARC (CCL18)は、CCL18遺伝子にコードされる89アミノ酸の CCモチーフを有するケモカインの１種であり、恒常性あるいは血漿ケモカインとして分類される。脂肪細胞、肺、リンパ球などに発現している。UniProt番号P55774。
PARC (CCL18) is a type of chemokine having a CC motif of 89 amino acids encoded by the CCL18 gene, and is classified as homeostatic or plasma chemokine. It is expressed in adipocytes, lungs, lymphocytes, etc. UniProt number P55774.
LD78-betaは、CCL3L1遺伝子およびそのコピーCCL3L3遺伝子にコードされる93アミノ酸の CCモチーフの炎症性ケモカインである。骨髄やリンパ球に発現する。HIV共受容体CCR5やサイレント受容体CCBP2に結合し、HIVの感染制御に関わることが知られている。UniProt番号P16619。
LD78-beta is an inflammatory chemokine with a CC motif of 93 amino acids encoded by the CCL3L1 gene and its copy CCL3L3 gene. It is expressed in bone marrow and lymphocytes. It is known to bind to the HIV co-receptor CCR5 and the silent receptor CCBP2 and to be involved in the control of HIV infection. UniProt number P16619.
PAPP-A (パパリシン)は、PAPPA遺伝子にコードされる1627アミノ酸の分泌型メタロプロテアーゼである。妊婦胎盤に特異的に発現することが知られており、動脈瘤における発現も報告されている。UniProt番号Q13219。
PAPP-A (paparicin) is a secretory metalloprotease of 1627 amino acids encoded by the PAPPA gene. It is known to be specifically expressed in the placenta of pregnant women, and its expression in aneurysms has also been reported. UniProt number Q13219.
アポリポプロテインAI（Apo-AI）は、APOAI遺伝子によりコードされる267アミノ酸の分泌タンパク質である。肝蔵および消化管で強い発現が認められ、血中HDLの主用な構成成分として知られている。UniProt番号P02647。
Apolipoprotein AI (Apo-AI) is a secretory protein of 267 amino acids encoded by the APOAI gene. It is strongly expressed in the liver and gastrointestinal tract and is known as the main component of blood HDL. UniProt number P02647.
カリスタチンは、カリクレインインヒビターやセルピンA4とも呼ばれ、SERPINA4遺伝子にコードされる427アミノ酸の分泌型セリンプロテアーゼインヒビターである。肝臓で産生され、血液中に豊富に存在し、組織カリクレインやカリジノゲナーゼに結合しその活性を阻害する。UniProt番号P29622。
Calistatin, also called kallikrein inhibitor or serpin A4, is a secretory serine protease inhibitor of 427 amino acids encoded by the SERPINA4 gene. Produced in the liver and abundant in blood, it binds to tissue kallikrein and caridinogenase and inhibits its activity. UniProt number P29622.
補体C3b（C3b）は、C3遺伝子にコードされる補体C3の分解産物である。補体C3は肝臓にて産生され、血液中でC3aとC3bに分解され、C3bは補体C5の分解を制御することで、生体の免疫に関与している。UniProt番号P01024。
Complement C3b (C3b) is a degradation product of complement C3 encoded by the C3 gene. Complement C3 is produced in the liver and decomposed into C3a and C3b in the blood, and C3b is involved in the immunity of the living body by controlling the decomposition of complement C5. UniProt number P01024.
カーボニックアンヒドラーゼ６は、CA6遺伝子にコードされる308アミノ酸の金属酵素で、二酸化炭素と水を炭酸水素イオンと水素イオンに変換する反応を触媒する。唾液腺に強く発現する。UniProt番号P23280。
Carbonic amphidrase 6 is a 308 amino acid metal enzyme encoded by the CA6 gene, which catalyzes the reaction of converting carbon dioxide and water into bicarbonate ions and hydrogen ions. It is strongly expressed in the salivary glands. UniProt number P23280.
本発明の間質性肺炎の検査方法において、被験者は、間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の発症が疑われる哺乳動物であるが、発症の危険性が考えられるすべての哺乳動物を対象としてもよい。典型的にはヒトである。被験者由来の試料、発現の測定については、上述した通りである。発現を測定するタンパク質又は遺伝子は１種類でもよいし、複数種類であってもよいことについても、上述した通りである。
In the method for testing interstitial pneumonia of the present invention, the subject is a mammal suspected of developing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia), but there is a risk of developing it. It may cover all possible mammals. It is typically human. The sample derived from the subject and the measurement of expression are as described above. As described above, the protein or gene whose expression is measured may be one type or a plurality of types.
ストラテフィン、NPS-PLA2、PARC (CCL18)、LD78-beta及びPAPP-A (パパリシン)及び補体C3bからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定し、その発現レベルが高い場合に、間質性肺炎（びまん性肺胞傷害やその他の間質性肺炎）を発症している可能性が高いと判定し、前記レベルが低い場合に、間質性肺炎を発症している可能性が低いと判定することができる。また、これらとは逆に、Apo-AI、カリスタチン及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定し、その発現レベルが低い場合に、間質性肺炎（びまん性肺胞傷害やその他の間質性肺炎）を発症している可能性が高いと判定し、前記レベルが高い場合に、間質性肺炎を発症している可能性が低いと判定することができる。
The expression of at least one protein selected from the group consisting of stratefin, NPS-PLA2, PARC (CCL18), LD78-beta and PAPP-A (paparicin) and complement C3b was measured in a sample derived from the subject. If the expression level is high, it is determined that there is a high possibility of developing interstitial pneumonia (diffuse alveolar injury or other interstitial pneumonia), and if the level is low, interstitial pneumonia is likely to occur. It can be determined that the possibility of developing is low. On the contrary, when the expression level of at least one protein selected from the group consisting of Apo-AI, calistatin and carbonic anhydrase 6 is measured in a sample derived from a subject and the expression level is low. It is determined that there is a high possibility of developing interstitial pneumonia (diffuse alveolar injury or other interstitial pneumonia), and if the above level is high, it is possible that interstitial pneumonia has developed. It can be determined that the sex is low.
よって、本発明の間質性肺炎の検査方法は、間質性肺炎の診断（びまん性肺胞傷害及びその他の間質性肺炎の発症の有無の判定）を補助することができる。本発明は、間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c2. b2の測定値に基づき、間質性肺炎の発症の有無を判定すること
を含む前記方法を提供する。 Therefore, the method for testing interstitial pneumonia of the present invention can assist in the diagnosis of interstitial pneumonia (determination of the presence or absence of diffuse alveolar injury and other interstitial pneumonia). The present invention is a method for diagnosing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic anhydrase 6. To measure the expression of a protein in a sample derived from a subject, and
c2. Provided above are the methods comprising determining the presence or absence of the onset of interstitial pneumonia based on the measurements of b2.
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c2. b2の測定値に基づき、間質性肺炎の発症の有無を判定すること
を含む前記方法を提供する。 Therefore, the method for testing interstitial pneumonia of the present invention can assist in the diagnosis of interstitial pneumonia (determination of the presence or absence of diffuse alveolar injury and other interstitial pneumonia). The present invention is a method for diagnosing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
c2. Provided above are the methods comprising determining the presence or absence of the onset of interstitial pneumonia based on the measurements of b2.
本発明の一つの例として、間質性肺炎（びまん性肺胞傷害やその他の間質性肺炎）の診断は、以下のような基準で行うことができる。被験者から採取した血清もしくは血漿（血液試料）における上記タンパク質の少なくとも１個の発現を測定し、健常人から採取した血液試料におけるそれと比較した結果、予め設定されたカットオフ値や基準値よりも高い（ストラテフィン、NPS-PLA2、PARC (CCL18)、LD78-beta及びPAPP-A (パパリシン)及び補体C3b）値、あるいは低い（Apo-AI、カリスタチン及びカーボニックアンヒドラーゼ６）値が確認された場合、被験者は間質性肺炎を発症していると評価する。あるいは、医薬品投与中の患者において、上記タンパク質の少なくとも１個の血液試料中濃度がカットオフ値よりも高くなった場合（ストラテフィン、NPS-PLA2、PARC (CCL18)、LD78-beta及びPAPP-A (パパリシン)及び補体C3b）、もしくは低くなった場合（Apo-AI、カリスタチン及びカーボニックアンヒドラーゼ６）、当該医薬品による間質性肺炎（薬剤性間質性肺炎）の発生を疑うことができる。上記の予め設定するカットオフ値は、当業者が適宜設定することができる。例えば、間質性肺炎を発症していない健常者の定量値の95％信頼区間を基準値としたり、ROC曲線からカットオフ値を設定したりすることができる。一実施態様として、後述の実施例のROC曲線解析から、以下のカットオフ値を設定することができる。例えば健常人との鑑別のためのカットオフ値は、ストラテフィン：0.2～1.0 ng/mL（好ましくは、0.5 ng/mL）、カリスタチン：7.5～14.5 μg/mL（好ましくは、10 μg/mL）、NPS-PLA2：3～12 ng/mL（好ましくは、6 ng/mL）、PARC (CCL18)：17～60 ng/mL（好ましくは、35 ng/mL）。また、医薬品投与患者における間質性肺炎の発生を検出するためのカットオフ値は、間質性肺炎非発症例と比較することで、ストラテフィン：0.2～1.7 ng/mL（好ましくは、0.6 ng/mL）、カリスタチン：7.5～14 μg/mL（好ましくは、11 μg/mL）、NPS-PLA2：7.5～14 ng/mL（好ましくは、9 ng/mL）、PARC (CCL18)：20～60 ng/mL（好ましくは、36 ng/mL）と設定できる。あるいは、過去の測定値と比較して、ストラテフィン、NPS-PLA2、PARC (CCL18)、LD78-beta、PAPP-A (パパリシン)及び補体C3bの一つが上昇の傾向を辿った場合、あるいはApo-AI、カリスタチンやカーボニックアンヒドラーゼ６が減少の傾向を辿った場合、間質性肺炎の発症の可能性を疑う。
As an example of the present invention, the diagnosis of interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia) can be made based on the following criteria. As a result of measuring the expression of at least one of the above proteins in serum or plasma (blood sample) collected from a subject and comparing it with that in a blood sample collected from a healthy person, it is higher than a preset cutoff value or reference value. (Stratephine, NPS-PLA2, PARC (CCL18), LD78-beta and PAPP-A (paparicin) and complement C3b) or low (Apo-AI, calistatin and carbonic anhydrase 6) levels were confirmed. If so, the subject is assessed as developing interstitial pneumonia. Alternatively, if the concentration of at least one of the above proteins in a blood sample is higher than the cutoff value in a patient receiving a drug (Stratefin, NPS-PLA2, PARC (CCL18), LD78-beta and PAPP-A). (Paparicin) and complement C3b), or when it becomes low (Apo-AI, calistatin and carbonic anhydrase 6), the occurrence of interstitial pneumonia (drug-induced interstitial pneumonia) due to the drug may be suspected. it can. The above-mentioned preset cutoff value can be appropriately set by those skilled in the art. For example, the 95% confidence interval of the quantitative value of a healthy person who does not develop interstitial pneumonia can be used as a reference value, or the cutoff value can be set from the ROC curve. As one embodiment, the following cutoff values can be set from the ROC curve analysis of Examples described later. For example, the cut-off values for differentiation from healthy subjects are Stratefin: 0.2 to 1.0 ng / mL (preferably 0.5 ng / mL) and Calistatin: 7.5 to 14.5 μg / mL (preferably 10 μg / mL). , NPS-PLA2: 3 to 12 ng / mL (preferably 6 ng / mL), PARC (CCL18): 17 to 60 ng / mL (preferably 35 ng / mL). In addition, the cut-off value for detecting the occurrence of interstitial pneumonia in drug-administered patients was compared with those who did not develop interstitial pneumonia, and Stratefin: 0.2 to 1.7 ng / mL (preferably 0.6 ng). / mL), calistatin: 7.5-14 μg / mL (preferably 11 μg / mL), NPS-PLA2: 7.5-14 ng / mL (preferably 9 ng / mL), PARC (CCL18): 20-60 It can be set to ng / mL (preferably 36 ng / mL). Alternatively, if one of Stratefin, NPS-PLA2, PARC (CCL18), LD78-beta, PAPP-A (Paparicin) and complement C3b follows an upward trend compared to past measurements, or Apo -If AI, calistatin and carbonic anhydrase 6 follow a decreasing trend, suspect the possibility of developing interstitial pneumonia.
また、カリスタチン、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６の少なくとも一つの測定により間質性肺炎の発症が疑われた患者において、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質が、基準値あるいはカットオフ値よりも高い場合は、びまん性肺胞傷害を強く疑うことができ、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTが、いずれも基準値内あるいはカットオフ値以下の場合は、その他の間質性肺炎である可能性を疑うことができる。
In addition, the onset of interstitial pneumonia was suspected by at least one measurement of calistatin, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6. Diffuse if at least one protein selected from the group consisting of stratephin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T is higher than the baseline or cutoff value in the patient. Sexual alveolar injury can be strongly suspected, and if stratephin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T are all within the norm or below the cutoff value, during the rest. You can suspect that you have pneumonia.
