Pneumonia testing methods and pneumonia diagnostic kits

Abstract

This invention provides a pneumonia testing method that can distinguish between interstitial pneumonia and severe lung injury (diffuse alveolar injury). [Solution] Mucin 4 (MUC4) levels are measured, and based on the results, interstitial pneumonia and severe lung injury (diffuse alveolar injury) are determined. The measurement result is the serum concentration of MUC4, and the concentration threshold is set at 600-1000 ng / ml to determine interstitial pneumonia and severe lung injury. Furthermore, non-interstitial lung disease and interstitial lung disease may also be determined. In this case, the concentration threshold is set at 150-400 ng / ml to determine non-interstitial lung disease and interstitial lung disease. Furthermore, any combination of bacterial pneumonia, interstitial pneumonia, and severe lung injury may be determined based on the measurement results. It is also possible to combine this with the results of KL-6 measurements.

Description

Technical Field

[0001] The present invention particularly relates to a pneumonia inspection method and a pneumonia diagnostic kit for determining the type of pneumonia.

Background Art

[0002] Interstitial pneumonia is a general term for diseases mainly characterized by inflammation and fibrosis in the alveolar septum, and its etiology and clinical manifestations are diverse. For the diagnosis of interstitial pneumonia, although there are methods such as bronchoalveolar lavage and lung biopsy (cryobiopsy and VATs biopsy), all of them are invasive and difficult to perform in severely ill patients. In addition, interstitial pneumonia generally shows a clinical course of chronic progression and severe lung disorders with acute exacerbation, in which respiratory failure rapidly progresses within a short period. The pathological image of acute exacerbation is diffuse alveolar damage (DAD), which is severe neutrophilic inflammation. Also, the pathological image of acute respiratory distress syndrome (ARDS), which is a severe lung disorder, is also diffuse alveolar damage, and they are common. The clinical courses of these diseases are also common, and the effect of antibiotics is minimal and the prognosis is poor. Therefore, biomarkers are needed to distinguish severe lung disorders such as acute exacerbation of interstitial pneumonia and ARDS from bacterial pneumonia and chronic interstitial pneumonia.

[0003] Here, KL-6 is cited as a serum biomarker for interstitial pneumonia that has been conventionally used in clinical practice. KL-6 is known to show high values in interstitial pneumonia caused by various etiologies such as idiopathic interstitial pneumonias (IIPs) including idiopathic pulmonary fibrosis, collagen disease-related interstitial pneumonia, and radiation pneumonitis. Conventionally, KL-6 has been shown to be highly useful in the diagnosis of interstitial pneumonia and the evaluation of disease activity. That is, among the biomarkers for interstitial pneumonia, KL-6 has relatively high sensitivity and specificity, and it is said that the value of KL-6 correlates with the prognosis.

[0004] On the other hand, Patent Document 1 describes a method for determining the risk of developing severe lung injury, which includes detecting gene polymorphisms present in the Mucin 4 (MUC4) gene. This technology provides a method and a testing kit for determining the risk of developing severe lung injury caused by the administration of anticancer drugs, etc. It also provides a method for determining the risk of developing side effects of anticancer drug therapy. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2014 / 133055 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] In this context, KL-6, a biomarker conventionally used in pneumonia testing, shows elevated levels in various interstitial pneumonias, making it less useful for differential diagnosis of interstitial pneumonias with different etiologies and pathologies. Specifically, while KL-6 shows elevated levels in ARDS and acute exacerbations of interstitial pneumonia, its specificity is not high. Furthermore, MUC4 in Patent Document 1 was only used to assess the risk of pneumonia as a side effect of anticancer drugs, and its use as a biomarker in patients with interstitial pneumonia was not shown. Therefore, there was a need for a pneumonia testing method that could reflect the severe pathology of interstitial pneumonia and distinguish it from severe lung injury.

[0007] This invention has been made in view of these circumstances and aims to resolve the above-mentioned problems. [Means for solving the problem]

[0008] The present invention provides a pneumonia testing method characterized by measuring Mucin4 (MUC4) and determining interstitial pneumonia and severe lung injury based on the results of the measurement. The present invention provides a pneumonia testing method characterized in that the measurement result is the serum concentration of MUC4, and the concentration threshold is set at 600 to 1000 ng / ml to determine interstitial pneumonia and severe lung injury. The pneumonia testing method of the present invention is characterized by its ability to determine early on the possibility of interstitial pneumonia progressing to severe lung injury when the serum concentration of MUC4 is higher than the threshold. The present invention provides a pneumonia testing method characterized by measuring Mucin4 (MUC4) and determining whether the patient has non-interstitial lung disease or interstitial lung disease based on the results of the measurement. The present invention provides a pneumonia testing method characterized in that the measurement result is the serum concentration of MUC4, and a concentration threshold of 150 to 400 ng / ml is used to determine whether the patient has non-interstitial lung disease or interstitial lung disease. The present invention provides a pneumonia testing method characterized by measuring Mucin4 (MUC4) and determining any combination of bacterial pneumonia, interstitial pneumonia, and severe lung injury based on the results of the measurement. The present invention's pneumonia testing method is characterized by determining the type of pneumonia by combining the results of the measurement for MUC4 and the results of the measurement for KL-6. The pneumonia diagnostic kit of the present invention is characterized by containing a reagent for measuring Mucin4 (MUC4) for the pneumonia testing method described above. [Effects of the Invention]

