WO2016133324A1 - Method for diagnosing inflammatory respiratory diseases using outer membrane vesicles derived from bacteria - Google Patents

Abstract

The present invention relates to a method for diagnosing inflammatory respiratory diseases using outer membrane vesicles derived from bacteria and, more specifically, to a method for diagnosing asthma, chronic obstructive pulmonary disease, and lung cancer by measuring specific antibodies to outer membrane vesicles derived from bacteria existing in indoor dust or to proteins derived from the outer membrane vesicles. The outer membrane vesicles derived from Staphylococcus spp., Acinetobacter spp., Pseudomonas spp., and Enterobacter spp., which exist in large quantities in dust in the indoor air, may cause inflammatory respiratory diseases, and thus the present invention can be favorably used as a method for diagnosing asthma, chronic obstructive pulmonary disease, and lung cancer, by measuring specific antibodies to outer membrane vesicles/proteins derived from the bacteria in patient samples.

Description

Diagnosis of Respiratory Inflammatory Disease Using Bacteria-derived Extracellular Vesicles

The present invention relates to a method for diagnosing respiratory inflammatory disease using bacterial-derived extracellular vesicles, and more specifically to measuring asthma and chronic obstruction by measuring specific antibodies against bacterial-derived extracellular vesicles or proteins derived from these vesicles in indoor dust. Lung diseases, and methods for diagnosing lung cancer.

Indoor dust includes Bacillus sp . , Staphylococcus aureus , Staphylococcus epidermidis , and Pseudomonas. A variety of bacteria, including Gram-negative and Gram-positive bacteria, exist, including stutzeri , Streptomycete s, Corynebacteriaceae , and Escherichia coli . Substances derived from these bacteria, such as lipopolysaccharide (LPS) or peptidoglycan, are known to induce the production of inflammatory cytokines in immune cells and lung epidermal cells.

Gram-negative bacteria constantly secrete extracellular vesicles (also called outer membrane vesicles). Nanometer-sized extracellular vesicles secreted by bacteria are substances that are secreted to exchange information between cells, which are ultrafine dust. Extracellular vesicles secreted by Gram-negative bacteria are spherical phospholipid bilayers, 20-200 nm in size. The extracellular vesicles have outer membrane proteins that can control the inflammatory response of the host as well as LPS. In addition, gram-positive bacteria have been reported to secrete extracellular vesicles to the outside, and proteome analysis has reported that endoplasmic reticulum contains proteins that induce inflammation. Therefore, inhalation of extracellular vesicles in indoor dust can cause inflammatory reactions in alveolar macrophages as well as airway epithelial cells.

On the other hand, respiratory inflammatory diseases can be largely classified into rhinitis and sinusitis occurring in the upper airway, asthma and bronchitis in the lower respiratory tract, bronchiolitis and emphysema in the small airways. Clinically, the disease causes airway obstruction, asthma is characterized by reversible airway obstruction, there are 350 million people worldwide, about 2.3 million patients in Korea. Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airway obstruction. Chronic obstructive bronchitis, chronic obstructive bronchiolitis, emphysema and the like are the causes of chronic obstructive pulmonary disease. In relation to the causes of inflammatory respiratory diseases, protein antigens (allergens) present in indoor air are known to be important in asthma, and stimulating factors such as smoking are known to be important in the cause of chronic obstructive pulmonary disease.

The fact that further inflamed causing cancer has been raised for a long time, most recently by the Th17 immune response to the derived toxins from the bacteria that inhabit the intestine has been reported that the occurrence of colorectal cancer, also known as Helicobacter pylori that symbiotic above (Helicobacter pylori) Chronic gastritis is caused by bacteria, and it has been reported as an important risk factor of gastric cancer. In relation to the pathogenesis of chronic obstructive pulmonary disease and lung cancer, it is suggested that the two diseases are caused by the same cause factor, and smoking is important as the cause of both diseases. It has been reported that the disease itself is an important risk factor for the development of lung cancer. In addition, the fact that there are a large number of chronic obstructive pulmonary disease and lung cancer occurring in non-smokers can be seen that there are factors that cause these two diseases in addition to smoking.

However, in the development of chronic obstructive pulmonary disease and lung cancer in addition to asthma, the effects of bacteria-derived extracellular vesicles contained in indoor dust and the causes of the above diseases are identified and used to identify diseases derived from extracellular vesicles in indoor dust. No method of diagnosis has been reported.