本発明の間質性肺炎の検査方法は、間質性肺炎（びまん性肺胞傷害及びその他の間質性肺炎）の病勢診断にも利用できる。ストラテフィン、NPS-PLA2、PARC (CCL18)、LD78-beta、PAPP-A (パパリシン)及び補体C3bからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現レベルが高い場合、あるいは、Apo-AI、カリスタチン及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現レベルが低い場合に、間質性肺炎の急性期にある可能性が高いと判定することができる。例えば、間質性肺炎の回復期にある患者の定量値の95％信頼区間を基準値としたり、ROC曲線からカットオフ値を設定したりすることができる。一実施態様として、後述の実施例のROC曲線解析から、例えば以下のカットオフ値を設定することができる。ストラテフィン：0.2～1.7 ng/mL（好ましくは、1 ng/mL）、カリスタチン：7.5～14.5 ng/mL（好ましくは、10 ng/mL）、NPS-PLA2：4～28（好ましくは、8 ng/mL）、PARC (CCL18)：16～60 ng/mL（好ましくは、33 ng/mL）。
The method for testing interstitial pneumonia of the present invention can also be used for pathological diagnosis of interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia). High expression levels in subject-derived samples for at least one protein selected from the group consisting of stratefin, NPS-PLA2, PARC (CCL18), LD78-beta, PAPP-A (paparicin) and complement C3b. Alternatively, at least one protein selected from the group consisting of Apo-AI, calistatin and carbonic anhydrase 6 is in the acute phase of interstitial pneumonia when the expression level in the sample derived from the subject is low. It can be determined that the possibility is high. For example, the 95% confidence interval of the quantitative value of a patient in the recovery phase of interstitial pneumonia can be used as a reference value, or the cutoff value can be set from the ROC curve. As one embodiment, for example, the following cutoff value can be set from the ROC curve analysis of the examples described later. Stratefin: 0.2-1.7 ng / mL (preferably 1 ng / mL), calistatin: 7.5-14.5 ng / mL (preferably 10 ng / mL), NPS-PLA2: 4-28 (preferably 8 ng) / mL), PARC (CCL18): 16-60 ng / mL (preferably 33 ng / mL).
本発明は、間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c2. b2の測定値に基づき、間質性肺炎の病勢を判定すること
を含む前記方法を提供する。 The present invention is a method for diagnosing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic amphidrase 6. To measure the expression of a protein in a sample derived from a subject, and
c2. Provided above are methods that include determining the pathology of interstitial pneumonia based on b2 measurements.
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c2. b2の測定値に基づき、間質性肺炎の病勢を判定すること
を含む前記方法を提供する。 The present invention is a method for diagnosing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
c2. Provided above are methods that include determining the pathology of interstitial pneumonia based on b2 measurements.
さらに、間質性肺炎（びまん性肺胞傷害及びその他の間質性肺炎）の急性期にある可能性が高いと判定された被験者由来の試料において、ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、１回又は異なる時期に複数回測定し、基準値内に入った場合に、治療により間質性肺炎から回復したと判定し、前記レベルが基準値から外れたままの場合は、治療により間質性肺炎から回復していない、あるいは、回復が不十分であると判定することができる。本発明の方法は、間質性肺炎の病勢診断の他、予後の検査、治療効果の確認にも利用できる。
In addition, in samples from subjects determined to be more likely to be in the acute phase of interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia), Stratefin, calistatin, NPS-PLA2, PARC ( At least one protein selected from the group consisting of CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6, once or multiple times at different times. If the measurement is within the standard value, it is determined that the patient has recovered from interstitial pneumonia by treatment, and if the level remains outside the standard value, the patient has not recovered from interstitial pneumonia by treatment. Alternatively, it can be determined that the recovery is inadequate. The method of the present invention can be used not only for diagnosing the pathology of interstitial pneumonia, but also for prognosis examination and confirmation of therapeutic effect.
被験者が間質性肺炎を発症している可能性が高いと評価された場合には、薬剤性が疑われる場合は、被疑薬をすみやかに中止する。次いで、喀痰や血清の感染症検査、画像検査、気管支肺胞洗浄検査や病理検査などの各種検査を行い、間質性肺炎の病型の診断を確定させる。非特異性間質性肺炎や器質化肺炎、好酸球性肺炎の場合には、症状や重症度に応じて、ステロイド治療の施行を検討する。ステロイドの投与量はプレドニゾロン換算で0.5～1.0 mg/kg/日から開始し、2か月程度で漸減・終了する。びまん性肺胞傷害の場合は、上述しているように、パルス療法を含むステロイド治療に加え、重症例では免疫抑制薬（シクロスポリン、タクロリムスなど）や好中球エラスターゼ阻害薬（シベレスタット）の投与、ポリミキシンB固定化線維カラム（PMX）療法などを組み合わせた集学的治療を考慮する。
If the subject is evaluated to have a high possibility of developing interstitial pneumonia, and if drug-induced is suspected, the suspected drug should be discontinued immediately. Next, various tests such as sputum and serum infection tests, imaging tests, bronchoalveolar lavage tests, and pathological tests are performed to confirm the diagnosis of the type of interstitial pneumonia. In the case of nonspecific interstitial pneumonia, organizing pneumonia, and eosinophilic pneumonia, steroid treatment should be considered according to the symptoms and severity. The dose of steroid starts from 0.5 to 1.0 mg / kg / day in terms of prednisolone, and gradually decreases and ends in about 2 months. For diffuse alveolar injury, as described above, in addition to steroid therapy including pulse therapy, immunosuppressive drugs (cyclosporine, tacrolimus, etc.) and neutrophil elastase inhibitors (sivelestat) are administered in severe cases. , Consider multidisciplinary therapy combined with polymyxin B-immobilized fiber column (PMX) therapy.
米国では、薬剤性の間質性肺炎の発生頻度が日本ほど高くなく（RESPIRATORY INVESTIGATION, Dec 2013, 51(4):260-77）、間質性肺炎としては、薬剤性でなく特発性間質性肺炎がまず挙げられる。米国呼吸器学会（American Thoracic Society）では診断（病型等）に関するStatementは発表しているものの（AM J RESPIR CRIT CARE MED. Sep 2013;188(6):733-48）、一般的な病型に関する治療ガイドラインを有しておらず、基本的に各医療施設の方針に委ねられている。例えば、Mayo clinicでは、一般的な間質性肺炎の治療として、薬物治療、酸素療法、肺のリハビリテーション、肺移植が行われている（https://www.mayoclinic.org/diseases-conditions/interstitial-lung-disease/diagnosis-treatment/drc-20353113）。薬物治療としては、1) コルチコステロイド薬（プレドニゾロン等）及びその免疫抑制薬との薬剤と併用：間質性肺炎の進行を遅くする、または安定させると期待される、2) 特発性肺線維症治療薬（ピルフェニドンとニンテダニブ：間質性肺炎の進行を遅くすると期待される）。3）胃酸分泌抑制薬（ランソプラゾールやオメプラゾール等：逆流性食道炎の防止により胃液の誤嚥を低下させ、肺損傷悪化を抑制する）、がある。酸素療法は、肺の損傷を防ぐことはできないが、呼吸を楽にする、血中酸素濃度の低下による合併症を予防・軽減する、心臓の右側の血圧低下、が期待される。肺のリハビリテーションは、日常動作の容易性などQOLを改善する。肺移植は、他の治療選択肢がない重症間質性肺炎患者にとり、最後の手段である。
In the United States, the incidence of drug-induced interstitial pneumonia is not as high as in Japan (RESPIRATORY INVESTIGATION, Dec 2013, 51 (4): 260-77), and interstitial pneumonia is not drug-induced but idiopathic interstitium. Sexual pneumonia is mentioned first. Although the American Thoracic Society has announced a statement regarding diagnosis (disease type, etc.) (AM J RESPIR CRIT CARE MED. Sep 2013; 188 (6): 733-48), general disease types It does not have treatment guidelines for, and is basically left to the policy of each medical facility. For example, Mayoclinic offers general treatments for interstitial pneumonia such as drug treatment, oxygen therapy, lung rehabilitation, and lung transplantation (https://www.mayoclinic.org/diseases-conditions/interstitial). -lung-disease / diagnosis-treatment / drc-20353113). Pharmacotherapy includes 1) concomitant use of corticosteroids (prednisolone, etc.) and their immunosuppressive drugs: expected to slow or stabilize the progression of interstitial pneumonia, 2) idiopathic lung fibers. Drugs for illness (pirfenidone and nintedanib: expected to slow the progression of interstitial pneumonia). 3) There are gastric acid secretion inhibitors (lansoprazole, omeprazole, etc .: prevention of reflux esophagitis reduces aspiration of gastric juice and suppresses exacerbation of lung damage). Oxygen therapy cannot prevent lung damage, but is expected to ease breathing, prevent and reduce complications due to decreased blood oxygen levels, and reduce blood pressure on the right side of the heart. Lung rehabilitation improves quality of life, including ease of daily living. Lung transplantation is a last resort for patients with severe interstitial pneumonia who have no other treatment options.
一方、間質性肺炎の一種であるIPFに関しては、患者数も多く予後不良のため、臨床研究結果に基づき、米国呼吸器学会より治療ガイドラインが発表されている（AMERICAN JOURNAL OF RESPIRATORY AND CRINICAL CARE MEDICINE, JULY 2015. 192(2):e3-19）。IPFの治療薬として条件付きで推奨されている医薬品としては、ニンテダニブ（血管内皮細胞増殖因子受容体（VEGFR）、線維芽細胞増殖因子受容体（FGFR）、血小板由来成長因子受容体（PDGFR）等のチロシンキナーゼに対する阻害薬）、ピルフェニドン（抗繊維化作用）、胃酸分泌抑制薬（逆流性食道炎の防止による肺損傷悪化抑制）、がある。しかし、ワルファリン（抗凝固薬）、イマチニブ（血小板由来成長因子（PDGF）の選択的チロシンキナーゼ阻害剤）、プレドニゾン・アザチオプリン・N-アセチルシステインの組み合わせやアンブリセンタン（選択的エンドセリン受容体拮抗薬）はその有効性に疑問が持たれており、使用することは推奨されていないが、その使用を排除するものではない。
On the other hand, regarding IPF, which is a type of interstitial pneumonia, the number of patients is large and the prognosis is poor. Therefore, treatment guidelines have been published by the American Respiratory Society based on clinical research results (AMERICAN JOURNAL OF RESPIRATORY AND CRINICAL CARE MEDICINE). , JULY 2015. 192 (2): e3-19). Nintedanib (vascular endothelial cell growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), etc.) are conditionally recommended as therapeutic agents for IPF. (Inhibitor against tyrosine kinase), pirfenidone (anti-fibroblasting effect), gastric acid secretion inhibitor (suppressing exacerbation of lung damage by preventing reflux esophagitis). However, warfarin (an anticoagulant), imatinib (a selective tyrosine kinase inhibitor of platelet-derived growth factor (PDGF)), a combination of prednisone, azathiopurine and N-acetylcysteine, and ambrisentan (a selective endothelin receptor antagonist) Its effectiveness has been questioned and its use is not recommended, but it does not preclude its use.
本発明は、間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断及び治療方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c2. b2の測定値に基づき、間質性肺炎の発症の有無を判定すること、及び
d2. 間質性肺炎を発症している可能性が高いと判定された被験者に間質性肺炎の治療を施すこと
を含む前記方法を提供する。 The present invention is a method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic amphidrase 6. To measure the expression of a protein in a sample derived from a subject,
c2. Determine the presence or absence of interstitial pneumonia based on the measured value of b2, and
d2. Provided by the above method, which comprises treating an interstitial pneumonia in a subject determined to have a high possibility of developing interstitial pneumonia.
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c2. b2の測定値に基づき、間質性肺炎の発症の有無を判定すること、及び
d2. 間質性肺炎を発症している可能性が高いと判定された被験者に間質性肺炎の治療を施すこと
を含む前記方法を提供する。 The present invention is a method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
c2. Determine the presence or absence of interstitial pneumonia based on the measured value of b2, and
d2. Provided by the above method, which comprises treating an interstitial pneumonia in a subject determined to have a high possibility of developing interstitial pneumonia.
また、本発明は、間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断及び治療方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c2. b2の測定値に基づき、間質性肺炎の病勢を判定すること、及び
d2. 間質性肺炎の急性期にある可能性が高いと判定された被験者に間質性肺炎の治療を施すこと
を含む前記方法を提供する。 The present invention is a method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b andcarbonic amphidrase 6. To measure the expression of a protein in a sample derived from a subject,
c2. Judging the pathology of interstitial pneumonia based on the measured values of b2, and
d2. Provided by the above method, which comprises treating an interstitial pneumonia with a subject determined to be likely to be in the acute phase of interstitial pneumonia.
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c2. b2の測定値に基づき、間質性肺炎の病勢を判定すること、及び
d2. 間質性肺炎の急性期にある可能性が高いと判定された被験者に間質性肺炎の治療を施すこと
を含む前記方法を提供する。 The present invention is a method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and
c2. Judging the pathology of interstitial pneumonia based on the measured values of b2, and
d2. Provided by the above method, which comprises treating an interstitial pneumonia with a subject determined to be likely to be in the acute phase of interstitial pneumonia.
また、本発明は、ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することができる試薬を含む、間質性肺炎（びまん性肺胞傷害やその他の間質性肺炎）の検査のためのキットを提供する。
The present invention is also selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6. A kit for testing interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia) containing a reagent capable of measuring the expression of at least one protein in a sample derived from a subject. provide.
一つの例として、本発明のキットは、上記タンパク質を特異的に認識できる抗体を試薬として含む。抗体はマイクロタイタープレートや磁気ビーズ、セルロース膜や基板に固定されていてもよい。キットには、さらに、被験者由来の試料を採取するための器具、抗凝固剤、上記タンパク質を検出するための試薬一式、取扱説明書などが含まれてもよい。取扱説明書には、キットの使用方法の他、間質性肺炎の評価及び／又は鑑別基準なども記載しておくとよい。
As an example, the kit of the present invention contains an antibody capable of specifically recognizing the above protein as a reagent. The antibody may be immobilized on a microtiter plate, magnetic beads, a cellulose membrane or a substrate. The kit may further include an instrument for collecting a sample derived from a subject, an anticoagulant, a set of reagents for detecting the protein, an instruction manual, and the like. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or discrimination criteria for interstitial pneumonia.
別の一例として、本発明のキットは、上記タンパク質のmRNAと特異的にハイブリダイズできる核酸プローブを試薬として含む。核酸プローブは基板に固定されていてもよい。キットには、さらに、生体試料を採取するための器具、抗凝固剤、被験者由来の試料からRNAを抽出するための試薬類、RNAを検出するための試薬類、取扱説明書などが含まれてもよい。取扱説明書には、キットの使用方法の他、間質性肺炎の評価及び／又は鑑別基準なども記載しておくとよい。
As another example, the kit of the present invention contains a nucleic acid probe that can specifically hybridize with the mRNA of the above protein as a reagent. The nucleic acid probe may be fixed to the substrate. The kit also includes equipment for collecting biological samples, anticoagulants, reagents for extracting RNA from samples derived from subjects, reagents for detecting RNA, instruction manuals, etc. May be good. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or discrimination criteria for interstitial pneumonia.