[0009] According to the present invention, a pneumonia testing method is provided that can distinguish between interstitial pneumonia and severe lung injury by using Mucin 4 (MUC4) as a biomarker, performing measurements related to it, and determining interstitial pneumonia and severe lung injury based on the measurement results. [Brief explanation of the drawing]

[0010] [Figure 1] This graph shows the results of cell experiments that support the embodiments of the present invention. [Figure 2] This is a graph showing the serum concentration of MUC4 according to an embodiment of the present invention. [Figure 3] It is a graph showing the serum concentration of KL-6 according to an embodiment of the present invention. [Figure 4] It is a graph showing a plot of the correlation coefficient between MUC4 and KL-6 according to an embodiment of the present invention. [Figure 5] It is a graph showing the comparison result of the serum concentration of MUC4 for non-interstitial lung disease and interstitial lung disease according to an embodiment of the present invention. [Figure 6] It is a graph showing the comparison result of the serum concentration of KL-6 for non-interstitial lung disease and interstitial lung disease according to an embodiment of the present invention. [Figure 7] It is a graph showing the ROC curve of MUC4 for distinguishing non-interstitial lung disease and interstitial lung disease according to an embodiment of the present invention. [Figure 8] It is a graph showing the comparison result of the serum concentration of MUC4 for the bacterial pneumonia group, chronic interstitial pneumonia, and severe lung injury according to an embodiment of the present invention. [Figure 9] It is a graph showing the comparison result of the serum concentration of KL-6 for the bacterial pneumonia group, chronic interstitial pneumonia, and severe lung injury according to an embodiment of the present invention. [Figure 10] It is a graph showing the ROC curves of KL-6 and MUC4 for distinguishing chronic interstitial pneumonia and severe lung injury (acute exacerbation) according to an embodiment of the present invention. [Figure 11] It is a graph showing the result of examining whether chronic interstitial pneumonia according to an embodiment of the present invention develops severe lung injury (acute exacerbation) early with KL-6 and MUC4.

Mode for Carrying Out the Invention

[0011] <Embodiment> Conventionally, serum KL-6 levels have been used as biomarkers for interstitial pneumonia and diffuse alveolar damage, but their sensitivity and specificity have been low. KL-6 is the sugar chain portion of Mucin 1 (MUC1), a membrane-bound mucin expressed in bronchial epithelial cells and type II alveolar epithelial cells in healthy lungs. Serum KL-6 levels are values measured by detecting the antibody titer against the sugar chain portion of this MUC1 using the ELISA method. Although the mechanism by which KL-6 shows high levels in patients with interstitial pneumonia has not been clarified, mechanisms such as increased permeability and leakage on the alveolar cavity side and the vascular bed side due to the progression of interstitial pneumonia, and overexpression of KL-6 in type II alveolar epithelial cells that are damaged and regenerated due to lung injury are considered.

[0012] In contrast, the present inventors intensively searched for biomarkers specific to interstitial pneumonia and severe lung injury (diffuse alveolar damage). And it has been reported that severe lung injury (diffuse alveolar damage) is more likely to occur in Japanese people compared to other countries such as Europe and the United States. Therefore, it was hypothesized that Japanese people have gene mutations that cause diffuse alveolar damage. For this reason, the genes of diffuse alveolar damage patients and healthy people including people from foreign countries and Japanese people were compared and studied, and attention was focused on Mucin 4 (MUC4) as the causative gene. Therefore, first, a cell experiment using human normal airway epithelial cells was conducted, stimulated with neutrophil elastase, and after 24 hours, it was confirmed that the expression of the mRNAs of MUC1 and MUC4 was enhanced, and the increase in MUC4 was particularly significant. That is, since severe lung injury is severe neutrophil inflammation, it was considered that MUC4 reacts more sensitively to neutrophilic inflammation, that is, severe lung injury (diffuse alveolar damage).

[0013] Therefore, the present inventors repeatedly conducted experiments on MUC4 to confirm whether serum MUC levels (concentrations) are biomarkers for differentiating interstitial pneumonia from healthy people and patients with non-interstitial pneumonia lung diseases. Furthermore, it was confirmed whether MUC4 can be a biomarker for severe lung injury (diffuse alveolar damage) such as ARDS and acute exacerbation of interstitial pneumonia, and the present invention was completed. Hereinafter, the pneumonia examination method and diagnostic kit according to the present embodiment will be described in detail.

[0014] [Testing Method] The interstitial pneumonia testing method according to this embodiment is characterized by measuring Mucin 4 (MUC4) and determining interstitial pneumonia and severe lung injury (diffuse alveolar injury) based on the measurement results.