In order to solve the above problems, the present invention by identifying the bacteria that can act as a causative agent of asthma, chronic obstructive pulmonary disease, lung cancer in bacteria-derived extracellular vesicles isolated from the indoor dust through a metagenome analysis Was completed.

Accordingly, an object of the present invention is to provide a method for diagnosing asthma, chronic obstructive pulmonary disease, and lung cancer using bacterial extracellular vesicles or proteins derived from the endoplasmic reticulum contained in indoor dust.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a method for diagnosing a respiratory inflammatory disease, comprising the following steps.

(A) reacting a patient sample with extracellular vesicles or proteins derived from indoor dust bacteria;

(B) reacting the reactant with an anti-human IgG antibody to determine the amount of IgG antibody in the sample; And

(C) determining the respiratory inflammatory disease when the amount of IgG antibody in the sample is more than doubled compared to the normal person.

In one embodiment, the extracellular vesicles, and proteins Staphylococci (Staphylococcus), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas) and Enterobacter (Enterobacter) derived from one or more bacteria selected from the group consisting of It may be.

In another embodiment, the Staphylococcus aureus is Staphylococcus aureus , the Acinetobacter is Acinetobacter baumannii , and the Pseudomonas aeruginosa is Pseudomonas aeruginosa , May be Enterobacter aerogenes .

In another embodiment of the present invention, the patient sample may be selected from the group consisting of blood and sputum.

In another embodiment of the invention, the IgG antibody may be IgG1 or IgG4.

In another embodiment of the present invention, the extracellular vesicles are characterized in that the average diameter of 10-300 nm.

In another embodiment of the present invention, the respiratory inflammatory disease may be selected from the group consisting of asthma, chronic obstructive pulmonary disease, and lung cancer.

In addition, the present invention provides a diagnostic use of respiratory inflammatory diseases of indoor dust bacteria-derived extracellular vesicles.

Staphylococcus aureus present in a large quantity in dust in the room air (Staphylococcus), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas), and Enterobacter (Enterobacter) in germ cells derived from outside the ER because it can lead to respiratory inflammatory diseases, the present invention It can be usefully used as a method for diagnosing asthma, chronic obstructive pulmonary disease and lung cancer by measuring the antibody-specific extracellular vesicles or specific antibodies to the vesicle-derived proteins in a patient sample.

1 is a result of quantifying the endoplasmic reticulum after separating the extracellular vesicles by collecting indoor dust seasonally.

2 is a result of observing the morphology of the extracellular vesicles separated from the indoor dust seasonally through an electron microscope.

3 is a result of measuring the size of the extracellular vesicles separated from the indoor dust seasonally by dynamic light scattering method.

4 is a result of analyzing the distribution of bacteria and extracellular vesicles separated from the indoor dust seasonally at the phylum level through a metagenomic analysis.

5 is a result of analyzing the distribution of bacteria and extracellular vesicles separated from the indoor dust seasonally at the genus level through a metagenomic analysis.

FIG. 6 shows the distribution and the positive rate of extracellular vesicle-specific IgG antibodies in indoor dust measured from blood and sputum samples of control, asthma, chronic obstructive pulmonary disease (COPD), lung cancer patients (Lung cancer). Is the result.

Figure 7 shows the number of sensitized to each bacteria-derived extracellular vesicles in the control group (Asthma), chronic obstructive pulmonary disease (COPD), lung cancer patients (positive) showed positive extracellular vesicle-specific IgG antibody This figure shows the distribution of.

FIG. 8 shows the number of sensitized bacteria-derived extracellular vesicles in normal, control, asthma, chronic obstructive pulmonary disease (COPD), and lung cancer patients with positive extracellular vesicle-specific IgG1 antibodies. Figure shows the distribution.

FIG. 9 shows the number of sensitized bacteria-derived extracellular vesicles in normal, control, asthma, chronic obstructive pulmonary disease (COPD), and lung cancer patients with positive extracellular vesicle-specific IgG4 antibodies. Figure shows the distribution.

The present invention relates to a method for diagnosing asthma, chronic obstructive pulmonary disease, and lung cancer using a bacterial-derived extracellular vesicle or a protein derived from the endoplasmic reticulum present in indoor dust causing respiratory inflammatory disease.