さらに別の一例として、本発明のキットは上記タンパク質のmRNAを鋳型として合成されるcDNAを特異的に増幅できる少なくとも１対の核酸プライマーを試薬として含む。キットには、さらに、被験者由来の試料を採取するための器具、抗凝固剤、被験者由来の試料からRNAを抽出するための試薬類、RNAを検出するための試薬類、取扱説明書などが含まれるとよい。取扱説明書には、キットの使用方法の他、間質性肺炎の評価及び／又は鑑別基準なども記載しておくとよい。
As yet another example, the kit of the present invention contains at least one pair of nucleic acid primers capable of specifically amplifying cDNA synthesized using the above protein mRNA as a reagent. The kit also includes equipment for collecting subject-derived samples, anticoagulants, reagents for extracting RNA from subject-derived samples, reagents for detecting RNA, instruction manuals, and the like. It is good to be. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or discrimination criteria for interstitial pneumonia.
本発明のキットには、この他、標準タンパク質、バッファー、基質（抗体が酵素標識されている場合）、反応停止液、洗浄液、反応容器などを含めてもよい。
The kit of the present invention may also include a standard protein, a buffer, a substrate (when the antibody is enzyme-labeled), a reaction stop solution, a washing solution, a reaction vessel, and the like.
本発明のキットは、疾病を診断するための医薬品として用いることができる。
The kit of the present invention can be used as a pharmaceutical product for diagnosing a disease.
The kit of the present invention can be used as a pharmaceutical product for diagnosing a disease.
以下、実施例により本発明を更に詳細に説明する。
〔実施例１〕
（１）検体
解析に用いたヒト間質性肺炎試料については、4箇所の拠点病院（信州大学、日本医科大学、千葉大学、広島大学）、国立医薬品食品衛生研究所、木原財団、アステラス製薬、及び第一三共において、各研究倫理委員会の承認を得て収集・解析した。 Hereinafter, the present invention will be described in more detail with reference to Examples.
[Example 1]
(1) Regarding human interstitial pneumonia samples used for sample analysis, four base hospitals (Shinshu University, Japan Medical University, Chiba University, Hiroshima University), National Institute of Pharmaceutical and Food Sanitation, Kihara Foundation, Astellas Pharma, And Daiichi Sankyo collected and analyzed with the approval of each research ethics committee.
〔実施例１〕
（１）検体
解析に用いたヒト間質性肺炎試料については、4箇所の拠点病院（信州大学、日本医科大学、千葉大学、広島大学）、国立医薬品食品衛生研究所、木原財団、アステラス製薬、及び第一三共において、各研究倫理委員会の承認を得て収集・解析した。 Hereinafter, the present invention will be described in more detail with reference to Examples.
[Example 1]
(1) Regarding human interstitial pneumonia samples used for sample analysis, four base hospitals (Shinshu University, Japan Medical University, Chiba University, Hiroshima University), National Institute of Pharmaceutical and Food Sanitation, Kihara Foundation, Astellas Pharma, And Daiichi Sankyo collected and analyzed with the approval of each research ethics committee.
医薬品による間質性肺炎の発症が疑われた患者に対して薬剤性間質性肺炎の急性期（最悪期付近）および回復期に採血を上記の拠点病院にて行った。入院時は早朝空腹時に、外来時は随時、採血した。各拠点病院において、患者の同意の下、血漿採取用の7 mLのEDTA-2K採血管を用いて採血を行い、速やかに混和後、3,000 rpm×10分（15℃～20℃）遠心分離を行った。採取した血漿は-80℃にて凍結保存した。
For patients suspected of developing interstitial pneumonia due to drugs, blood was collected at the above-mentioned base hospital during the acute phase (near the worst phase) and convalescent phase of drug-induced interstitial pneumonia. Blood was collected early in the morning on an empty stomach at the time of admission and at any time during the outpatient department. At each base hospital, with the consent of the patient, blood is collected using a 7 mL EDTA-2K blood collection tube for plasma collection, and after prompt mixing, centrifugation is performed at 3,000 rpm x 10 minutes (15 ° C to 20 ° C). went. The collected plasma was cryopreserved at -80 ° C.
薬剤性間質性肺炎の患者検体において、画像診断において、びまん性肺胞傷害（DAD）パターンを呈する症例の急性期に採取された検体8例、回復期で採取された検体3例が存在し、解析に供した。器質化肺炎（OP）パターンの症例については、急性期16例、回復期13例、非特異性間質性肺炎（NSIP）パターンの症例は、急性期18例、回復期16例、それら以外の間質性肺炎病型は急性期2例、回復期１例を用いた。健常人検体としては、ノイエス株式会社にて収集されたボランティア健常成人（間質性肺炎発症患者群との年齢構成比率を揃えた24例）の血漿検体を用いた。
Among the patient specimens of drug-induced interstitial pneumonia, there are 8 specimens collected in the acute phase and 3 specimens collected in the convalescent phase in the cases showing the diffuse alveolar injury (DAD) pattern in the diagnostic imaging. , Used for analysis. About the cases of organizing pneumonia (OP) pattern, 16 cases of acute phase, 13 cases of convalescent phase, and the cases of nonspecific interstitial pneumonia (NSIP) pattern are 18 cases of acute phase, 16 cases of convalescent phase, and others. For the interstitial pneumonia type, 2 cases in the acute phase and 1 case in the convalescent phase were used. As a healthy person sample, a plasma sample of volunteer healthy adults (24 cases having the same age composition ratio with the group of patients with interstitial pneumonia) collected by Neues Co., Ltd. was used.
（２）測定手法
各検体のプロテオーム解析は、SomaLogic社のSOMAscanシステムを用いて行った。当該システムは、アプタマー（一本鎖核酸による人工リガンド）により、1,310種のタンパク質を検出する測定法である。血漿は米国SomaLogic社に凍結状態で送付し、測定に供した。 (2) Measurement method Proteome analysis of each sample was performed using the SOMAscan system of SomaLogic. The system is a measurement method that detects 1,310 types of proteins using aptamers (artificial ligands made from single-stranded nucleic acids). The plasma was sent frozen to SomaLogic in the United States for measurement.
各検体のプロテオーム解析は、SomaLogic社のSOMAscanシステムを用いて行った。当該システムは、アプタマー（一本鎖核酸による人工リガンド）により、1,310種のタンパク質を検出する測定法である。血漿は米国SomaLogic社に凍結状態で送付し、測定に供した。 (2) Measurement method Proteome analysis of each sample was performed using the SOMAscan system of SomaLogic. The system is a measurement method that detects 1,310 types of proteins using aptamers (artificial ligands made from single-stranded nucleic acids). The plasma was sent frozen to SomaLogic in the United States for measurement.
（３）間質性肺炎バイオマーカー候補タンパク質の選定
1,310種のタンパク質のSOMAscanデータセットを用いて、臨床で特に重要とされるDADとOPで変動するタンパク質を探索した。SOMAscan測定で得られた各プローブの蛍光シグナル強度に基づき、全症例の回復期群（総回復期群、計33例）と健常人群（24例）を統合した計57例のコントロール群と比較して、DAD急性期群（9例）あるいはOP急性期群（16例）で、効果量Hedge’s g値が0.8以上、Fold change値が2倍以上の変動を示すタンパク質から、上位10位以内に入るものを抽出した。抽出したタンパク質の各病型における変動パターンから、DADの急性期では増加するが他の病型では変化が認められない、DAD特異検出しうるタンパク質が６種類見いだされた（図１、表１）。また、DADだけでなくOPやNSIPにおいても増加あるいは減少するタンパク質も９種類見つかった（図２、表２）。 (3) Selection of candidate proteins for interstitial pneumonia biomarkers Using the SOMAscan dataset of 1,310 proteins, we searched for proteins that fluctuate with DAD and OP, which are of particular clinical importance. Based on the fluorescence signal intensity of each probe obtained by SOMAscan measurement, it was compared with the control group of 57 cases in which the convalescent group (total convalescent group, 33 cases in total) and the healthy subject group (24 cases) of all cases were integrated. In the DAD acute phase group (9 cases) or OP acute phase group (16 cases), the proteins showing fluctuations in the effect size Hedge's g value of 0.8 or more and the Fold change value of 2 times or more are in the top 10 Extracted things. From the variation patterns of the extracted proteins in each disease type, 6 types of DAD-specific detectable proteins were found that increased in the acute phase of DAD but did not change in other disease types (Fig. 1, Table 1). .. In addition, 9 types of proteins that increase or decrease not only in DAD but also in OP and NSIP were found (Fig. 2, Table 2).
1,310種のタンパク質のSOMAscanデータセットを用いて、臨床で特に重要とされるDADとOPで変動するタンパク質を探索した。SOMAscan測定で得られた各プローブの蛍光シグナル強度に基づき、全症例の回復期群（総回復期群、計33例）と健常人群（24例）を統合した計57例のコントロール群と比較して、DAD急性期群（9例）あるいはOP急性期群（16例）で、効果量Hedge’s g値が0.8以上、Fold change値が2倍以上の変動を示すタンパク質から、上位10位以内に入るものを抽出した。抽出したタンパク質の各病型における変動パターンから、DADの急性期では増加するが他の病型では変化が認められない、DAD特異検出しうるタンパク質が６種類見いだされた（図１、表１）。また、DADだけでなくOPやNSIPにおいても増加あるいは減少するタンパク質も９種類見つかった（図２、表２）。 (3) Selection of candidate proteins for interstitial pneumonia biomarkers Using the SOMAscan dataset of 1,310 proteins, we searched for proteins that fluctuate with DAD and OP, which are of particular clinical importance. Based on the fluorescence signal intensity of each probe obtained by SOMAscan measurement, it was compared with the control group of 57 cases in which the convalescent group (total convalescent group, 33 cases in total) and the healthy subject group (24 cases) of all cases were integrated. In the DAD acute phase group (9 cases) or OP acute phase group (16 cases), the proteins showing fluctuations in the effect size Hedge's g value of 0.8 or more and the Fold change value of 2 times or more are in the top 10 Extracted things. From the variation patterns of the extracted proteins in each disease type, 6 types of DAD-specific detectable proteins were found that increased in the acute phase of DAD but did not change in other disease types (Fig. 1, Table 1). .. In addition, 9 types of proteins that increase or decrease not only in DAD but also in OP and NSIP were found (Fig. 2, Table 2).
（４）びまん性肺胞傷害に特異的なタンパク質のバイオマーカー性能
図1は、DADマーカー候補タンパク質６種類（ストラテフィン、シスタチンF、NPS-PLA2、IL-1Ra、ネトリン-1及びトロポニンT）の血漿中の変動パターンをボックスプロットにて示したものである。いずれのタンパク質も健常人に比べてDADの急性期で顕著な増加を示し、回復期で健常人と同程度まで減少するが、OPやNSIPにおける変動は小さかった。これらのタンパク質はDAD特異診断に有用なマーカーとなり得る。 (4) Biomarker performance of proteins specific for diffuse alveolar injury Figure 1 shows the 6 types of DAD marker candidate proteins (Stratefin, Cystatin F, NPS-PLA2, IL-1Ra, Netrin-1 and Troponin T). The fluctuation pattern in plasma is shown by a box plot. Both proteins showed a marked increase in DAD in the acute phase compared to healthy subjects and decreased to the same extent as in healthy subjects in the recovery phase, but the fluctuations in OP and NSIP were small. These proteins can be useful markers for DAD-specific diagnosis.
図1は、DADマーカー候補タンパク質６種類（ストラテフィン、シスタチンF、NPS-PLA2、IL-1Ra、ネトリン-1及びトロポニンT）の血漿中の変動パターンをボックスプロットにて示したものである。いずれのタンパク質も健常人に比べてDADの急性期で顕著な増加を示し、回復期で健常人と同程度まで減少するが、OPやNSIPにおける変動は小さかった。これらのタンパク質はDAD特異診断に有用なマーカーとなり得る。 (4) Biomarker performance of proteins specific for diffuse alveolar injury Figure 1 shows the 6 types of DAD marker candidate proteins (Stratefin, Cystatin F, NPS-PLA2, IL-1Ra, Netrin-1 and Troponin T). The fluctuation pattern in plasma is shown by a box plot. Both proteins showed a marked increase in DAD in the acute phase compared to healthy subjects and decreased to the same extent as in healthy subjects in the recovery phase, but the fluctuations in OP and NSIP were small. These proteins can be useful markers for DAD-specific diagnosis.
これらDAD特異的タンパク質のバイオマーカー性能を比較するために、DAD患者と健常人および回復期群との識別（患者の病勢診断性能）、DAD以外の間質性肺炎患者との識別（DAD診断性能）に関するROC解析を実施した。表１に、各タンパク質のROC解析から得られるAUCの値をまとめた。DAD急性期群と健常群を比較すると、ほとんどのタンパク質がAUC 0.95以上と良好な識別精度を示した。回復期群に対する識別性能は、ほとんどがAUC 0.8以上の値を示し、中でもストラテフィン（同 0.91）とシスタチンF(同 0.90)が最も高かった。既存の間質性肺炎マーカーKL-6の臨床検査値に基づくAUC値は 0.78であり、上記タンパク質よりも低い値を示した。DAD診断性能（DAD急性期とそれ以外の間質性肺炎急性期との識別）については、ストラテフィン (同 0.90)とシスタチンF (同 0.91) のAUC値が最も高く、それ以外のタンパク質もAUC 0.8以上と、既存マーカーKL-6（同 0.67）よりも優れたDAD診断性能を示した。
In order to compare the biomarker performance of these DAD-specific proteins, discrimination between DAD patients and healthy subjects and convalescent groups (patient pathological diagnosis performance), and discrimination between patients with interstitial pneumonia other than DAD (DAD diagnostic performance) ) Was performed. Table 1 summarizes the AUC values obtained from the ROC analysis of each protein. Comparing the DAD acute phase group and the healthy group, most of the proteins showed good discrimination accuracy with AUC 0.95 or higher. Most of the discrimination performances for the convalescent group showed a value of AUC 0.8 or higher, and among them, Stratefin (0.91) and Cystatin F (0.90) were the highest. The AUC value based on the clinical laboratory test value of the existing interstitial pneumonia marker KL-6 was 0.78, which was lower than that of the above protein. Regarding the DAD diagnostic performance (distinguishing between the acute phase of DAD and the acute phase of interstitial pneumonia other than that), the AUC values of Stratefin (0.90) and Sistatin F (0.91) are the highest, and other proteins are also AUC. The DAD diagnostic performance was 0.8 or higher, which was superior to the existing marker KL-6 (0.67).