[0015] In this embodiment, MUC4 is a membrane-bound mucin expressed in the bronchial epithelium, possessing a structure similar to MUC1. Both MUC1 and MUC4 are bound to the cell membrane and possess intracellular and extracellular domains, with the extracellular domain being composed of high molecular weight glycoproteins. However, MUC4 has a larger glycosylation structure than MUC1, and is expressed exclusively in the bronchial epithelium, unlike MUC1, which is expressed in both the alveoli and bronchial epithelium in healthy lungs. Therefore, MUC4 exhibits different dynamics from MUC1.

[0016] Specifically, in this embodiment, the serum concentration of MUC4 may be measured as a biomarker for testing, and the results of this measurement may be used to distinguish between interstitial pneumonia and severe lung injury (diffuse alveolar damage) with a concentration threshold of 600 to 1000 ng / ml. More specifically, as shown in the examples described later, if the concentration threshold is set to 800 ng to 900 ng / ml, it becomes possible to distinguish between interstitial pneumonia and severe lung injury (diffuse alveolar damage) with a specificity of 0.8 or higher and a sensitivity of 0.83 or higher.

[0017] Furthermore, if the serum concentration of MUC4 is higher than the threshold, it is possible to determine early on the possibility of interstitial pneumonia progressing to severe lung injury (diffuse alveolar injury). More specifically, as shown in the examples described later, by setting the threshold higher, such as 800 ng to 900 ng / ml, it is possible to determine whether the patient currently has interstitial pneumonia or severe lung injury (diffuse alveolar injury), as well as to predict and estimate whether the symptoms of interstitial pneumonia will progress to severe lung injury. In this embodiment, "early stage" may refer to the stage in which tests are performed before a diagnosis of pneumonia is made, the initial stage in which pneumonia is diagnosed by general tests, or the stage before invasive tests such as the bronchoalveolar lavage or lung biopsy described above are performed. Furthermore, the threshold values ​​for distinguishing between interstitial pneumonia and severe lung injury (diffuse alveolar injury) may be different from the threshold values ​​for determining the likelihood of symptom progression.

[0018] Here, since MUC4 has an extracellular domain composed of sugar chains similar to MUC1, it can be transferred from the alveolar lumen into the bloodstream, similar to KL-6 / MUC1, and can be detected in serum. Serum MUC4 is presumed to show elevated levels in interstitial pneumonia and diffuse alveolar damage, similar to KL-6. Furthermore, as shown in the examples described later, MUC4 is more specific to diffuse alveolar damage, so by measuring MUC4, it becomes possible to distinguish between interstitial pneumonia and diffuse alveolar damage based on the results of this measurement. In other words, it becomes possible to diagnose interstitial pneumonia and severe lung injury (diffuse alveolar damage) at an early stage. In other words, the pneumonia testing method according to this embodiment can determine with high reliability whether interstitial pneumonia or severe lung injury (diffuse alveolar injury) is present, even at an early stage. This provides important information for determining the treatment plan.

[0019] Furthermore, as a method for examining interstitial pneumonia according to this embodiment, Mucin 4 (MUC4) may be measured to determine whether it is a non-interstitial lung disease or an interstitial lung disease. In this case, bacterial pneumonia may be defined as a "non-interstitial lung disease" in this embodiment. In addition, idiopathic interstitial pneumonia, collagen disease lung, and severe lung injury (diffuse alveolar injury) can be defined as "interstitial lung diseases."

[0020] More specifically, by measuring the serum concentration of MUC4 as a biomarker for testing, and setting the concentration threshold to 150-400 ng / ml, it is possible to distinguish and diagnose non-interstitial lung disease from interstitial lung disease. Even more specifically, as described in the examples below, if the concentration threshold is set to 200-300 ng / ml, it is possible to distinguish and diagnose non-interstitial lung disease with good specificity of 0.9 or higher and sensitivity of 0.9 or higher.

[0021] Furthermore, as a method for testing interstitial pneumonia according to this embodiment, any combination of bacterial pneumonia, interstitial pneumonia, and severe lung injury (diffuse alveolar injury) may be determined based on the results of measurements related to MUC4. In this case, as shown in the examples described later, the p-values ​​for each combination of bacterial pneumonia group versus chronic interstitial pneumonia, bacterial pneumonia versus severe lung injury (diffuse alveolar injury), and chronic interstitial pneumonia versus severe lung injury (diffuse alveolar injury) are 0.001% or less, which is significant, so it is possible to set an appropriate threshold to distinguish and judge each disease.

[0022] More specifically, when testing for statistical significance from measured serum MUC4 concentrations, the concentration is compared to that of a sample from a healthy individual, and a statistical test is performed to determine, for example, whether it is 5% significant (p<0.05). This test can be performed using methods such as the t-test, F-test, or chi-squared test, depending on the amount and nature of the data. If statistical significance is found, the patient can be distinguished and judged as having the type of pneumonia to be diagnosed. Conversely, if statistical significance is not found, the patient can be distinguished and judged as not having the type of pneumonia to be diagnosed. Based on these test results, it is possible to set thresholds to be used during actual examinations. Alternatively, it is possible to diagnose the patient as having a different type of pneumonia by combining it with other indicators.