In the present invention, the characteristics of bacteria-derived extracellular vesicles present in indoor dust causing inflammatory respiratory diseases were evaluated and the causative factors (bacteria) of extracellular vesicles were identified.

In one embodiment of the present invention, the DNA of the extracellular vesicles separated from the indoor dust was separated and then subjected to metagenomic analysis to analyze the distribution of bacteria and bacteria-derived extracellular vesicles. Bacterial Door (phylum level) analysis of the level, if any season indoor dust has accounted for the Gram-negative proteobacteria (Proteobacteria) statement bacteria and Gram-positive bacteria of Firmicutes (Firmicutes) Most doors bacteria. More specifically, in (genus level) at the level when the Gram-negative bacteria in indoor dust proteobacteria (proteobacteria) Pseudomonas (Pseudomonas) belonging to the door, Acinetobacter (Acinetobacter) genus, Enterobacter (Enterobacter), A Le Clerc cyano (Leclercia) in the bacteria, gram-positive when the Staphylococcus (Staphylococcus) in bacteria belonging to the Firmicutes (Firmicutes) gate were analyzed to achieve a state in all seasons (see example 3).

In another embodiment of the present invention, samples from three patients and normal groups suffering from the disease to evaluate the sensitization of bacteria-derived extracellular vesicles present in indoor dust and its association with asthma, chronic obstructive pulmonary disease, and lung cancer The amount of extracellular vesicle-specific IgG antibody was obtained. Correlation analysis revealed that 4.4% of normal subjects, 13.6% of asthmatic patients, 29.3% of patients with chronic obstructive pulmonary disease, and 54.9% of lung cancer patients were sensitized to extracellular vesicles (see Example 4). .

In addition, as a result of evaluating the association between extracellular vesicle sensitization or smoking and multiple diseases through multiple logistic regression analysis, the incidence of asthma was 3.3 times higher than that of non-sensitized individuals when sensitized to extracellular vesicles. Smokers were less likely to develop than non-smokers. In chronic obstructive pulmonary disease, the risk of extracellular vesicle sensitization was increased by 8.0 times and that of smokers was increased by 3.7 times. In the case of lung cancer, the incidence of sensitization to extracellular vesicles was increased by 38.7 times compared with the case without sensitization, and by 2.7 times when smoking. Therefore, it was confirmed that the sensitization to extracellular vesicles has a significantly higher risk than that of smoking and induces the onset of the disease (see Example 5).

In another embodiment of the invention, the above-described one staphylococci (Staphylococcus), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas), Enterobacter each of Staphylococcus aureus for the (Enterobacter) in bacteria isolated from Example 3 ( Staphylococcus aureus ), Acinetobacter extracellular vesicles were isolated from baumannii ), Pseudomonas aeruginosa , and Enterobacter aerogenes , and the extracellular vesicle specific IgG, IgG1, IgG4 antibodies of each group were measured. The association of extracellular vesicle sensitization with Staphylococcus aureus, asthma, chronic obstructive pulmonary disease, and lung cancer was evaluated to increase the risk of developing 3.0, 12.6, and 11.2 times, respectively. Acinetobacter The risk of asthma, chronic obstructive pulmonary disease, and lung cancer was increased by 2.2, 6.3, and 8.0 times, respectively. Pseudomonas aeruginosa ) 6.2 times each when sensitized to extracellular vesicles. The risk of asthma, chronic obstructive pulmonary disease, and lung cancer increased by 4.5 and 17.5 times, and Enterobacter When sensitized to aerogenes- derived extracellular vesicles, the risk of asthma, chronic obstructive pulmonary disease, and lung cancer was increased by 2.4, 2.8, and 8.3 times (see Example 6).

In another embodiment of the present invention, the association between the number of sensitization and asthma, chronic obstructive pulmonary disease, lung cancer in the four bacteria-derived extracellular vesicles. Anti-vesicle-specific IgG antibodies were measured for at least three bacterial-derived vesicles in asthma, chronic obstructive pulmonary disease, and lung cancer patients. For IgG1, all four bacterial-derived vesicles were found in chronic obstructive pulmonary disease and lung cancer patients only. Increased. In the case of IgG4, it was confirmed that two or three bacteria-derived vesicles increased in lung cancer patients (see Example 7).