（５）コンビネーションアッセイによるびまん性肺胞傷害の診断性能の向上
DAD診断性能が高い上位のタンパク質の中で、相関が低いもの同士を組み合わせれば、さらにDAD診断性能の向上が期待される。図３AおよびBは、相関関係の低かったシスタチンFとストラテフィン、およびネトリン－１とIL-1Raを例に、DAD診断性能に対するコンビネーションアッセイの効果を検討した結果である。各タンパク質のSOMAscan測定値を標準化した値の和を用いることにより、DAD検体の検出効率が向上し、シスタチンFとストラテフィンの組み合わせを用いた際のAUC値は0.98に、ネトリン－１とIL-1RaのAUC値は0.97にまで上昇した。これらに加えて、シスタチンFとネトリン－１やストラテフィンとネトリン－１の組み合わせなどもまた、AUC 0.95程度にまでDAD診断性能が向上することが示された。 (5) Improvement of diagnostic performance of diffuse alveolar injury by combination assay By combining high-ranking proteins with high DAD diagnostic performance with low correlation, further improvement of DAD diagnostic performance is expected. FIGS. 3A and 3B show the results of examining the effect of the combination assay on the DAD diagnostic performance using cystatin F and stratefin, which had low correlation, and netrin-1 and IL-1Ra as examples. By using the sum of the standardized SOMAscan measurements of each protein, the detection efficiency of DAD samples was improved, and the AUC value when the combination of cystatin F and stratefin was 0.98, netrin-1 and IL- The AUC value of 1Ra rose to 0.97. In addition to these, the combination of cystatin F and netrin-1 and stratefin and netrin-1 was also shown to improve the DAD diagnostic performance to about AUC 0.95.
DAD診断性能が高い上位のタンパク質の中で、相関が低いもの同士を組み合わせれば、さらにDAD診断性能の向上が期待される。図３AおよびBは、相関関係の低かったシスタチンFとストラテフィン、およびネトリン－１とIL-1Raを例に、DAD診断性能に対するコンビネーションアッセイの効果を検討した結果である。各タンパク質のSOMAscan測定値を標準化した値の和を用いることにより、DAD検体の検出効率が向上し、シスタチンFとストラテフィンの組み合わせを用いた際のAUC値は0.98に、ネトリン－１とIL-1RaのAUC値は0.97にまで上昇した。これらに加えて、シスタチンFとネトリン－１やストラテフィンとネトリン－１の組み合わせなどもまた、AUC 0.95程度にまでDAD診断性能が向上することが示された。 (5) Improvement of diagnostic performance of diffuse alveolar injury by combination assay By combining high-ranking proteins with high DAD diagnostic performance with low correlation, further improvement of DAD diagnostic performance is expected. FIGS. 3A and 3B show the results of examining the effect of the combination assay on the DAD diagnostic performance using cystatin F and stratefin, which had low correlation, and netrin-1 and IL-1Ra as examples. By using the sum of the standardized SOMAscan measurements of each protein, the detection efficiency of DAD samples was improved, and the AUC value when the combination of cystatin F and stratefin was 0.98, netrin-1 and IL- The AUC value of 1Ra rose to 0.97. In addition to these, the combination of cystatin F and netrin-1 and stratefin and netrin-1 was also shown to improve the DAD diagnostic performance to about AUC 0.95.
（６）間質性肺炎全病型の急性期で変動するタンパク質のバイオマーカー性能
図２に示すように、PARC (CCL18)、LD78-betaのケモカインは、DAD、NSIP、OPのいずれの病型においても、急性期における増加傾向と回復期の減少傾向が見られた。また、胎盤特異的タンパク質PAPP-A (パパリシン)や補体C3bも、これらと同様の変動パターンを示し、これらとは逆に、Apo-AI、カーボニックアンヒドラーゼ６（CA6）、カリスタチンは、いずれの病型においても、急性期で減少する傾向が見られ、回復期では健常群と同程度にまで戻る傾向が示された（図２）。 (6) Biomarker performance of proteins that fluctuate in the acute phase of all types of interstitial pneumonia As shown in Fig. 2, the chemokine of PARC (CCL18) and LD78-beta is a type of DAD, NSIP, or OP. In addition, there was an increasing tendency in the acute phase and a decreasing tendency in the recovery phase. The placenta-specific protein PAPP-A and complement C3b also show similar fluctuation patterns, and conversely, Apo-AI, carbonic anhydrase 6 (CA6), and calistatin are used. In all types of disease, there was a tendency for the disease to decrease in the acute phase, and in the convalescent phase, it tended to return to the same level as in the healthy group (Fig. 2).
図２に示すように、PARC (CCL18)、LD78-betaのケモカインは、DAD、NSIP、OPのいずれの病型においても、急性期における増加傾向と回復期の減少傾向が見られた。また、胎盤特異的タンパク質PAPP-A (パパリシン)や補体C3bも、これらと同様の変動パターンを示し、これらとは逆に、Apo-AI、カーボニックアンヒドラーゼ６（CA6）、カリスタチンは、いずれの病型においても、急性期で減少する傾向が見られ、回復期では健常群と同程度にまで戻る傾向が示された（図２）。 (6) Biomarker performance of proteins that fluctuate in the acute phase of all types of interstitial pneumonia As shown in Fig. 2, the chemokine of PARC (CCL18) and LD78-beta is a type of DAD, NSIP, or OP. In addition, there was an increasing tendency in the acute phase and a decreasing tendency in the recovery phase. The placenta-specific protein PAPP-A and complement C3b also show similar fluctuation patterns, and conversely, Apo-AI, carbonic anhydrase 6 (CA6), and calistatin are used. In all types of disease, there was a tendency for the disease to decrease in the acute phase, and in the convalescent phase, it tended to return to the same level as in the healthy group (Fig. 2).
これらのタンパク質について、間質性肺炎全般を検出する性能を、ROC解析により比較すると、DADとOPの患者急性期を健常群と識別する性能は、全てのタンパク質で、AUC値0.9以上の性能を示した（表２）。さらに、DADで顕著な上昇が見られたストラテフィンとNPS-PLA2においても、AUC値はそれぞれ0.87、0.95であり、DADとOPの患者急性期を健常群と良好に識別できる性能を示した。また、NSIPを含む全間質性肺炎の急性期の患者に対しては、AUC値は若干低下するものの、補体C3bを除くタンパク質がAUC 値0.89以上で健常人と鑑別した。一方、回復期群に対する識別性能は、DADとOPの患者急性期および全間質性肺炎の患者急性期、いずれの場合においても、健常人と比較した時よりも劣る傾向にあったものの、既存マーカーKL-6やSP-Dと同等、又はより高いAUC値を示した（表2）。以上の結果により、表２に示した９種のタンパク質は、病型を問わず、間質性肺炎を検出するバイオマーカーとなり得ることが示された。
Comparing the ability of these proteins to detect interstitial pneumonia in general by ROC analysis, the ability to distinguish the acute phase of patients with DAD and OP from the healthy group is that all proteins have an AUC value of 0.9 or higher. It is shown (Table 2). Furthermore, the AUC values of Stratefin and NPS-PLA2, which showed a marked increase in DAD, were 0.87 and 0.95, respectively, showing the ability to distinguish the acute phase of DAD and OP patients from the healthy group. In addition, in patients with acute phase of all interstitial pneumonia including NSIP, although the AUC value decreased slightly, proteins other than complement C3b had an AUC value of 0.89 or more, which differentiated them from healthy subjects. On the other hand, the discrimination performance for the convalescent group tended to be inferior to that of healthy subjects in both the acute phase of patients with DAD and OP and the acute phase of patients with total interstitial pneumonia, although it was existing. The AUC value was equal to or higher than that of the markers KL-6 and SP-D (Table 2). From the above results, it was shown that the nine proteins shown in Table 2 can be biomarkers for detecting interstitial pneumonia regardless of the disease type.
表１.びまん性肺胞傷害特異的なタンパク質とバイオマーカー性能.
SOMAscan解析においてDADに特徴的な変動がみられたタンパク質６種類について、DAD急性期 (DAD-Ac)と健常群(HC)、総回復期群(All-R)、DAD以外の間質性肺炎群 (non-DAD-Ac)との識別性能をROC曲線解析から算出されるAUCの値で示した。既存マーカーKL-6のAUC値は、検体採取時に測定された臨床検査値より算出した。
Table 1. Diffuse alveolar injury-specific proteins and biomarker performance.
Interstitial pneumonia other than DAD acute phase (DAD-Ac), healthy group (HC), total recovery phase group (All-R), and DAD for 6 types of proteins that showed characteristic fluctuations in DAD in SOMAscan analysis. The discrimination performance from the group (non-DAD-Ac) is shown by the AUC value calculated from the ROC curve analysis. The AUC value of the existing marker KL-6 was calculated from the clinical laboratory test values measured at the time of sample collection.
SOMAscan解析においてDADに特徴的な変動がみられたタンパク質６種類について、DAD急性期 (DAD-Ac)と健常群(HC)、総回復期群(All-R)、DAD以外の間質性肺炎群 (non-DAD-Ac)との識別性能をROC曲線解析から算出されるAUCの値で示した。既存マーカーKL-6のAUC値は、検体採取時に測定された臨床検査値より算出した。
Interstitial pneumonia other than DAD acute phase (DAD-Ac), healthy group (HC), total recovery phase group (All-R), and DAD for 6 types of proteins that showed characteristic fluctuations in DAD in SOMAscan analysis. The discrimination performance from the group (non-DAD-Ac) is shown by the AUC value calculated from the ROC curve analysis. The AUC value of the existing marker KL-6 was calculated from the clinical laboratory test values measured at the time of sample collection.
表２．間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）を検出するタンパク質のバイオマーカー性能.
SOMAscan解析において薬物性間質性肺炎全般で変動が見られたタンパク質７種類およびストラテフィンとNPS-PLA2について、健常群(HC)や回復期群(All-Re)との識別能を比較するためのROC曲線解析を行い、算出されるAUC値を示した。既存マーカーKL-6およびSP-DのAUC値は、検体採取時に測定された臨床検査値より算出した。DADはびまん性肺胞傷害、OPは器質化肺炎、Allは間質性肺炎全般、Acは急性期を示す。
Table 2. Biomarker performance of proteins that detect general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia).
To compare the distinguishing ability of 7 types of proteins and Stratefin and NPS-PLA2, which were found to fluctuate in general drug-induced interstitial pneumonia in SOMAscan analysis, from the healthy group (HC) and convalescent group (All-Re). ROC curve analysis was performed and the calculated AUC value was shown. The AUC values of the existing markers KL-6 and SP-D were calculated from the clinical laboratory test values measured at the time of sample collection. DAD indicates diffuse alveolar injury, OP indicates organizing pneumonia, All indicates interstitial pneumonia in general, and Ac indicates acute phase.
SOMAscan解析において薬物性間質性肺炎全般で変動が見られたタンパク質７種類およびストラテフィンとNPS-PLA2について、健常群(HC)や回復期群(All-Re)との識別能を比較するためのROC曲線解析を行い、算出されるAUC値を示した。既存マーカーKL-6およびSP-DのAUC値は、検体採取時に測定された臨床検査値より算出した。DADはびまん性肺胞傷害、OPは器質化肺炎、Allは間質性肺炎全般、Acは急性期を示す。
To compare the distinguishing ability of 7 types of proteins and Stratefin and NPS-PLA2, which were found to fluctuate in general drug-induced interstitial pneumonia in SOMAscan analysis, from the healthy group (HC) and convalescent group (All-Re). ROC curve analysis was performed and the calculated AUC value was shown. The AUC values of the existing markers KL-6 and SP-D were calculated from the clinical laboratory test values measured at the time of sample collection. DAD indicates diffuse alveolar injury, OP indicates organizing pneumonia, All indicates interstitial pneumonia in general, and Ac indicates acute phase.
（７）ELISA測定系を用いた検証
表１および表２に示したタンパク質のバイオマーカー性能を検証するために、サンドイッチELISAキットを用いて、多数検体の解析を行った。 (7) Verification using ELISA measurement system In order to verify the biomarker performance of the proteins shown in Tables 1 and 2, a large number of samples were analyzed using a sandwich ELISA kit.
表１および表２に示したタンパク質のバイオマーカー性能を検証するために、サンドイッチELISAキットを用いて、多数検体の解析を行った。 (7) Verification using ELISA measurement system In order to verify the biomarker performance of the proteins shown in Tables 1 and 2, a large number of samples were analyzed using a sandwich ELISA kit.