[0023] Furthermore, as a method for testing interstitial pneumonia according to this embodiment, it is also possible to determine the type of pneumonia by combining the results of measurements related to MUC4 and KL-6.

[0024] As shown in the examples described later, serum samples were collected from patients with interstitial pneumonia and severe lung injury, as well as from sarcoidosis patients and healthy adults as normal controls. When the serum concentrations of KL-6 and MUC4 were measured, the serum concentration of MUC4 correlated with that of KL-6. Furthermore, although the serum concentration of MUC4 was higher in patients with interstitial pneumonia and severe lung injury (diffuse alveolar injury) than in the control group, this did not necessarily coincide with KL-6 in all cases. Therefore, by testing KL-6 and MUC4 in combination, a more specific diagnosis of interstitial pneumonia and severe lung injury (diffuse alveolar injury) becomes possible.

[0025] [Pneumonia diagnostic kit] The pneumonia diagnostic kit according to an embodiment of the present invention is a kit for testing pneumonia and is characterized by containing a reagent for measuring Mucin 4 (MUC4), which is a biomarker for the above-mentioned diagnosis.

[0026] For such MUC4 measurement reagents, for example, when measuring serum concentration, reagents for measuring the concentration (ng / ml) of MUC4 in the patient's serum using methods such as CLEIA or ELISA, as well as reagent-fixed paper, instruments, equipment, and discrimination devices may be prepared. In this case, the color or light intensity may change depending on the concentration, and this color or light intensity may be applied to a threshold to distinguish and determine the type of pneumonia. Furthermore, a configuration is also possible to measure the concentration of MUC4 mRNA in cells collected from patients. In this case, the system may include reagents, instruments, equipment, and devices corresponding to detection methods such as Northern blotting, microarrays, QCM sensor measurement, and real-time PCR. That is, the reagents for testing according to this embodiment include probes and primers for MUC4 detection, various enzymes, buffers, washing solutions, lysis solutions, etc. In addition to these, the system may also include materials and equipment for detecting MUC4 using the methods described above.

[0027] Furthermore, the pneumonia testing kit of this embodiment may include a program for analyzing the diagnostic results, processing the data, and visualizing the results on a computer, as well as a device and system equipped with such a computer. Such a device and system can be developed by those skilled in the art using common techniques and methods. Such a device and system enables high-throughput testing and facilitates patient diagnosis.

[0028] The pneumonia testing kit according to this embodiment allows a person other than a physician to perform the above-described testing method on a sample such as serum or cells obtained from a patient for testing, as a non-medical testing procedure. Based on the results and assessments obtained from this test, a physician may ultimately make a diagnosis of the disease.

[0029] [Medicinal composition for interstitial pneumonia] Based on the experiments in the following examples, the inventors have hypothesized that MUC4 is one of the causative genes for severe lung injury (diffuse alveolar injury). Therefore, it can be understood by those skilled in the art that it is possible to provide a pharmaceutical product (pharmaceutical composition) for interstitial pneumonia using a drug corresponding to the mechanism of action of MUC4. In this case, the pharmaceutical composition for interstitial pneumonia of this embodiment is characterized by comprising a composition that increases or decreases the expression level of genes in the gene pathway related to MUC4. Specifically, the pharmaceutical composition of this embodiment may contain a functional activity modifier for MUC4. This functional activity modifier is a composition that has the effect of regulating the expression of MUC4.

[0030] In this embodiment, the functional activity modifier for MUC4 may be a functional activity inhibitor. This functional activity inhibitor can be any compound that affects pathways related to gene transcription, splicing, and other mRNA maturation, translation, metabolism, and degradation.

[0031] Specifically, the functional activity inhibitor for MUC4 according to this embodiment may be, for example, a peptide containing an antibody, a protein, a nucleic acid, a synthetic polymer such as PNA, or a small molecule compound that targets nucleic acids (nucleotides) such as DNA and RNA, which regulate transcription and modification in the biosynthetic pathway from genes, or proteins such as various enzymes (hereinafter referred to as "target proteins"). Of these, the nucleic acid may be antisense DNA or RNA, siRNA, shRNA, ribozyme, etc.

[0032] [Treatment method] The results obtained by the inspection method according to the embodiment of the present invention can be used for treatment by a physician to diagnose a disease. Furthermore, by obtaining a pneumonia medicine according to the results of the inspection method according to the embodiment of the present invention, a therapeutic effect can be expected. Furthermore, the pharmaceutical composition according to this embodiment can also be used in veterinary medicine to treat animals. These animals are not particularly limited and broadly include mammals that have an analog of MUC4 in their genome. Specifically, for example, they may be rodents such as mice, rats, ferrets, hamsters, guinea pigs, or rabbits, dogs, cats, sheep, pigs, cattle, horses, or non-human transgenic primates. In other words, the interstitial pneumonia drug according to the embodiment of the present invention can be used not only for the treatment of humans, but also for the treatment of various animals, and for promoting the growth of livestock, etc.