The term 'metagenome' used in the present invention, also referred to as 'gunoelectric', refers to the sum total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil and animal intestine. It is used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms that are not. In particular, the metagenome does not refer to one genome or genome, but to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species. Technically, rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.

The present invention comprises the steps of reacting a patient sample with extracellular vesicles or proteins derived from the indoor dust bacteria; Reacting the reaction with an anti-human IgG antibody to determine the amount of IgG antibody in the sample; And it provides a method for diagnosing respiratory inflammatory disease comprising the step of determining the respiratory inflammatory disease when the amount of IgG antibody in the sample is increased more than two times compared to the normal person.

As used herein, the term “extracellular vesicles” refers to nanoscale nanovesicles secreted by various bacteria. The extracellular vesicles present in the indoor dust are caused by bacteria living in the room, house dust mites, wheels, pets, humans, etc., or by various kinds of bacteria living in and around structures including structures. The extracellular vesicles may be produced, but the average diameter may be 10-300 nm, but is not limited thereto.

In the present invention, a protein contained in the extracellular vesicles may be used as an antigen instead of the extracellular vesicles, but is not limited thereto.

The cell outside the ER and the ER-derived protein of the present invention are Staphylococcus aureus (Staphylococcus), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas) and Enterobacter (Enterobacter) may be derived from one strain or more kinds selected from the group consisting of Preferably, staphylococcus aureus is Staphylococcus aureus , the acinetobacter is Acinetobacter Baumani ( Acinetobacter) baumannii ), and the pseudomonas is Pseudomonas aeruginosa ), the enterobacter may be Enterobacter aerogenes , but is not limited thereto.

The patient sample of the present invention may be selected from the group consisting of blood and sputum, but is not limited thereto.

The antibody of the present invention may be IgG, IgA, or IgE, IgG antibody may be IgG1 or IgG4, but is not limited thereto.

The respiratory inflammatory disease of the present invention may be selected from the group consisting of asthma, chronic obstructive pulmonary disease, and lung cancer, but is not limited thereto.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example  One. Extracellular  Vesicular Extract

To analyze the characteristics, concentrations, and metagenomics of extracellular vesicles in indoor air, the bed mattresses of two apartments in Seoul by spring, summer, fall, and winter seasons. The experiment was performed to isolate and identify extracellular vesicles by collecting dust.

Specifically, after collecting the dust present in the bed mattress using a vacuum cleaner, the dust present in the filter of the vacuum cleaner was transferred to a clean glass bottle to measure the mass. 5 g of indoor dust was placed in a beaker containing 200 ml of PBS (phosphate-buffered saline) and dissolved at 4 ° C. for 12 hours. Afterwards, the large amount of foreign matter is first filtered out, the filtered solution is divided into high speed centrifuge tubes, and high speed centrifugation is performed at 10,000 xg for 15 minutes at 4 ° C. Performed twice in succession. After passing about 180 mL of the supernatant through a membrane filter having a pore diameter of 0.45 μm, the filtered supernatant was divided into a 70 mL ultracentrifuge tube, and then stored at 4 ° C., 100,000 xg. The ultracentrifugation (ultracetrifugation) was performed for 4 hours at. The supernatant was discarded and the extracellular vesicles were extracted by dissolving the precipitate under the tube with PBS.

As a result, as shown in Figure 1, the dust separated from the two mattress bed mattress in spring, summer, autumn and winter contains 115.6, 83.8, 71.3, 96.1 ㎍ (protein / g dust) extracellular vesicles, respectively It was confirmed.

Example  2. Separated from indoor dust Extracellular  Characterization of endoplasmic reticulum

The extracellular vesicles were observed by transmission electron microscopy (TEM) to evaluate the properties of the extracellular vesicles separated by the method of Example 1. After diluting the extracellular vesicles separated from the bed mattress dust to a concentration of 10 μg / ml, 10 μl was placed on 400-mesh copper grids. After staining with 2% uranyl acetate (uranyl acetate) and blowing water was observed under 100 kV using a JEM1011 microscope (JEOL, Peabody, MA, USA).

As a result, as shown in Figure 2, it was confirmed that the round endoplasmic reticulum contained in the dust separated in the spring, summer, autumn, winter.

Next, the size of the extracellular vesicles separated from the bed mattress dust was measured by dynamic light scattering. After diluting the extracellular endoplasmic reticulum to 10 ㎍ / ㎖ concentration, 1 mL into the cuvette (cuvette) and measured the degree of light scattering at 633 nm wavelength using Zetasizer Nano S (Malvern Instrument Ltd. Worcestershire, UK) (30 X 10 times / sample).