７－１）検体
検体は、SOMAscan解析で用いた症例とは別の新たに収集した症例を追加し、薬剤性間質性肺炎の急性期の検体としては、DADパターンが１８例、OPや過敏性肺炎（HP）パターンが優勢な診断ではあるが、DADパターンの併存も認められるDAD混在型（OP＞DAD、HP＞DAD、HP=DADなど）が９例存在した。これ以降、DADパターンとDAD混在型(DAD mixed)を合わせて、「DAD確認症例」と記した。また、OPパターン30例、NSIPパターン22例、その他のパターン（好酸球性肺炎、過敏性肺炎など）6例、およびこれらの回復期（All-R）55例と健常ボランティア77例、さらに、薬剤性間質性肺炎を発症していない化学療法中の肺がん症例（非発症例）、肺がん症例、特発性間質性肺炎、膠原病肺、COPD、非結核性抗酸菌症、気管支喘息、感染性肺疾患（真菌・細菌性感染症）などを加えて合計401例、またはその一部の血清あるいは血漿検体をELISAキットによる検証に用いた。 7-1) Specimen As the sample, a newly collected case other than the case used in the SOMAscan analysis was added, and as a sample in the acute phase of drug-induced interstitial pneumonia, 18 cases of DAD pattern, OP and hypersensitivity Although the diagnosis was predominantly the pneumonitis (HP) pattern, there were 9 cases of mixed DAD (OP> DAD, HP> DAD, HP = DAD, etc.) in which the DAD pattern was also observed. From this point onward, the DAD pattern and the DAD mixed type are combined and described as "DAD confirmed cases". In addition, OP pattern 30 cases, NSIP pattern 22 cases, other patterns (eosinophilic pneumonitis, hypersensitivity pneumonitis, etc.) in 6 cases, these convalescent (All-R) 55 cases and healthy volunteers 77 cases, and more. Lung cancer cases (non-onset cases) during chemotherapy without drug-induced interstitial pneumonitis, lung cancer cases, idiopathic interstitial pneumonitis, collagen disease lung, COPD, non-tuberculous mycobacteriosis, bronchial asthma, A total of 401 cases, including infectious lung diseases (fungal / bacterial infections), or some serum or plasma samples thereof were used for verification using the ELISA kit.
検体は、SOMAscan解析で用いた症例とは別の新たに収集した症例を追加し、薬剤性間質性肺炎の急性期の検体としては、DADパターンが１８例、OPや過敏性肺炎（HP）パターンが優勢な診断ではあるが、DADパターンの併存も認められるDAD混在型（OP＞DAD、HP＞DAD、HP=DADなど）が９例存在した。これ以降、DADパターンとDAD混在型(DAD mixed)を合わせて、「DAD確認症例」と記した。また、OPパターン30例、NSIPパターン22例、その他のパターン（好酸球性肺炎、過敏性肺炎など）6例、およびこれらの回復期（All-R）55例と健常ボランティア77例、さらに、薬剤性間質性肺炎を発症していない化学療法中の肺がん症例（非発症例）、肺がん症例、特発性間質性肺炎、膠原病肺、COPD、非結核性抗酸菌症、気管支喘息、感染性肺疾患（真菌・細菌性感染症）などを加えて合計401例、またはその一部の血清あるいは血漿検体をELISAキットによる検証に用いた。 7-1) Specimen As the sample, a newly collected case other than the case used in the SOMAscan analysis was added, and as a sample in the acute phase of drug-induced interstitial pneumonia, 18 cases of DAD pattern, OP and hypersensitivity Although the diagnosis was predominantly the pneumonitis (HP) pattern, there were 9 cases of mixed DAD (OP> DAD, HP> DAD, HP = DAD, etc.) in which the DAD pattern was also observed. From this point onward, the DAD pattern and the DAD mixed type are combined and described as "DAD confirmed cases". In addition, OP pattern 30 cases, NSIP pattern 22 cases, other patterns (eosinophilic pneumonitis, hypersensitivity pneumonitis, etc.) in 6 cases, these convalescent (All-R) 55 cases and healthy volunteers 77 cases, and more. Lung cancer cases (non-onset cases) during chemotherapy without drug-induced interstitial pneumonitis, lung cancer cases, idiopathic interstitial pneumonitis, collagen disease lung, COPD, non-tuberculous mycobacteriosis, bronchial asthma, A total of 401 cases, including infectious lung diseases (fungal / bacterial infections), or some serum or plasma samples thereof were used for verification using the ELISA kit.
７－２）ELISAキット
表１および２に示したタンパク質の中で、ストラテフィン、カリスタチン、NPS-PLA2、PARCに関してサンドイッチELISA法による測定を行った。ストラテフィンに関しては、抗ストラテフィンマウスモノクローナル抗体（シグマアルドリッチ社およびメルク社）を用いてELISA測定系を構築し、標準品には大腸菌リコンビナントヒトストラテフィン（NKMAX社）を使用した。NPS-PLA2 (Cayman社)、PARC（R&D社）、カリスタチン（R&D社）の検証には、市販の研究用のELISAキットを用いた。これら自製ELISAおよび市販ELISAキットに関しては、健常人血清・血漿を用いた部分的な分析バリデーションを行い、変動係数（CV値）15%内の精度で測定可能であること、添加回収率が80-120%内であること、希釈直線性および併行精度を事前に確認したうえで、患者検体の測定を行った。既存マーカーのSP-DおよびKL-6は、それぞれヤマサ醤油社製と東ソー社製の臨床検査試薬により測定した。乳酸デヒドロゲナーゼ（LDH）およびC反応性タンパク質（CRP）の値は採血時の臨床検査の値を解析に用いた。 7-2) ELISA Kit Among the proteins shown in Tables 1 and 2, stratefin, calistatin, NPS-PLA2, and PARC were measured by the sandwich ELISA method. Regarding stratefin, an ELISA measurement system was constructed using anti-stratefin mouse monoclonal antibodies (Sigma-Aldrich and Merck), and Escherichia coli recombinant human stratefin (NKMAX) was used as a standard product. Commercially available research ELISA kits were used to verify NPS-PLA2 (Cayman), PARC (R & D), and calistatin (R & D). For these self-made ELISA and commercially available ELISA kits, partial analysis validation using healthy person's serum / plasma is performed, and the measurement can be performed with an accuracy within the coefficient of variation (CV value) of 15%, and the addition recovery rate is 80-. The patient sample was measured after confirming in advance that it was within 120%, dilution linearity, and parallel accuracy. The existing markers SP-D and KL-6 were measured with clinical test reagents manufactured by Yamasa Soy Sauce and Tosoh, respectively. The values of lactate dehydrogenase (LDH) and C-reactive protein (CRP) were analyzed from the values of clinical tests at the time of blood sampling.
表１および２に示したタンパク質の中で、ストラテフィン、カリスタチン、NPS-PLA2、PARCに関してサンドイッチELISA法による測定を行った。ストラテフィンに関しては、抗ストラテフィンマウスモノクローナル抗体（シグマアルドリッチ社およびメルク社）を用いてELISA測定系を構築し、標準品には大腸菌リコンビナントヒトストラテフィン（NKMAX社）を使用した。NPS-PLA2 (Cayman社)、PARC（R&D社）、カリスタチン（R&D社）の検証には、市販の研究用のELISAキットを用いた。これら自製ELISAおよび市販ELISAキットに関しては、健常人血清・血漿を用いた部分的な分析バリデーションを行い、変動係数（CV値）15%内の精度で測定可能であること、添加回収率が80-120%内であること、希釈直線性および併行精度を事前に確認したうえで、患者検体の測定を行った。既存マーカーのSP-DおよびKL-6は、それぞれヤマサ醤油社製と東ソー社製の臨床検査試薬により測定した。乳酸デヒドロゲナーゼ（LDH）およびC反応性タンパク質（CRP）の値は採血時の臨床検査の値を解析に用いた。 7-2) ELISA Kit Among the proteins shown in Tables 1 and 2, stratefin, calistatin, NPS-PLA2, and PARC were measured by the sandwich ELISA method. Regarding stratefin, an ELISA measurement system was constructed using anti-stratefin mouse monoclonal antibodies (Sigma-Aldrich and Merck), and Escherichia coli recombinant human stratefin (NKMAX) was used as a standard product. Commercially available research ELISA kits were used to verify NPS-PLA2 (Cayman), PARC (R & D), and calistatin (R & D). For these self-made ELISA and commercially available ELISA kits, partial analysis validation using healthy person's serum / plasma is performed, and the measurement can be performed with an accuracy within the coefficient of variation (CV value) of 15%, and the addition recovery rate is 80-. The patient sample was measured after confirming in advance that it was within 120%, dilution linearity, and parallel accuracy. The existing markers SP-D and KL-6 were measured with clinical test reagents manufactured by Yamasa Soy Sauce and Tosoh, respectively. The values of lactate dehydrogenase (LDH) and C-reactive protein (CRP) were analyzed from the values of clinical tests at the time of blood sampling.
７－３）ELISAによる測定結果
各ELISAキットで定量した、健常人、薬剤性間質性肺炎発症患者の急性期と回復期、および非発症群における各バイオマーカーの濃度を表３に示した。またそれらに加えて、様々な肺疾患における既存マーカーおよび4種のバイオマーカーの変動パターンをボックスプロットとして図４に示した。いずれのタンパク質も、SOMAscanの結果を再現する形で、健常人に比べて、薬剤性間質性肺炎の急性期で増加（カリスタチンにおいては減少）し、特にストラテフィンとカリスタチンは、DAD確認症例（DADおよびDAD混在型）にて大きな変動を示した（いずれも g = 2.6, p <0.0001）。 7-3) Measurement results by ELISA Table 3 shows the concentrations of each biomarker in the acute and convalescent stages of healthy subjects and patients with drug-induced interstitial pneumonia, and in the non-onset group, as quantified by each ELISA kit. In addition to them, the variation patterns of existing markers and four biomarkers in various lung diseases are shown as box plots in FIG. Both proteins increased in the acute phase of drug-induced interstitial pneumonia (decreased in calistatin) compared to healthy subjects in a form that reproduced the results of SOMAscan, and especially stratefin and calistatin were DAD-confirmed cases (DAD confirmed cases). Large fluctuations were shown in DAD and mixed DAD type) (both g = 2.6, p <0.0001).
各ELISAキットで定量した、健常人、薬剤性間質性肺炎発症患者の急性期と回復期、および非発症群における各バイオマーカーの濃度を表３に示した。またそれらに加えて、様々な肺疾患における既存マーカーおよび4種のバイオマーカーの変動パターンをボックスプロットとして図４に示した。いずれのタンパク質も、SOMAscanの結果を再現する形で、健常人に比べて、薬剤性間質性肺炎の急性期で増加（カリスタチンにおいては減少）し、特にストラテフィンとカリスタチンは、DAD確認症例（DADおよびDAD混在型）にて大きな変動を示した（いずれも g = 2.6, p <0.0001）。 7-3) Measurement results by ELISA Table 3 shows the concentrations of each biomarker in the acute and convalescent stages of healthy subjects and patients with drug-induced interstitial pneumonia, and in the non-onset group, as quantified by each ELISA kit. In addition to them, the variation patterns of existing markers and four biomarkers in various lung diseases are shown as box plots in FIG. Both proteins increased in the acute phase of drug-induced interstitial pneumonia (decreased in calistatin) compared to healthy subjects in a form that reproduced the results of SOMAscan, and especially stratefin and calistatin were DAD-confirmed cases (DAD confirmed cases). Large fluctuations were shown in DAD and mixed DAD type) (both g = 2.6, p <0.0001).
各バイオマーカーの疾患特異性を比較すると、SP-DとKL-6は、間質性肺炎マーカーとして知られるように、DADやDAD混在型だけでなくOPやNSIPの各急性期症例でも高い値を示し、さらに多くの特発性間質性肺炎や膠原病肺症例においても高値傾向を示した（図４）。CRPは、DADとDAD混在型の急性期症例で高値傾向が見られるほか、感染性の肺疾患においても特徴的な高い値を示した。LDHは他に比べて特異性が低い傾向にあった。これらの既存バイオマーカーに対し、ストラテフィンはDADおよびDAD混在型で特徴的な増加が見られるが、他の病型においては軽度の上昇にとどまるという、SOMAscan解析結果と同様の傾向を示した。また、ストラテフィンはCRPとは異なり、感染性肺疾患では高値検体は見られなかった。一方、カリスタチン、NPS-PLA2、PARCについては、薬剤性間質性肺炎全般で変動を示す他、感染性疾患や特発性の間質性肺炎や膠原病肺等でも変動するという特徴がみられた。
Comparing the disease specificity of each biomarker, SP-D and KL-6 are high in each acute phase case of OP and NSIP as well as DAD and DAD mixed type, as known as interstitial pneumonia marker. It also showed a tendency of high value in more cases of idiopathic interstitial pneumonia and collagen disease lung disease (Fig. 4). CRP tended to be high in acute cases with mixed DAD and DAD, and was also characteristically high in infectious lung disease. LDH tended to be less specific than others. In contrast to these existing biomarkers, stratefin showed a characteristic increase in DAD and mixed DAD type, but only a slight increase in other types, showing a tendency similar to the SOMAscan analysis result. In addition, unlike CRP, Stratefin did not show high-value samples in infectious lung disease. On the other hand, calistatin, NPS-PLA2, and PARC showed fluctuations in drug-induced interstitial pneumonia in general, as well as infectious diseases, idiopathic interstitial pneumonia, and collagen disease lungs. ..
７－４）DADに対するバイオマーカーとしての検証
図5および表4は、ELISA測定結果に基づいて、各タンパク質のDADに対するバイオマーカー性能を比較した結果である。DAD急性期（確認）症例（DADおよびDAD混在型）に対する健常人との鑑別性能、総回復期との鑑別性能（病勢判別能）、その他の間質性肺炎病型との鑑別性能（DAD診断性能）および非発症群との鑑別性能について、ROC曲線解析（図５A～D）から算出されるAUC値を表４に示した。 7-4) Verification as a biomarker for DAD Fig. 5 and Table 4 show the results of comparing the biomarker performance of each protein for DAD based on the ELISA measurement results. Differentiation performance from healthy subjects for DAD acute phase (confirmation) cases (mixed DAD and DAD type), differentiation performance from total convalescent phase (disease discrimination ability), and differentiation performance from other interstitial pneumonia disease types (DAD diagnosis) Table 4 shows the AUC values calculated from ROC curve analysis (FIGS. 5A to D) for performance) and differentiation performance from the non-symptomatic group.