[0033] By configuring it as described above, the following effects can be obtained. Traditionally, acute exacerbations of interstitial pneumonia, acute respiratory distress syndrome (ARDS), and severe drug-induced lung injury have been pathologically classified as diffuse lung injury and are considered a group of diseases with a poor prognosis. In these diseases, patients often have a poor condition, and pathological diagnosis has been difficult. In other words, it has been difficult to differentiate these pneumonias from typical bacterial pneumonia or heart failure in the early stages. Specifically, imaging diagnosis of interstitial pneumonia is difficult in the early stages, and diagnosis has been particularly difficult for respiratory physicians other than those specializing in interstitial pneumonia. Furthermore, while KL-6 and SP-D have traditionally been used as biomarkers for interstitial pneumonia and severe lung injury (diffuse alveolar injury), their sensitivity and specificity have not been high. In other words, although KL-6 levels rise slightly in severe conditions such as acute exacerbation of interstitial pneumonia and severe lung injury, and are therefore used as an indicator, in reality, levels often do not rise significantly, making it less useful. For example, since serum KL-6 levels also rise in lung cancer, it was difficult to diagnose interstitial pneumonia or lung injury in lung cancer patients even if their KL-6 levels were high. In recent years, research has been conducted to search for other acute-phase biomarkers besides KL-6, and while IL-6, IL-8, CC16, VEGF, Angiopoietin-2, D-dimer, and ICAM-1 have been reported as biomarkers for severe lung injury, none have been established as biomarkers that strongly reflect the pathophysiology of acute-phase severe lung injury. On the other hand, while MUC4 has long been known to be expressed in the bronchial epithelium of healthy lungs, its role in healthy lungs and its relationship to lung diseases were unknown. For this reason, MUC4 has not been shown to be useful as a biomarker in patients with interstitial pneumonia.

[0034] In contrast, the pneumonia testing method according to this embodiment makes it possible to use MUC4 as a useful biomarker specific to interstitial pneumonia and acute-phase conditions. That is, by using the results of MUC4 measurements as a biomarker in diagnostic tests and disease progression assessments for interstitial pneumonia, it becomes useful in the diagnosis of interstitial pneumonia and severe lung injury (diffuse alveolar injury). In other words, MUC4 is significantly elevated in severe conditions such as acute exacerbation of interstitial pneumonia and severe lung injury, making it useful as a biomarker for interstitial lung disease. In addition, the results of MUC4 measurements can be used as a useful biomarker for the clinical diagnosis of interstitial pneumonia and severe lung injury (diffuse alveolar injury) in routine clinical practice, possessing both high applicability and minimal invasiveness. Furthermore, the results of MUC4 measurements can be used as a biomarker for the early diagnosis and prognosis prediction of severe lung injury (diffuse alveolar injury). Cases with high serum MUC4 concentrations have a poor prognosis, and MUC4 can also be used as a prognostic marker.

[0035] In the above embodiment, an example was described in which various types of pneumonia are distinguished and diagnosed based on the serum concentration of MUC4. However, it is also possible to differentiate and diagnose pneumonia based on the concentration of MUC4 in body fluids other than serum. For example, it is possible to differentiate and diagnose pneumonia based on the concentration of MUC4 mRNA in sample cells during invasive diagnostic procedures.

[0036] Furthermore, the diagnostic method of this embodiment can also be used as a preliminary diagnostic method before diagnosing pneumonia. That is, as mentioned above, in interstitial pneumonia, conventional treatments were sometimes too late. Therefore, early diagnosis of interstitial pneumonia enables accurate early treatment and improves treatment effectiveness.

[0037] Furthermore, as a pharmaceutical composition for interstitial pneumonia, it is also possible to perform gene therapy by targeting the genes or pathways that MUC4 regulates, in relation to the mechanism of action of MUC4.

[0038] Furthermore, the pharmaceutical product according to the embodiment of the present invention can be used in combination with other compositions. The composition of the present invention may be administered, sprayed, applied, etc., simultaneously with other compositions.

[0039] In addition, MUC4 functional activity modifiers can be used for non-pharmaceutical applications. Furthermore, MUC4 activators may be used as functional activity modifiers. This makes it possible to use them in experiments and models to study the mechanism of action in interstitial pneumonia in animals. [Examples]

[0040] Below, the interstitial pneumonia-specific biomarker according to embodiments of the present invention will be described in more detail as examples based on specific experiments. However, these examples are merely illustrative and not limiting.

[0041] [Materials and Methods] (cell culture) NHBE cells purchased from Lonza (Switzerland) were placed in 1 x 10⁶ well plates. 4 Cells were seeded at a density of cells / well and cultured in Pneumacult Ex plus (Stem Cell Technologies), a non-serum medium for bronchial epithelial cell proliferation, under conditions of 5% CO2 and 37°C. After culturing to subconfluence, the medium was changed to a 1:1 mixture of bronchial epithelial cell basal medium (Lonza) and Dulbecco's Modified Eagle's Medium (Life Technologies), in accordance with previous reports showing increased MUC4 expression mediated by neutrophil elastase in NHBE cells. 100 U / ml of penicillin G and 100 μg / ml of streptomycin sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the medium.