As a result, as shown in Figure 3, the size of the extracellular vesicles separated from the bed mattress dust in spring, summer, autumn and winter was 30-220, 20-100, 30-220 and 50-250 nm, respectively.

Example  3. Germs and germ origin present in indoor dust Extracellular  Endoplasmic reticulum Metage Metagenome analysis

DNA was extracted from bacteria and extracellular vesicles present in the indoor dust and subjected to metagenomic analysis to analyze the distribution of bacteria and bacteria-derived extracellular vesicles at the phylum level and genus level.

Specifically, DNA was extracted from the bacterial and extracellular vesicles present in the indoor dust using a DNA extraction kit (Bioneer Inc., Daejeon, Korea), and the DNA library was prepared according to the GS FLX titanium library prep guide. Subsequently, the production library was quantified using Picogreen assay (Invitrogen, Carlsbad, CA, USA), and emulsion PCR (emulsion PCR; emPCR) using GSFLX titanium emPCR Kit (454 Life Sciences, Basel, Switzerland) for amplification of the production library. It was performed by. The production library was fixed on the beads, mixed with an amplification mix and oil, mixed vigorously in Tissue Lyser II (Qiagen, Limburg, Netherlands) to form “micro-reactors”, and the fluid was dispensed on 96-well plates. PCR amplification reactions were performed. For DNA sequencing of the isolated DNA, the DNA isolated by the above method was amplified by a polymerase chain reaction, and then the bead containing the amplified library was separated through the filter by breaking the emulsion state. Then, only beads having amplified libraries were purified using biotinylated primer A (complementary to adapter A), which can be attached to streptavidin-coated magnetic beads (streptavidin-coated magnetic beads). Next, heat DNA double helix separation was used to create single-stranded template DNA fragments attached to the beads. Subsequently, sequencing primers were attached to the isolated template single-stranded DNA, and the number of beads was measured using a particle counter (Beckman Coulter, Brea, CA, USA). Sequencing was performed using the Genome Sequencer FLX titanium sequencer (454 Life Sciences), and each sample was placed in a portion of a 70-75-mm PicoTiter plate (454 Life Sciences) equipped with an eight lane gasket.

All sequences were compared using the BLASTN and EzTaxon databases. Similar sequences, having a value above 100 bit score and below 1.0 E-value were considered partial 16s rRNA. Non 16S rRNA sequences were found to be less than 1%. Read sequences were classified taxonomically using EzTaxon database. For each sequence reading, the five most similar sequences from the database were extracted from the BLASTN program according to the bit score. In these five sequences, the similarity was measured using the ClustalW program, and the sequence with the highest similarity to the read sequence was used for classification. By classification, 94% of genus and 75% of inquiries were classified according to similarity criteria.

As a result of analyzing the distribution of bacteria and bacteria-derived extracellular vesicles by each sample at the phylum level, as shown in FIG. 4, in the case of bacteria (Bacteria), gram-negative bacteria in all four seasons ( Proteobacteria) ) More than 95% of the bacteria were gram-positive bacteria, followed by Firmicutes and Actinobacteria . When the dust within the extracellular vesicles (EV), an extracellular vesicles derived from the four seasons all Gram-negative bacteria of proteobacteria (Proteobacteria) statement bacteria accounted for more than 90%, Gram-positive bacteria of Firmicutes (Firmicutes) statement bacteria-derived cells Outer vesicles came next.

In addition, the distribution of bacteria and bacteria-derived extracellular vesicles present in the dust was analyzed at the genus level. As a result, as shown in FIG. 5, the bacteria (Bacteria) derived in the case of Pseudomonas (Pseudomonas) genus Acinetobacter (Acinetobacter) belonging to the Proteobacteria door in the case of gram-negative genus Enterobacter (Enterobacter), A Le Clerc cyano (Leclercia) in bacteria is achieved when the main yirueotgo, Gram-positive bacteria in the main all four seasons, Staphylococcus (Staphylococcus) in bacteria belonging to the Firmicutes door in all seasons. When evaluating the dust bacteria-derived extracellular vesicles distribution, also Pseudomonas (Pseudomonas) genus Acinetobacter (Acinetobacter) genus Enterobacter (Enterobacter), A Le Clerc cyano (Leclercia) belonging to the Proteobacteria door in the case of gram-negative bacteria If the bacteria in the yirueotgo a week in all seasons, gram-positive, show that the Staphylococcus aureus (Staphylococcus) in bacteria belonging to the Firmicutes door forming a week in all seasons.