図5および表4は、ELISA測定結果に基づいて、各タンパク質のDADに対するバイオマーカー性能を比較した結果である。DAD急性期（確認）症例（DADおよびDAD混在型）に対する健常人との鑑別性能、総回復期との鑑別性能（病勢判別能）、その他の間質性肺炎病型との鑑別性能（DAD診断性能）および非発症群との鑑別性能について、ROC曲線解析（図５A～D）から算出されるAUC値を表４に示した。 7-4) Verification as a biomarker for DAD Fig. 5 and Table 4 show the results of comparing the biomarker performance of each protein for DAD based on the ELISA measurement results. Differentiation performance from healthy subjects for DAD acute phase (confirmation) cases (mixed DAD and DAD type), differentiation performance from total convalescent phase (disease discrimination ability), and differentiation performance from other interstitial pneumonia disease types (DAD diagnosis) Table 4 shows the AUC values calculated from ROC curve analysis (FIGS. 5A to D) for performance) and differentiation performance from the non-symptomatic group.
いずれのタンパク質も、健常人とDAD急性期（確認）症例との識別性能は良好であった (AUC 0.96以上、図５Ａ)。DAD急性期（確認）症例の病勢判別（総回復期群との鑑別、図5Ｂ）においては、ストラテフィン（AUC 0.92）とカリスタチン（同0.91）が最も高い性能を示し、PARC (同0.86)とNPS-PLA2 (同0.83)は、SP-D (0.86)やCRP(0.88)と同等程度の性能であった。DAD診断能（DAD急性期（確認）症例とその他の間質性肺炎病型との鑑別性）については、ストラテフィンが最も優れており (AUC 0.88)、次いでカリスタチン（同0.82）であり、KL-6やSP-D、LDH、CRP（同0.77以下）よりも高かった（表4、図５Ｃ）。また、間質性肺炎の非発症例との鑑別においては、ストラテフィンとカリスタチンが高い性能を示した（図５Ｄ）。
各ROC曲線解析の結果から、各タンパク質のカットオフ値をYouden’s Index［感度-（100-特異度）］が最大となる濃度（感度と特異度が最も高くなる値）を指標にして求めると、健常人とDAD急性期（確認）症例の鑑別においては、ストラテフィン：0.7 ng/mL、カリスタチン：9.2 μg/mL、PARC：35 ng/mL、NPS-PLA2：7.0 ng/mLであった。DADの病勢判別のためのカットオフ値は、ストラテフィン：2.3 ng/mL、カリスタチン：8.6 μg/mL、PARC：32 ng/mL、NPS-PLA2：16 ng/mLであり、DAD診断（他の病型との鑑別）のためのカットオフ値は、ストラテフィン：2.4 ng/mL、カリスタチン：8.6 μg/mL、PARC：57 ng/mL、NPS-PLA2：63 ng/mLであった。非発症例との鑑別のためのカットオフ値は、ストラテフィン：2.4 ng/mL、カリスタチン：11 μg/mL、PARC：35 ng/mL、NPS-PLA2：32 ng/mLであった。
薬剤性間質性肺炎の診断においては感染性肺疾患との鑑別は、治療方針を決定するためにも重要である。DAD急性期（確認）症例と感染性肺疾患（真菌、細菌性）との鑑別性能を比較すると、今回解析したタンパク質の中では、ストラテフィンが最も高いAUCの値（0.99）を示し、SP-D（同 0.92）やKL-6（同 0.89）を上回る性能を示した（図５E）。この時のストラテフィンのカットオフ値（Youden’s Index最大値を示す濃度）は1.4 ng/mLであった。 All proteins had good discrimination performance between healthy subjects and acute DAD (confirmed) cases (AUC 0.96 or higher, FIG. 5A). Stratefin (AUC 0.92) and calistatin (0.91) showed the highest performance in determining the pathology of acute DAD (confirmed) cases (differentiation from the total recovery group, Fig. 5B), with PARC (0.86). NPS-PLA2 (0.83) had the same performance as SP-D (0.86) and CRP (0.88). In terms of DAD diagnostic ability (distinguishability between DAD acute phase (confirmed) cases and other interstitial pneumonia types), stratefin is the best (AUC 0.88), followed by calistatin (0.82), and KL. It was higher than -6, SP-D, LDH, and CRP (0.77 or less) (Table 4, Fig. 5C). In addition, Stratefin and calistatin showed high performance in differentiating from non-onset cases of interstitial pneumonia (Fig. 5D).
From the results of each ROC curve analysis, the cutoff value of each protein is calculated using the concentration at which the Youden's Index [sensitivity- (100-specificity)] is maximized (the value at which the sensitivity and specificity are highest) as an index. In the differentiation between healthy subjects and DAD acute phase (confirmed) cases, Stratefin: 0.7 ng / mL, calistatin: 9.2 μg / mL, PARC: 35 ng / mL, NPS-PLA2: 7.0 ng / mL. The cut-off values for determining the pathology of DAD are Stratefin: 2.3 ng / mL, Calistatin: 8.6 μg / mL, PARC: 32 ng / mL, NPS-PLA2: 16 ng / mL, and DAD diagnosis (others). The cut-off values for differentiation from the disease type) were Stratefin: 2.4 ng / mL, calistatin: 8.6 μg / mL, PARC: 57 ng / mL, and NPS-PLA2: 63 ng / mL. The cut-off values for differentiation from non-symptomatic cases were Stratefin: 2.4 ng / mL, calistatin: 11 μg / mL, PARC: 35 ng / mL, and NPS-PLA2: 32 ng / mL.
In the diagnosis of drug-induced interstitial pneumonia, differentiation from infectious lung disease is also important for determining treatment policy. Comparing the differentiation performance between DAD acute phase (confirmed) cases and infectious lung diseases (fungal, bacterial), stratefin showed the highest AUC value (0.99) among the proteins analyzed this time, and SP- It showed better performance than D (0.92) and KL-6 (0.89) (Fig. 5E). At this time, the cutoff value of Stratefin (concentration indicating the maximum value of Youden's Index) was 1.4 ng / mL.
各ROC曲線解析の結果から、各タンパク質のカットオフ値をYouden’s Index［感度-（100-特異度）］が最大となる濃度（感度と特異度が最も高くなる値）を指標にして求めると、健常人とDAD急性期（確認）症例の鑑別においては、ストラテフィン：0.7 ng/mL、カリスタチン：9.2 μg/mL、PARC：35 ng/mL、NPS-PLA2：7.0 ng/mLであった。DADの病勢判別のためのカットオフ値は、ストラテフィン：2.3 ng/mL、カリスタチン：8.6 μg/mL、PARC：32 ng/mL、NPS-PLA2：16 ng/mLであり、DAD診断（他の病型との鑑別）のためのカットオフ値は、ストラテフィン：2.4 ng/mL、カリスタチン：8.6 μg/mL、PARC：57 ng/mL、NPS-PLA2：63 ng/mLであった。非発症例との鑑別のためのカットオフ値は、ストラテフィン：2.4 ng/mL、カリスタチン：11 μg/mL、PARC：35 ng/mL、NPS-PLA2：32 ng/mLであった。
薬剤性間質性肺炎の診断においては感染性肺疾患との鑑別は、治療方針を決定するためにも重要である。DAD急性期（確認）症例と感染性肺疾患（真菌、細菌性）との鑑別性能を比較すると、今回解析したタンパク質の中では、ストラテフィンが最も高いAUCの値（0.99）を示し、SP-D（同 0.92）やKL-6（同 0.89）を上回る性能を示した（図５E）。この時のストラテフィンのカットオフ値（Youden’s Index最大値を示す濃度）は1.4 ng/mLであった。 All proteins had good discrimination performance between healthy subjects and acute DAD (confirmed) cases (AUC 0.96 or higher, FIG. 5A). Stratefin (AUC 0.92) and calistatin (0.91) showed the highest performance in determining the pathology of acute DAD (confirmed) cases (differentiation from the total recovery group, Fig. 5B), with PARC (0.86). NPS-PLA2 (0.83) had the same performance as SP-D (0.86) and CRP (0.88). In terms of DAD diagnostic ability (distinguishability between DAD acute phase (confirmed) cases and other interstitial pneumonia types), stratefin is the best (AUC 0.88), followed by calistatin (0.82), and KL. It was higher than -6, SP-D, LDH, and CRP (0.77 or less) (Table 4, Fig. 5C). In addition, Stratefin and calistatin showed high performance in differentiating from non-onset cases of interstitial pneumonia (Fig. 5D).
From the results of each ROC curve analysis, the cutoff value of each protein is calculated using the concentration at which the Youden's Index [sensitivity- (100-specificity)] is maximized (the value at which the sensitivity and specificity are highest) as an index. In the differentiation between healthy subjects and DAD acute phase (confirmed) cases, Stratefin: 0.7 ng / mL, calistatin: 9.2 μg / mL, PARC: 35 ng / mL, NPS-PLA2: 7.0 ng / mL. The cut-off values for determining the pathology of DAD are Stratefin: 2.3 ng / mL, Calistatin: 8.6 μg / mL, PARC: 32 ng / mL, NPS-PLA2: 16 ng / mL, and DAD diagnosis (others). The cut-off values for differentiation from the disease type) were Stratefin: 2.4 ng / mL, calistatin: 8.6 μg / mL, PARC: 57 ng / mL, and NPS-PLA2: 63 ng / mL. The cut-off values for differentiation from non-symptomatic cases were Stratefin: 2.4 ng / mL, calistatin: 11 μg / mL, PARC: 35 ng / mL, and NPS-PLA2: 32 ng / mL.
In the diagnosis of drug-induced interstitial pneumonia, differentiation from infectious lung disease is also important for determining treatment policy. Comparing the differentiation performance between DAD acute phase (confirmed) cases and infectious lung diseases (fungal, bacterial), stratefin showed the highest AUC value (0.99) among the proteins analyzed this time, and SP- It showed better performance than D (0.92) and KL-6 (0.89) (Fig. 5E). At this time, the cutoff value of Stratefin (concentration indicating the maximum value of Youden's Index) was 1.4 ng / mL.
７－５）間質性肺炎全般に対するバイオマーカーとしての検証
表５は、間質性肺炎全般に対して、健常人、あるいは回復期との鑑別性能を比較した結果である。ストラテフィン、カリスタチン、NPS-PLA2、PARCは、健常人との高い鑑別性能（AUC 0.86以上）を示した（図６Ａ）。間質性肺炎全般における病勢判別能（総回復期群との比較）は、カリスタチン（AUC 0.76）とPARC（同0.74）が、SP-D（同0.77）と同様であり、KL-6 (同0.62)を上回った（図６Ｂ）。非発症との鑑別では、カリスタチンが（AUC 0.81）が優れていた（図６C）。
各タンパク質のカットオフ値をYouden’s Index最大値となる濃度で求めると、健常人との鑑別においては、ストラテフィン：0.5 ng/mL、カリスタチン：10 μg/mL、PARC：35 ng/mL、NPS-PLA2：6 ng/mLであり、間質性肺炎全般の病勢判別のカットオフ値は、ストラテフィン：1 ng/mL、カリスタチン：10 μg/mL、PARC：33 ng/mL、NPS-PLA2：8 ng/mLであった。非発症例との鑑別のためのカットオフ値は、ストラテフィン：0.56 ng/mL、カリスタチン：11 μg/mL、PARC：36 ng/mL、NPS-PLA2：9 ng/mLであった。
また、臨床上重要とされる間質性肺炎と感染性肺疾患との鑑別においては、ストラテフィン（AUC 0.84）がSP-D（同 0.84）やKL-6 (同 0.83)と同等の性能を示した（図６D）。この時のストラテフィンのカットオフ値（Youden’s Index最大値を示す濃度）は 0.8 ng/mLであった。 7-5) Verification as a biomarker for interstitial pneumonia in general Table 5 shows the results of comparing the discrimination performance of interstitial pneumonia in general with healthy subjects or in the convalescent period. Stratefin, calistatin, NPS-PLA2, and PARC showed high discrimination performance (AUC 0.86 or higher) from healthy subjects (Fig. 6A). The ability to discriminate disease in general interstitial pneumonia (compared to the total recovery phase group) is similar to that of SP-D (0.77) for calistatin (AUC 0.76) and PARC (0.74), and KL-6 (same). It exceeded 0.62) (Fig. 6B). In differentiating from non-onset, calistatin (AUC 0.81) was superior (Fig. 6C).
When the cut-off value of each protein is calculated at the concentration that becomes the maximum value of Youden's Index, in the differentiation from healthy subjects, Stratefin: 0.5 ng / mL, calistatin: 10 μg / mL, PARC: 35 ng / mL, NPS- PLA2: 6 ng / mL, and the cut-off value for disease discrimination of interstitial pneumonia in general is Stratefin: 1 ng / mL, calistatin: 10 μg / mL, PARC: 33 ng / mL, NPS-PLA2: 8 It was ng / mL. The cut-off values for differentiation from non-symptomatic cases were stratefin: 0.56 ng / mL, calistatin: 11 μg / mL, PARC: 36 ng / mL, and NPS-PLA2: 9 ng / mL.
In addition, in the differentiation between clinically important interstitial pneumonia and infectious lung disease, Stratefin (AUC 0.84) has the same performance as SP-D (0.84) and KL-6 (0.83). Shown (Fig. 6D). At this time, the cutoff value of Stratefin (concentration indicating the maximum value of Youden's Index) was 0.8 ng / mL.