[0042] (Addition of neutrophil elastase) Neutrophil elastase was purchased from Elastin Products Company, INC (USA). 0.1 mg of neutrophil elastase, which was stored refrigerated in powder form, was dissolved in 100 μL of double distilled water containing NaOAc (0.05 M) and NaCl (0.1 M) as specified by the manufacturer, aliquoted, and stored at -20°C until use in the experiment. The refrigerated neutrophil elastase solution was diluted with culture medium to prepare culture media containing neutrophil elastase at concentrations of 0 nM, 20 nM, 40 nM, 60 nM, 80 nM, and 100 nM. The culture medium used was a 1:1 mixture of bronchial epithelial cell medium (Lonza) and Dulbecco's Modified Eagle's Medium (Life Technologies Corporation). To ensure consistent conditions regarding buffer influence, buffer was added to each group's culture medium so that it contained the same concentration of buffer as when 100 nM neutrophil elastase was added. Confluent NHBE cells were cultured for 24 hours in a medium containing neutrophil elastase at various concentrations.

[0043] (Real-time PCR) The changes in MUC1 and MUC4 mRNA expression levels induced by neutrophil elastase were quantified using real-time polymerase chain reaction (PCR). Cell lysates and cDNA synthesis were performed using the TaqMan® Gene Expression Cells-to-Ct kit (Thermo Fisher Scientific) according to the manufacturer's instructions. MUC1 (Hs00159357_m1), MUC4 (Hs00366414_m1), and β-actin (Hs99999903_m1) were used as probes for the TaqMan® Gene Expression Assay. Ct values ​​obtained by measuring with QuantStudio™12K Flex real-time PCR systems (Applied Biosystems) were normalized to β-actin, and the changes in MUC1 and MUC4 mRNA expression levels induced by neutrophil elastase and other mediators were quantified.

[0044] (Case inclusion criteria) Control group: Healthy adult volunteers aged 20 to under 80 were recruited as the "healthy individuals" group. Patients with bacterial pneumonia who received treatment at Saitama Medical Center, Jichi Medical University, and who gave their consent were included as the "bacterial pneumonia" group. Patients with idiopathic interstitial pneumonia (IIPs), such as idiopathic pulmonary fibrosis, were classified as the "idiopathic interstitial pneumonia" group, and patients with collagen vascular disease-associated interstitial pneumonia (CVD-IP) were classified as the "collagen disease lung" group. Patients with acute respiratory failure that worsened within one month against a background of idiopathic interstitial pneumonia or collagen disease-related interstitial pneumonia (acute exacerbation of interstitial pneumonia), and patients with acute respiratory distress syndrome (ARDS) who did not have interstitial pneumonia as an underlying disease but presented with widespread ground-glass opacities in both lung fields, experienced acute progression of respiratory failure, and whose other diseases such as heart failure and infections had been ruled out, were included as "severe lung injury."The diagnosis of interstitial pneumonia was made in accordance with the following representative domestic and international guidelines: An Official ATS / ERS / JRS / ALAT Guideline (2018), Interstitial Pneumonia with Autoimmune Features: An ATS / ERS Research Statement (2015), Update of the International Multidisciplinary Classification of the Idiopathic Interstitial Pneumonias: An Official ATS / ERS Statement (2013), Idiopathic Pulmonary Fibrosis: Evidence-based Guidelines for Diagnosis and Management: An Official ATS / ERS / JRS / ALAT Statement (2011), and Idiopathic Interstitial Pneumonia Diagnosis and Treatment Guidelines 2022 Revised 4th Edition (edited by the Japanese Respiratory Society Diffuse Lung Disease Diagnosis and Treatment Guideline Creation Committee), and Interstitial Lung Disease Diagnosis and Treatment Guidelines 2020 (Japanese Respiratory Society / Japanese College of Rheumatology). In accordance with the guidelines, interstitial pneumonia patients who received detailed examination and treatment at our hospital's outpatient and inpatient facilities were included. This study was approved by the Ethics Committee of Saitama Medical Center, Jichi Medical University (Clinical S24-045).

[0045] (Measurement method) Serum KL-6 levels were measured at the Clinical Laboratory Department of Saitama Medical Center, Jichi Medical University, using serum collected as needed. The test reagent used was the Lumipulse Pressed KL-6 kit (manufactured by Sekisui Medical Co., Ltd.), and the levels were measured using a two-step sandwich chemiluminescent enzyme immunoassay (CLEIA method). Serum MUC4 concentrations were measured in the laboratory at Saitama Medical Center, Jichi Medical University, using serum collected at times when clinical needs arose. A commercially available research-use ELISA kit based on the sandwich method, the Human-4 / MUC4 ELISA Kit (Assay Genis, Catalogue Code: HUFI00902), was purchased and used for the measurements. The measurable range was 0.313–20 ng / ml, and serum concentrations were measured using serum samples diluted with sample buffer. Preliminary experiments showed that human serum concentrations were approximately 100–1200 ng / ml, and were diluted 64–128 times.