Example  4. Asthma, Chronic Obstructive Pulmonary Disease Of ER-specific IgG Antibodies in Indoor Dust in Patients with Lung Cancer

In order to evaluate the association between bacterial-derived extracellular vesicle sensitization and asthma, chronic obstructive pulmonary disease, and lung cancer in indoor dust, samples were prepared from blood and sputum in four groups. The first group consisted of 294 Asthma patients who had undergone a medical diagnosis based on reversible airway obstruction.The second group had a 1-second expiratory volume / total lung capacity (FEV1 / FVC) after bronchodilator use regardless of smoking history. The study consisted of 242 patients with chronic obstructive pulmonary disease (COPD). The third group consisted of patients diagnosed with lung cancer at Dankook University Hospital from November 2003 to March 2011. The fourth group was the control group. It consists of 90 members of the public. The four groups are shown in detail in Table 1 below.

Subjects Control Asthma COPD Lung cancer Number of subjects 90 294 242 325 Age (years) * 51 ± 10 55 ± 15 66 ± 7 66 ± 9 Male,% 57 42 88 84 Cigarette smoker,% 50 27 85 75

* Mean ± standard deviation

In order to evaluate the sensitization of extracellular vesicles present in indoor dust, vesicle-specific IgG antibodies were measured in samples obtained from the patients and controls. To measure the amount of extracellular vesicle-specific IgG antibodies, 50 ng of extracellular vesicles were placed in each well of a 96-well plate for one day. For quantification of extracellular vesicle-specific IgG antibodies, anti-human IgG antibodies were placed in place of extracellular vesicles. The next day, 5% skim milk powder filled the antibody and extracellular vesicle seating gap, and the obtained sample was reacted by diluting 1000-fold in 5% skim milk powder. Next, the horseradish peroxidase conjugated secondary antibody (Horseradish peroxidase-conjugated anti-IgG) (abcam, Cambridge, UK) was diluted in 5% skim milk solution, and then reacted with an IgG antibody through a microplate reader. The amount was measured. Sensitization to extracellular vesicles in dust was defined as positive when more than 95% of vesicle-specific IgG antibody values present in normal samples.

As a result, as shown in Fig. 6, 4.4% in normal people, 13.6% in asthma patients, 29.3% in patients with chronic obstructive pulmonary disease, 54.9% in lung cancer patients.

Example  5. indoor dust Extracellular  For endoplasmic reticulum Sensitization  asthma, Chronic Obstructive Pulmonary Disease Association of lung cancer

Multiple logistic regression analysis was performed to investigate the sensitization of bacteria-derived extracellular vesicles in indoor dust and their association with asthma, chronic obstructive pulmonary disease and lung cancer. As a result of evaluating the association between sensitization to extracellular vesicles in smoking or indoor dust and asthma, as shown in Table 2 below, the risk of asthma is increased when sensitized to extracellular vesicles in indoor dust compared to non-sensitized persons. On the other hand, smoking increased by 3.3 times compared to nonsmokers, resulting in a decrease in asthma.

Univariable analysis Multivariable analysis odds ratio 95% confidence interval p- value † odds ratio 95% confidence interval p- value † Aging 1.02 1.00-1.04 0.023 1.03 1.01-1.05 0.002 Female gender 1.8 1.12-2.92 0.015 0.7 0.3-1.5 0.352 Cigarette smoking 0.4 0.2-0.6 <0.001 0.3 0.1-0.6 0.001 Anti-dust EV IgG (+) * 3.4 1.2-9.7 0.024 3.3 1.1-10.0 0.039

High serum IgG against dust EVs (extracellular vesicles)

† Linear logistic regression analysis vs. control subjects

In addition, when analyzing the association between sensitization to the endoplasmic reticulum in smoking or indoor dust and chronic obstructive pulmonary disease, as shown in Table 3 below, people who are not sensitized to the occurrence of disease when sensitized to extracellular vesicles in the dust It was 8.0 times higher than that of non-smokers and 3.7 times higher than non-smokers.