表５は、間質性肺炎全般に対して、健常人、あるいは回復期との鑑別性能を比較した結果である。ストラテフィン、カリスタチン、NPS-PLA2、PARCは、健常人との高い鑑別性能（AUC 0.86以上）を示した（図６Ａ）。間質性肺炎全般における病勢判別能（総回復期群との比較）は、カリスタチン（AUC 0.76）とPARC（同0.74）が、SP-D（同0.77）と同様であり、KL-6 (同0.62)を上回った（図６Ｂ）。非発症との鑑別では、カリスタチンが（AUC 0.81）が優れていた（図６C）。
各タンパク質のカットオフ値をYouden’s Index最大値となる濃度で求めると、健常人との鑑別においては、ストラテフィン：0.5 ng/mL、カリスタチン：10 μg/mL、PARC：35 ng/mL、NPS-PLA2：6 ng/mLであり、間質性肺炎全般の病勢判別のカットオフ値は、ストラテフィン：1 ng/mL、カリスタチン：10 μg/mL、PARC：33 ng/mL、NPS-PLA2：8 ng/mLであった。非発症例との鑑別のためのカットオフ値は、ストラテフィン：0.56 ng/mL、カリスタチン：11 μg/mL、PARC：36 ng/mL、NPS-PLA2：9 ng/mLであった。
また、臨床上重要とされる間質性肺炎と感染性肺疾患との鑑別においては、ストラテフィン（AUC 0.84）がSP-D（同 0.84）やKL-6 (同 0.83)と同等の性能を示した（図６D）。この時のストラテフィンのカットオフ値（Youden’s Index最大値を示す濃度）は 0.8 ng/mLであった。 7-5) Verification as a biomarker for interstitial pneumonia in general Table 5 shows the results of comparing the discrimination performance of interstitial pneumonia in general with healthy subjects or in the convalescent period. Stratefin, calistatin, NPS-PLA2, and PARC showed high discrimination performance (AUC 0.86 or higher) from healthy subjects (Fig. 6A). The ability to discriminate disease in general interstitial pneumonia (compared to the total recovery phase group) is similar to that of SP-D (0.77) for calistatin (AUC 0.76) and PARC (0.74), and KL-6 (same). It exceeded 0.62) (Fig. 6B). In differentiating from non-onset, calistatin (AUC 0.81) was superior (Fig. 6C).
When the cut-off value of each protein is calculated at the concentration that becomes the maximum value of Youden's Index, in the differentiation from healthy subjects, Stratefin: 0.5 ng / mL, calistatin: 10 μg / mL, PARC: 35 ng / mL, NPS- PLA2: 6 ng / mL, and the cut-off value for disease discrimination of interstitial pneumonia in general is Stratefin: 1 ng / mL, calistatin: 10 μg / mL, PARC: 33 ng / mL, NPS-PLA2: 8 It was ng / mL. The cut-off values for differentiation from non-symptomatic cases were stratefin: 0.56 ng / mL, calistatin: 11 μg / mL, PARC: 36 ng / mL, and NPS-PLA2: 9 ng / mL.
In addition, in the differentiation between clinically important interstitial pneumonia and infectious lung disease, Stratefin (AUC 0.84) has the same performance as SP-D (0.84) and KL-6 (0.83). Shown (Fig. 6D). At this time, the cutoff value of Stratefin (concentration indicating the maximum value of Youden's Index) was 0.8 ng / mL.
７－６）陽性率の比較
表６は、ストラテフィン、カリスタチン、PARC、NPS-PLA2のそれぞれのカットオフ値を設定し、各間質性肺炎病型や各疾患に対する陽性率について、SP-DやKL-6と比較した結果である。KL-6とSP-Dについては臨床で用いられている基準値（500 U/mLおよび110 ng/mL）を、その他のタンパク質については、DAD急性期（確認）症例と非発症群を比較した際のROC曲線から（図５D）、感度と特異度が最も高くなる濃度をカットオフ値として設定した。
ストラテフィンにおいて、健常群では陽性例（＞2.4 ng/mL）は全く見られない一方で、DAD急性期（確認）症例（DADやDAD混在型）では92%と高い陽性率を示した。対して、OPやNSIPなど他の病型では20-33%と低かったことから、適切なカットオフ値を設定することで、ストラテフィンはDAD特異的な血清診断に有用である可能性があることが示唆された。また、ストラテフィンの総回復期群や非発症群における陽性率（11%、13%）は、KL-6（各々49%、30%）やSP-D（各々42%、27％）よりも低く、感染性肺疾患や非結核性抗酸菌症では陽性は見られなかった。 7-6) Comparison of positive rates Table 6 sets the cutoff values for stratefin, calistatin, PARC, and NPS-PLA2, and SP-D for each interstitial pneumonia type and positive rate for each disease. This is the result of comparison with KL-6. Clinically used reference values (500 U / mL and 110 ng / mL) were compared for KL-6 and SP-D, and DAD acute (confirmed) cases and non-symptomatic groups were compared for other proteins. From the ROC curve (Fig. 5D), the concentration with the highest sensitivity and specificity was set as the cutoff value.
In Stratefin, no positive cases (> 2.4 ng / mL) were found in the healthy group, while a high positive rate of 92% was shown in DAD acute phase (confirmed) cases (DAD and DAD mixed type). In contrast, other disease types such as OP and NSIP were as low as 20-33%, so by setting an appropriate cutoff value, Stratefin may be useful for DAD-specific serodiagnosis. It has been suggested. In addition, the positive rates (11% and 13%) of stratefin in the total recovery phase group and non-onset group were higher than those of KL-6 (49% and 30%, respectively) and SP-D (42% and 27%, respectively). It was low and was not positive for infectious lung disease or nontuberculous mycobacteriosis.
表６は、ストラテフィン、カリスタチン、PARC、NPS-PLA2のそれぞれのカットオフ値を設定し、各間質性肺炎病型や各疾患に対する陽性率について、SP-DやKL-6と比較した結果である。KL-6とSP-Dについては臨床で用いられている基準値（500 U/mLおよび110 ng/mL）を、その他のタンパク質については、DAD急性期（確認）症例と非発症群を比較した際のROC曲線から（図５D）、感度と特異度が最も高くなる濃度をカットオフ値として設定した。
ストラテフィンにおいて、健常群では陽性例（＞2.4 ng/mL）は全く見られない一方で、DAD急性期（確認）症例（DADやDAD混在型）では92%と高い陽性率を示した。対して、OPやNSIPなど他の病型では20-33%と低かったことから、適切なカットオフ値を設定することで、ストラテフィンはDAD特異的な血清診断に有用である可能性があることが示唆された。また、ストラテフィンの総回復期群や非発症群における陽性率（11%、13%）は、KL-6（各々49%、30%）やSP-D（各々42%、27％）よりも低く、感染性肺疾患や非結核性抗酸菌症では陽性は見られなかった。 7-6) Comparison of positive rates Table 6 sets the cutoff values for stratefin, calistatin, PARC, and NPS-PLA2, and SP-D for each interstitial pneumonia type and positive rate for each disease. This is the result of comparison with KL-6. Clinically used reference values (500 U / mL and 110 ng / mL) were compared for KL-6 and SP-D, and DAD acute (confirmed) cases and non-symptomatic groups were compared for other proteins. From the ROC curve (Fig. 5D), the concentration with the highest sensitivity and specificity was set as the cutoff value.
In Stratefin, no positive cases (> 2.4 ng / mL) were found in the healthy group, while a high positive rate of 92% was shown in DAD acute phase (confirmed) cases (DAD and DAD mixed type). In contrast, other disease types such as OP and NSIP were as low as 20-33%, so by setting an appropriate cutoff value, Stratefin may be useful for DAD-specific serodiagnosis. It has been suggested. In addition, the positive rates (11% and 13%) of stratefin in the total recovery phase group and non-onset group were higher than those of KL-6 (49% and 30%, respectively) and SP-D (42% and 27%, respectively). It was low and was not positive for infectious lung disease or nontuberculous mycobacteriosis.
NPS-PLA2もまたDAD優位な陽性率（79%）を示したが、感染性肺疾患でも陽性例が多く存在した（62%）。カリスタチンとPARCは、DAD急性期（確認）症例だけでなく、OPやNSIPでも陽性率が高く、総回復期群の陽性率がKL-6やSP-Dよりも低かった。これは、薬剤性間質性肺炎全般の病勢判別に、カリスタチンとPARCが有用である可能性を示唆する結果である。
NPS-PLA2 also showed a DAD-dominant positive rate (79%), but there were many positive cases of infectious lung disease (62%). Calistatin and PARC had high positive rates not only in acute DAD (confirmed) cases, but also in OP and NSIP, and the positive rate in the total recovery group was lower than that in KL-6 and SP-D. This is a result suggesting that calistatin and PARC may be useful for determining the pathological condition of drug-induced interstitial pneumonia in general.
表７は、KL-6あるいはSP-Dが陰性を示した薬剤性間質性肺炎症例に対して、他のタンパク質が陽性として判別検出できるかどうかを調べた結果である。薬剤性間質性肺炎全体において、急性期であるもののKL-6陰性（500 U/mL以下）だった検体は22例存在し、その中でSP-D陽性だったのが12例（55%）、ストラテフィン陽性だったのが8例（36%）存在した。これらに対し、カリスタチンは陽性（＜11 μg/mL）が19例（86%）と高く、PARCもまた、解析した19例中16例（84%）と高い陽性率を示した。カリスタチンとPARCは、ストラテフィンとは異なり、OPやNSIPに対しても陽性率が高かった。同様にして、SP-D陰性例（110 ng/mL以下）においても、カリスタチンとPARCは高い陽性率（79-81%）を示した。したがって、これらのタンパク質を組み合わせて使用することで、薬剤性間質性肺炎の検出効率がより高まる可能性が示唆された。
Table 7 shows the results of examining whether other proteins can be discriminated and detected as positive in drug-induced interstitial lung disease cases in which KL-6 or SP-D was negative. In all drug-induced interstitial pneumonia, there were 22 specimens that were KL-6 negative (500 U / mL or less) in the acute phase, and 12 of them were SP-D positive (55%). ), There were 8 cases (36%) who were positive for stratefin. In contrast, calistatin was positive (<11 μg / mL) in 19 cases (86%), and PARC was also positive in 16 cases (84%) out of 19 cases analyzed. Unlike Stratefin, calistatin and PARC also had higher positive rates for OP and NSIP. Similarly, in SP-D negative cases (110 ng / mL or less), calistatin and PARC showed a high positive rate (79-81%). Therefore, it was suggested that the combined use of these proteins may increase the detection efficiency of drug-induced interstitial pneumonia.
７－７）特発性間質性肺炎の急性増悪例の検出性能
特発性の間質性肺炎の急性増悪例も、病理組織像はDADである。特発性間質性肺炎の症例を病型ごとに分類して解析すると、図７に示すように、KL-6やSP-Dは、特発性間質性肺炎の急性増悪例（AE-IIPs, 特発性肺線維症（IPF）急性増悪と急性特発性肺炎を含む）で高い値を示すものの、IPFやNSIPなど、他の間質性肺炎病型でも陽性（SP-D ＞110 ng/mL、KL-6 ＞500 U/mL）が多かった。カリスタチンもまた、特発性間質性肺炎全般的に陽性例（カットオフ: ＜11 μg/mL）が多かった。これらに対し、ストラテフィンは、AE-IIPsで高値傾向が見られ、他の病型では陰性（2.4 ng/mL以下）が多かった。したがって、ストラテフィンは、薬剤性だけでなく、特発性の間質性肺炎に対してもDADマーカーとして有用であり、カリスタチンは特発性の間質性肺炎全般に対しても有用であることを示している。 7-7) Detecting performance of acute exacerbations of idiopathic interstitial pneumonia The histopathological image of acute exacerbations of idiopathic interstitial pneumonia is DAD. When the cases of idiopathic interstitial pneumonia are classified and analyzed according to the type of disease, as shown in FIG. 7, KL-6 and SP-D are cases of acute exacerbation of idiopathic interstitial pneumitis (AE-IIPs, High in idiopathic pulmonary fibrosis (IPF) including acute exacerbation and acute idiopathic pneumonia), but positive in other interstitial pneumonia types such as IPF and NSIP (SP-D> 110 ng / mL, KL-6> 500 U / mL) was the most common. Calistatin was also generally positive for idiopathic interstitial pneumonia (cutoff: <11 μg / mL). In contrast, Stratefin tended to be high in AE-IIPs, and was often negative (2.4 ng / mL or less) in other types. Therefore, it was shown that Stratefin is useful as a DAD marker not only for drug-induced but also for idiopathic interstitial pneumonia, and calistatin is also useful for idiopathic interstitial pneumonia in general. ing.
特発性の間質性肺炎の急性増悪例も、病理組織像はDADである。特発性間質性肺炎の症例を病型ごとに分類して解析すると、図７に示すように、KL-6やSP-Dは、特発性間質性肺炎の急性増悪例（AE-IIPs, 特発性肺線維症（IPF）急性増悪と急性特発性肺炎を含む）で高い値を示すものの、IPFやNSIPなど、他の間質性肺炎病型でも陽性（SP-D ＞110 ng/mL、KL-6 ＞500 U/mL）が多かった。カリスタチンもまた、特発性間質性肺炎全般的に陽性例（カットオフ: ＜11 μg/mL）が多かった。これらに対し、ストラテフィンは、AE-IIPsで高値傾向が見られ、他の病型では陰性（2.4 ng/mL以下）が多かった。したがって、ストラテフィンは、薬剤性だけでなく、特発性の間質性肺炎に対してもDADマーカーとして有用であり、カリスタチンは特発性の間質性肺炎全般に対しても有用であることを示している。 7-7) Detecting performance of acute exacerbations of idiopathic interstitial pneumonia The histopathological image of acute exacerbations of idiopathic interstitial pneumonia is DAD. When the cases of idiopathic interstitial pneumonia are classified and analyzed according to the type of disease, as shown in FIG. 7, KL-6 and SP-D are cases of acute exacerbation of idiopathic interstitial pneumitis (AE-IIPs, High in idiopathic pulmonary fibrosis (IPF) including acute exacerbation and acute idiopathic pneumonia), but positive in other interstitial pneumonia types such as IPF and NSIP (SP-D> 110 ng / mL, KL-6> 500 U / mL) was the most common. Calistatin was also generally positive for idiopathic interstitial pneumonia (cutoff: <11 μg / mL). In contrast, Stratefin tended to be high in AE-IIPs, and was often negative (2.4 ng / mL or less) in other types. Therefore, it was shown that Stratefin is useful as a DAD marker not only for drug-induced but also for idiopathic interstitial pneumonia, and calistatin is also useful for idiopathic interstitial pneumonia in general. ing.