[0046] (statistical analysis) The correlation between MUC4 and KL-6 was evaluated using the Pearson correlation coefficient. Since the Shapiro-Wilk normality test ruled out a normal distribution between the two groups, the Mann-Whitney U test was used for comparison. A p-value < 0.05 was considered statistically significant.

[0047] 〔result〕 (Cell experiments) First, as a supporting cell experiment (preliminary experiment), human normal airway epithelial cells were cultured, stimulated with neutrophil elastase, and Mucin1 (MUC1) or MUC4 (MUC4) mRNA was measured by real-time PCR 24 hours later.

[0048] Figure 1 shows these results (n=5). Figure 1(a) shows the results for MUC1, and (b) shows the results for MUC4. In both graphs, the horizontal axis represents the concentration of neutrophil elastase, and the vertical axis represents the mRNA expression level (multiple times) compared to the control (no neutrophil elastase stimulation). As a result, both MUC1 and MUC4 mRNA expression was upregulated in response to neutrophilic elastase stimulation, but the increase in MUC4 expression was particularly pronounced. In other words, MUC4 responded more sensitively than MUC1.

[0049] (MUC4 and KL-6 in serum) Next, serum MUC4 and KL-6 levels were measured in 20 control subjects (healthy individuals), 10 individuals with bacterial pneumonia, 27 individuals with autosomal interstitial pneumonia, 15 individuals with collagen disease-related interstitial pneumonia, and 10 individuals with severe lung injury (diffuse alveolar injury). Table 1 below shows the mean, standard deviation, maximum, and minimum values ​​of the results of this measurement.

[0050] [Table 1]

[0051] Figure 2 plots the MUC4 results from Table 1. The horizontal axis represents the groups of healthy individuals (control group), bacterial pneumonia, idiopathic interstitial pneumonia, collagen disease-associated lung disease (collagen disease-associated interstitial pneumonia), and severe lung injury (diffuse alveolar injury), while the vertical axis represents the serum concentration of MUC4 (ng / ml).

[0052] Figure 3 plots the results for KL-6 from Table 1. The horizontal axis represents each group, and the vertical axis represents the serum concentration (U / ml) of KL-6.

[0053] As a result, both MUC4 and KL-6 levels were low in the control group (healthy individuals) and the bacterial pneumonia group. On the other hand, both MUC4 and KL-6 levels were high in the idiopathic interstitial pneumonia group, the collagen disease lung group, and the severe lung injury (diffuse alveolar injury) group.

[0054] Next, we examined the correlation between MUC4 and KL-6. Figure 4 is a plot of the correlation coefficients between MUC4 (ng / ml) and KL-6 (U / ml). As a result, the correlation between MUC4 and KL-6 was moderate, with a Pearson correlation coefficient of 0.724 (95%: 0.60~0.81, p=1.496(x)10 -14 The result was <0.05).

[0055] (Diagnosis of non-interstitial lung disease and interstitial lung disease) Furthermore, the serum concentrations of MUC4 and KL-6 were compared between the "non-interstitial lung disease" group (healthy individuals + bacterial pneumonia group) and the "interstitial lung disease" group (idiopathic interstitial pneumonia group + collagen disease lung group + severe lung injury (diffuse alveolar injury) group).

[0056] Figure 5 shows a comparison of MUC4 (ng / ml) levels between the "non-interstitial lung disease" group and the "interstitial lung disease" group. p-value = 2.45e -13 (***)

[0057] Figure 6 similarly compares KL-6 (U / ml) levels in the "non-interstitial lung disease" group and the "interstitial lung disease" group. The p-value is also 6.17e. -14 (***)

[0058] As a result, both MUC4 and KL-6 differed between the "non-interstitial lung disease" group and the "interstitial lung disease" group, and these differences were statistically significant.

[0059] Figure 7 shows the threshold for distinguishing between the "non-interstitial lung disease" group and the "interstitial lung disease" group, examined using ROC curves. The horizontal axis represents specificity (1-Specificity), and the vertical axis represents sensitivity.

[0060] Analysis using the Youden method showed that interstitial lung disease could be well distinguished with a specificity of 0.962 and a sensitivity of 1.00 when a serum concentration of MUC4 of 237.18 ng / ml was used as the threshold.

[0061] (Diagnosis of bacterial pneumonia, chronic interstitial pneumonia, and severe lung injury) Next, we examined whether we could distinguish between the bacterial pneumonia group, the chronic interstitial pneumonia (idiopathic interstitial pneumonia + collagen disease lung) group, and the severe lung injury (diffuse alveolar injury) group.

[0062] Figure 8 shows the results of comparing the bacterial pneumonia group, the chronic interstitial pneumonia group, and the severe lung injury (diffuse alveolar injury) group in MUC4. The vertical axis shows the serum concentration (ng / ml) of each MUC4.

[0063] As a result, in MUC4, the p-value for bacterial pneumonia versus chronic interstitial pneumonia was 4.153e. -07 The results were as follows: The p-value for bacterial pneumonia versus severe lung injury was 0.00054. The p-value for chronic interstitial pneumonia versus severe lung injury was 0.00053. Thus, all of these findings were significant, and MUC4 was able to distinguish between the bacterial pneumonia group, the chronic interstitial pneumonia group, and the severe lung injury (diffuse alveolar injury) group.