Univariable analysis Multivariable analysis odds ratio 95% confidence interval p- value † odds ratio 95% confidence interval p- value † Aging 1.2 1.2-1.3 <0.001 1.2 1.1-1.2 <0.001 Female gender 0.2 0.1-0.3 <0.001 1.0 0.3-2.8 0.93 Cigarette smoking 5.5 3.2-9.5 <0.001 3.7 1.4-10.1 0.01 Anti-dust EV IgG (+) * 8.9 3.2-25.3 <0.001 8.0 2.0-32.5 0.003

High serum IgG against dust EVs (extracellular vesicles)

† Linear logistic regression analysis vs. control subjects

In addition, when evaluating the association between the sensitization of the endoplasmic reticulum in smoking or indoor dust and the occurrence of lung cancer, as shown in Table 4 below, when the extracellular vesicles in the dust are sensitized, compared with a person who is not at risk of developing lung cancer. It was 38.7 times higher and 2.7 times higher than the non-smoker.

Univariable analysis Multivariable analysis odds ratio 95% confidence interval p- value † odds ratio 95% confidence interval p- value † Aging 1.2 1.1-1.2 <0.001 1.2 1.1-1.2 <0.001 Female gender 0.2 0.1-0.4 <0.001 0.7 0.3-1.8 0.45 Cigarette smoking 3.1 1.9-5.0 <0.001 2.7 1.1-7.0 0.03 Anti-dust EV IgG (+) * 26.0 9.3-72.6 <0.001 38.7 10.4-144.3 <0.001

High serum IgG against dust EVs (extracellular vesicles)

† Linear logistic regression analysis vs. control subjects

Example  6. Asthma, Chronic Obstructive Pulmonary Disease In lung cancer patient samples Staphylococcus , Acinetobacter , Pseudomonas , Enterobacter  Bacterial origin Extracellular  Endoplasmic reticulum IgG , IgG1, IgG4  Antibody measurement

6-1. Each bacterium originated Extracellular  Endoplasmic reticulum separation and Extracellular  Endoplasmic reticulum IgG , IgG1 , IgG4 antibody measurement

Example 3 within the Staphylococcus aureus of the meta-house dust in genome analysis (Staphylococcus), and based on the findings that the Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas), Enterobacter (Enterobacter) in germ cells derived from outside the endoplasmic reticulum are large amounts present Specific IgG, IgG1, IgG4 antibodies against extracellular vesicles derived from the bacteria were measured in the four groups described in Example 4, namely, asthma, chronic obstructive pulmonary disease, lung cancer, and blood and sputum of normal persons. .

Bacteria corresponding to each genus, Staphylococcus aureus , Acinetobacter baumannii ), Pseudomonas aeruginosa ), and enterobacter aerogenes ( Enterobacter aerogenes ) were inoculated in a liquid nutrient medium and incubated at 37 ° C. under agitation / constant incubator until the absorbance was 1.5. Thereafter, centrifugation was performed at 4 ° C. at 10,000 × g for 20 minutes using a high-speed centrifuge to separate bacteria and supernatant, and only the supernatant was removed and passed once through a 0.45 μm membrane filter. Next, the supernatant was concentrated to 300 ml with a quickstand (GE healthcare) for concentration for high-speed centrifugation, and then passed once through a membrane filter (0.22 μm). This was divided into ultra high-speed centrifuge tubes (45 Ti, 70 ml, Beckman coulter), and ultra-high centrifugation was performed at 4 ° C. and 100,000 x g for 3 hours. Outer endoplasmic reticulum was isolated.

To measure the amount of extracellular vesicle specific antibodies, ie IgG, IgG1, IgG4, in patient samples, 50 ng of extracellular vesicles isolated from these bacteria were placed in each well of a 96-well plate for one day. Anti-human IgG antibodies, anti-human IgG1 antibodies, and anti-human IgG4 antibodies were deposited instead of the extracellular vesicles for the quantification of extracellular vesicle-specific IgG, IgG1, and IgG4, respectively. The next day, 5% skim milk powder filled the antibody and extracellular vesicle seating gap, and the obtained sample was reacted by diluting 1000-fold in 5% skim milk powder. Next, each secondary antibody (Horseradish peroxidase-conjugated anti-IgG, anti-IgG1, anti-IgG4) (abcam, Cambridge, UK) conjugated with horseradish peroxidase was diluted and reacted with 5% skim milk powder. Then, the amount of each IgG antibody was measured through a microplate reader. Sensitization to each bacterial-derived extracellular vesicles was defined as positive when showing at least 95% of the vesicle-specific IgG, IgG1, IgG4 antibody values present in normal human blood and sputum.