７－８）肺機能、炎症パラメータとの相関
図８は、患者の肺機能（血液酸素化能力）や炎症パラメータとストラテフィンとの相関を調べた結果である。血中ストラテフィン濃度は、血中酸素化能力の指標であるPaO2/FiO2比やSpO2/FiO2比と相関を示し（図8A、B）、組織の損傷や炎症の度合いの指標となるLDHやCRPとも相関を示した（図8C、D）。血中ストラテフィン濃度は、組織の損傷や炎症に伴う肺機能の低下に伴って変動することが示唆された。 7-8) Correlation with lung function and inflammatory parameters FIG. 8 shows the results of examining the correlation between the patient's lung function (blood oxygenation ability) and inflammatory parameters and Stratefin. Blood stratefin concentration correlates with PaO 2 / FiO 2 ratio and SpO 2 / FiO 2 ratio, which are indicators of blood oxygenation capacity (Figs. 8A and B), and is an indicator of the degree of tissue damage and inflammation. It also showed a correlation with LDH and CRP (Figs. 8C and D). It was suggested that the blood stratefin concentration fluctuates with the decrease of lung function due to tissue damage and inflammation.
図８は、患者の肺機能（血液酸素化能力）や炎症パラメータとストラテフィンとの相関を調べた結果である。血中ストラテフィン濃度は、血中酸素化能力の指標であるPaO2/FiO2比やSpO2/FiO2比と相関を示し（図8A、B）、組織の損傷や炎症の度合いの指標となるLDHやCRPとも相関を示した（図8C、D）。血中ストラテフィン濃度は、組織の損傷や炎症に伴う肺機能の低下に伴って変動することが示唆された。 7-8) Correlation with lung function and inflammatory parameters FIG. 8 shows the results of examining the correlation between the patient's lung function (blood oxygenation ability) and inflammatory parameters and Stratefin. Blood stratefin concentration correlates with PaO 2 / FiO 2 ratio and SpO 2 / FiO 2 ratio, which are indicators of blood oxygenation capacity (Figs. 8A and B), and is an indicator of the degree of tissue damage and inflammation. It also showed a correlation with LDH and CRP (Figs. 8C and D). It was suggested that the blood stratefin concentration fluctuates with the decrease of lung function due to tissue damage and inflammation.
表３．ELISAにより定量した各被験者群における各バイオマーカーの血中濃度。血中濃度は中央値（範囲）で示す。nは測定した患者検体数、g値とp値は健常群（HC）とDAD確認症例［DAD + DAD混合型］を比較した際の効果量 ヘッジのｇ値およびマンホイットニー検定のp値を示す。DADはびまん性肺胞傷害、OPは器質化肺炎、NSIPは非特異性間質肺炎、Allは間質性肺炎全般、no onsetは非発症、Acは急性期、Rは回復期を示す。
Table 3. Blood concentration of each biomarker in each subject group quantified by ELISA. Blood concentration is indicated by the median (range). n indicates the number of patient samples measured, g and p values indicate the g value of the effect size hedge and the p value of the Mann-Whitney test when comparing the healthy group (HC) and the DAD confirmed case [DAD + DAD mixed type]. .. DAD indicates diffuse alveolar injury, OP indicates organizing pneumonia, NSIP indicates nonspecific interstitial pneumonia, All indicates interstitial pneumonia in general, no onset indicates no onset, Ac indicates acute phase, and R indicates convalescent phase.
表４．ELISAにより検証したDADに対するバイオマーカー性能。DAD確認症例［DAD + DAD混合型］と健常群(HC)、総回復期群(All-R)、DAD以外の間質性肺炎群 (OP + NSIP + other)との識別性能、および非発症例群との鑑別性能を、ROC曲線解析から算出されるAUCの値で比較した。
Table 4. Biomarker performance for DAD verified by ELISA. Distinguishing performance between DAD confirmed cases [DAD + DAD mixed type] and healthy group (HC), total convalescent group (All-R), interstitial pneumonia group other than DAD (OP + NSIP + other), and non-onset The discrimination performance from the example group was compared with the AUC value calculated from the ROC curve analysis.
表5. ELISAにより検証した間質性肺炎全般に対するバイオマーカー性能。薬剤性間質性肺炎全病型（All-Ac）と健常群(HC)、および総回復期群(All-R)の識別性能をROC曲線解析から算出されるAUCの値で比較した。
Table 5. Biomarker performance for all interstitial pneumonia verified by ELISA. The discrimination performance of all drug-induced interstitial pneumonia types (All-Ac), healthy group (HC), and total convalescent group (All-R) was compared by the AUC value calculated from ROC curve analysis.
表６. 各間質性肺炎病型と各疾患群における各バイオマーカーの陽性率の比較
KL-6とSP-Dのカットオフ値は臨床で用いられている基準値（500 U/mLおよび110 ng/mL）を用いた。ストラテフィン、カリスタチン、PARC、NPS-PLA2のカットオフ値は、DAD急性期（確認）症例と非発症群を比較した際のYouden’s Index［感度-（100-特異度）］最大値から設定した値を用いた（段落番号[0092]記載）。
Table 6. Comparison of positive rates of each biomarker in each interstitial pneumonia type and each disease group
For the cutoff values of KL-6 and SP-D, the standard values used clinically (500 U / mL and 110 ng / mL) were used. The cutoff values for stratefin, calistatin, PARC, and NPS-PLA2 are the values set from the maximum Youden's Index [sensitivity- (100-specificity)] when comparing DAD acute phase (confirmed) cases with non-onset groups. Was used (described in paragraph number [0092]).
KL-6とSP-Dのカットオフ値は臨床で用いられている基準値（500 U/mLおよび110 ng/mL）を用いた。ストラテフィン、カリスタチン、PARC、NPS-PLA2のカットオフ値は、DAD急性期（確認）症例と非発症群を比較した際のYouden’s Index［感度-（100-特異度）］最大値から設定した値を用いた（段落番号[0092]記載）。
Table 6. Comparison of positive rates of each biomarker in each interstitial pneumonia type and each disease group
For the cutoff values of KL-6 and SP-D, the standard values used clinically (500 U / mL and 110 ng / mL) were used. The cutoff values for stratefin, calistatin, PARC, and NPS-PLA2 are the values set from the maximum Youden's Index [sensitivity- (100-specificity)] when comparing DAD acute phase (confirmed) cases with non-onset groups. Was used (described in paragraph number [0092]).
表７. KL-6およびSP-D陰性症例における各バイオマーカーの陽性率
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 Table 7. Positive rate of each biomarker in KL-6 and SP-D negative cases
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 Table 7. Positive rate of each biomarker in KL-6 and SP-D negative cases
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
本発明は、体外診断薬、臨床検査などに利用できる。
The present invention can be used for in vitro diagnostic agents, clinical tests, and the like.
Claims (16)
- ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することを含む、びまん性肺胞傷害の検査方法。 Diffuse alveolar expression, including measuring expression in a sample of subject origin, for at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T. How to test for alveolar injury.
- 発現を測定するタンパク質が、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質であり、測定値がびまん性肺胞傷害の病勢診断を補助する請求項１記載の方法。 The protein whose expression is measured is at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T, and the measured value is diffuse alveolar injury. The method according to claim 1, which assists in the diagnosis of the disease.
- 発現を測定するタンパク質が、ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質であり、測定値がびまん性肺胞傷害の特異的診断を補助する請求項１記載の方法。 The protein whose expression is measured is at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T, and the measured value is diffuse alveolar injury. The method according to claim 1, which assists in the specific diagnosis of.
- 発現を測定するタンパク質が、ストラテフィン、シスタチンF、NPS-PLA2、IL-1Ra、ネトリン-1、トロポニンT、又はそれらの組み合わせである請求項３記載の方法。 The method according to claim 3, wherein the protein whose expression is measured is stratefin, cystatin F, NPS-PLA2, IL-1Ra, netrin-1, troponin T, or a combination thereof.
- びまん性肺胞傷害の診断方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c1. b1の測定値に基づき、びまん性肺胞傷害の発症の有無を判定すること
を含む前記方法。 A method for diagnosing diffuse alveolar injury
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and
c1. The method comprising determining the presence or absence of the onset of diffuse alveolar injury based on the measurements of b1. - びまん性肺胞傷害の診断方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c1. b1の測定値に基づき、びまん性肺胞傷害の病勢を判定すること
を含む前記方法。 A method for diagnosing diffuse alveolar injury
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject, and
c1. The method comprising determining the pathology of diffuse alveolar injury based on measurements in b1. - びまん性肺胞傷害の診断及び治療方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c1. b1の測定値に基づき、びまん性肺胞傷害の発症の有無を判定すること、及び
d1. びまん性肺胞傷害を発症している可能性が高いと判定された被験者にびまん性肺胞傷害の治療を施すこと
を含む前記方法。 A method for diagnosing and treating diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
c1. Determine the presence or absence of diffuse alveolar injury based on the measured values of b1 and
d1. The method described above comprising treating a subject determined to have a high probability of developing diffuse alveolar injury with treatment for diffuse alveolar injury. - びまん性肺胞傷害の診断及び治療方法であって、
a1. 被験者から試料を得ること、
b1. ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c1. b1の測定値に基づき、びまん性肺胞傷害の病勢を判定すること、及び
d1. びまん性肺胞傷害の急性期にある可能性が高いと判定された被験者にびまん性肺胞傷害の治療を施すこと
を含む前記方法。 A method for diagnosing and treating diffuse alveolar injury.
a1. Obtaining a sample from a subject,
b1. Measuring the expression of at least one protein selected from the group consisting of Stratefin, NPS-PLA2, Cystatin F, IL-1Ra, Netrin-1 and Troponin T in a sample derived from the subject.
c1. Determining the pathology of diffuse alveolar injury based on b1 measurements, and
d1. The method described above comprising treating a subject determined to be likely to be in the acute phase of diffuse alveolar injury with treatment for diffuse alveolar injury. - ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することを含む、間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の検査方法。 At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6. A method for testing interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia), which comprises measuring the expression of a protein in a sample derived from a subject.
- 発現を測定するタンパク質が、ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質であり、測定値が間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の病勢診断を補助する請求項９記載の方法。 The protein whose expression is measured is from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6. The method of claim 9, wherein the method of claim 9 is at least one protein of choice and the measured values assist in the diagnosis of interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
- 間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c2. b2の測定値に基づき、間質性肺炎の発症の有無を判定すること
を含む前記方法。 A method for diagnosing general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6. To measure the expression of a protein in a sample derived from a subject, and
c2. The method comprising determining the presence or absence of the onset of interstitial pneumonia based on the measurements in b2. - 間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、及び
c2. b2の測定値に基づき、間質性肺炎の病勢を判定すること
を含む前記方法。 A method for diagnosing general interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6. To measure the expression of a protein in a sample derived from a subject, and
c2. The method comprising determining the pathology of interstitial pneumonia based on the measurements in b2. - 間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断及び治療方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c2. b2の測定値に基づき、間質性肺炎の発症の有無を判定すること、及び
d2. 間質性肺炎を発症している可能性が高いと判定された被験者に間質性肺炎の治療を施すこと
を含む前記方法。 A method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic amphidrase 6. To measure the expression of a protein in a sample derived from a subject,
c2. Determine the presence or absence of interstitial pneumonia based on the measured value of b2, and
d2. The method described above comprising treating a subject determined to have interstitial pneumonia with a high probability of developing interstitial pneumonia. - 間質性肺炎全般（びまん性肺胞傷害及びその他の間質性肺炎）の診断及び治療方法であって、
a2. 被験者から試料を得ること、
b2. ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定すること、
c2. b2の測定値に基づき、間質性肺炎の病勢を判定すること、及び
d2. 間質性肺炎の急性期にある可能性が高いと判定された被験者に間質性肺炎の治療を施すこと
を含む前記方法。 A method for diagnosing and treating interstitial pneumonia in general (diffuse alveolar injury and other interstitial pneumonia).
a2. Obtaining a sample from the subject,
b2. At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6. To measure the expression of a protein in a sample derived from a subject,
c2. Judging the pathology of interstitial pneumonia based on the measured values of b2, and
d2. The method comprising treating interstitial pneumonia in a subject determined to be likely to be in the acute phase of interstitial pneumonia. - ストラテフィン、NPS-PLA2、シスタチンF、IL-1Ra、ネトリン-1及びトロポニンTからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することができる試薬を含む、びまん性肺胞傷害の検査のためのキット。 A reagent that can measure the expression of at least one protein selected from the group consisting of stratefin, NPS-PLA2, cystatin F, IL-1Ra, netrin-1 and troponin T in a sample derived from a subject. A kit for testing for diffuse alveolar injury.
- ストラテフィン、カリスタチン、NPS-PLA2、PARC (CCL18)、LD78-beta、Apo-AI、PAPP-A (パパリシン)、補体C3b及びカーボニックアンヒドラーゼ６からなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現を測定することができる試薬を含む、間質性肺炎（びまん性肺胞傷害及びその他の間質性肺炎）の検査のためのキット。 At least one selected from the group consisting of stratefin, calistatin, NPS-PLA2, PARC (CCL18), LD78-beta, Apo-AI, PAPP-A (paparicin), complement C3b and carbonic anhydrase 6. A kit for testing interstitial pneumonia (diffuse alveolar injury and other interstitial pneumonia), which comprises a reagent capable of measuring the expression of a protein in a sample derived from a subject.
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| JP2015500988A (en) * | 2011-12-09 | 2015-01-08 | ミカーティス エヌ．ブイ． | LTBP2 as a biomarker for lung injury |
| JP2016519763A (en) * | 2013-03-15 | 2016-07-07 | インターミューン， インコーポレイテッド | Proteome IPF marker |
| JP2016079170A (en) * | 2014-10-10 | 2016-05-16 | 国立研究開発法人医薬基盤・健康・栄養研究所 | Prevention and treatment of diseases related to cell migration regulation and determination of disease activity and prognosis evaluation of diseases of lung stroma |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023082452A1 (en) * | 2021-11-09 | 2023-05-19 | 广州呼吸健康研究院 | Method for detecting content of kl-6 protein on exosome and application thereof |
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| JP7614477B2 (en) | 2025-01-16 |
| JPWO2021024856A1 (en) | 2021-02-11 |
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