[0064] Figure 9 shows the results of comparing KL-6 in the bacterial pneumonia group, the chronic interstitial pneumonia group, and the severe lung injury (diffuse alveolar injury) group. The vertical axis shows the serum concentration (U / ml) of MUC4 in each group.

[0065] As a result, in KL-6, the p-value for bacterial pneumonia versus chronic interstitial pneumonia was 3.8e. -10 The p-value for bacterial pneumonia versus severe lung injury was 3.2e. -05 The p-value for chronic interstitial pneumonia versus severe lung injury was 0.038. In other words, in the KL-6 group, a significant difference was observed between chronic interstitial pneumonia and severe lung injury (diffuse alveolar injury) at p<0.05, but not at p<0.01, suggesting that the difference between the two groups was small.

[0066] Figure 10 shows the threshold values ​​used to distinguish between chronic interstitial pneumonia (interstitial pneumonia) and severe lung injury (acute exacerbation, diffuse alveolar injury) for MUC4 and KL-6, obtained using ROC curves. The horizontal axis represents specificity (1-Specificity), and the vertical axis represents sensitivity.

[0067] The AUC of MUC4 was 0.89, while the AUC of KL-6 was 0.75. Therefore, MUC4 is considered a better marker than KL-6 for differentiating between (chronic) interstitial lung disease and severe lung injury. As a result, with a threshold of 811.52 ng / ml for MUC4, differentiation was possible with a specificity of 0.800 and a sensitivity of 0.833.

[0068] Figure 11 plots KL-6 and MUC4 levels in (chronic) interstitial pneumonia, with thresholds of 1100 U / ml for KL-6 and 800 ng / ml for MUC4, shown as solid and dashed lines. Although the observation period was short (less than one year), severe lung injury (acute exacerbation) developed in 2 out of 8 cases with high MUC4 levels and in 2 out of 20 cases with high KL-6 levels. In other words, in cases of (chronic) interstitial pneumonia, high MUC4 levels may allow for early detection and prediction of severe lung injury.

[0069] (Conclusion) MUC4 proved to be a superior marker for differentiating non-interstitial lung disease from interstitial lung disease compared to KL-6. Specifically, as shown in the ROC curve above, by setting a suitable threshold of 200-300 ng / ml, MUC4 could differentiate and diagnose non-interstitial lung disease from interstitial lung disease. Furthermore, it was considered difficult to distinguish between (chronic) interstitial lung disease and severe lung injury using KL-6. In contrast, as shown in the ROC curve above, MUC4 can differentiate and diagnose (distinguish) between (chronic) interstitial lung disease and severe lung injury, with a suitable threshold of 800-900 ng / ml.

[0070] Furthermore, it is believed that by using serum concentrations of MUC4 and KL-6 in combination, it will be possible to more reliably differentiate between bacterial pneumonia, chronic interstitial pneumonia, and severe lung injury, and utilize this information for diagnosis.

[0071] It goes without saying that the configuration and operation of the above embodiment are examples and can be modified as appropriate without departing from the spirit of the present invention. [Industrial applicability]

[0072] According to the present invention, by using MUC4 as an interstitial pneumonia-specific biomarker, it is expected that pharmaceuticals for the early diagnosis and treatment of interstitial pneumonia can be provided, and that this is industrially applicable.

Claims

1. Measurements were taken regarding Mucin 4 (MUC4). Based on the results of the above measurements, interstitial pneumonia and severe lung injury are diagnosed. A pneumonia testing method characterized by the following features.

2. The result of the above measurement is the serum concentration of MUC4. Interstitial pneumonia and severe lung injury are distinguished using a concentration threshold of 600-1000 ng / ml. The pneumonia testing method according to feature 1.

3. If the serum concentration of MUC4 is higher than the threshold, the possibility of interstitial pneumonia progressing to severe lung injury is determined early. The pneumonia testing method according to feature 2.

4. Measurements were taken regarding Mucin 4 (MUC4). Based on the results of the above measurements, a distinction is made between non-interstitial lung disease and interstitial lung disease. A pneumonia testing method characterized by the following features.

5. The result of the above measurement is the serum concentration of MUC4. The concentration threshold is set at 150–400 ng / ml to distinguish between non-interstitial lung disease and interstitial lung disease. The pneumonia testing method according to feature 4.

6. Measurements were taken regarding Mucin 4 (MUC4). Based on the results of the aforementioned measurements, a determination is made regarding any combination of bacterial pneumonia, interstitial pneumonia, and severe lung injury. A pneumonia testing method characterized by the following features.

7. The measurement results relating to the MUC4 described in any one of claims 1 to 6, The type of pneumonia is determined by combining the results of the KL-6 measurement with other relevant data. A pneumonia testing method characterized by the following features.

8. The present invention includes a reagent for performing measurements related to Mucin 4 (MUC4) for the pneumonia testing method described in any one of claims 1 to 6. A pneumonia diagnostic kit characterized by the following features.

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