6-2. Staphylococcus aureus ( Staphylococcus aureus Origin Extracellular  Endoplasmic reticulum Sensitization  asthma, Chronic Obstructive Pulmonary Disease Association of lung cancer

Multiple logistic regression analysis was performed to determine the association between Staphylococcus aureus-derived extracellular vesicle sensitization, asthma, chronic obstructive pulmonary disease, and lung cancer.

As a result, as shown in Table 5 below, in assessing the association between sensitization to Staphylococcus aureus-derived extracellular vesicles and asthma, the risk of asthma is 3.0-fold higher than that of non-sensitized individuals. It was. Analysis of the association between sensitization to Staphylococcus aureus-derived extracellular vesicles and chronic obstructive pulmonary disease showed a 12.6-fold increase in the incidence of vesicle sensitization compared to non-sensitized individuals. In addition, analysis of the association between sensitization to extracellular vesicles derived from Staphylococcus aureus and lung cancer showed a 11.2-fold increase in lung cancer incidence compared with sensitized ER.

Asthma COPD Lung cancer odds ratio (95% CI) p- value † odds ratio (95% CI) p- value † odds ratio (95% CI) p- value † Aging 1.0 (1.0-1.0) 0.003 1.2 (1.1-1.3) <0.001 1.2 (1.1-1.2) <0.001 Female gender 0.8 (0.4-1.6) 0.497 0.9 (0.3-2.8) 0.90 0.5 (0.2-1.2) 0.11 Cigarette smoking 0.3 (0.2-0.7) 0.005 5.6 (2.0-15.8) 0.001 2.0 (0.9-4.5) 0.11 Anti-staphylococcal EV IgG (+) * 3.0 (1.0-8.9) 0.048 12.6 (2.7-59.4) 0.001 11.2 (3.1-40.2) <0.001

High serum IgG against dust EVs (extracellular vesicles)

† Linear logistic regression analysis vs. control subjects

6-3. Acinetobacter Baumani ( Acinetobacter baumannii Origin Extracellular  Endoplasmic reticulum and asthma, Chronic Obstructive Pulmonary Disease Association of lung cancer

Multiple logistic regression analysis was performed to determine the association between extracellular vesicle sensitization and asthma, chronic obstructive pulmonary disease, and lung cancer derived from Acinetobacter Baumani.

As a result, as shown in Table 6 below, associating sensitization to acetobacter Baumani bacteria-derived extracellular vesicles and asthma, the risk of developing asthma when sensitized to endoplasmic reticulum, compared to those who are not sensitized It increased 2.2 times. The analysis of the association between sensitization to the extracellular vesicles derived from the Acinetobacter bacterium Baumani and the presence of chronic obstructive pulmonary disease showed a 6.3-fold increase in the incidence of non-sensitized individuals. In addition, analysis of the association between sensitization to extracellular vesicles derived from Acinetobacter Baumani and lung cancer showed a 8.0-fold increase in lung cancer incidence compared to sensitization to vesicles.

Asthma COPD Lung cancer odds ratio (95% CI) p- value † odds ratio (95% CI) p- value † odds ratio (95% CI) p- value † Aging 1.0 (1.0-1.0) 0.004 1.2 (1.1-1.2) <0.001 1.1 (1.1-1.2) <0.001 Female gender 0.7 (0.3-1.5) 0.375 0.7 (0.2-2.1) 0.541 0.5 (0.2-1.2) 0.099 Cigarette smoking 0.3 (0.1-0.6) 0.001 3.3 (1.2-9.2) 0.021 2.0 (0.9-4.6) 0.108 Anti-Acinetobacter EV IgG (+) * 2.2 (0.7-6.7) 0.158 6.3 (1.5-26.7) 0.013 8.0 (2.1-30.2) 0.002

High serum IgG against Acinetobacter EVs (extracellular vesicles)

† Linear logistic regression analysis vs. control subjects

6-4. Pseudomonas aeruginosa ( Pseudomonas aeruginosa Origin Extracellular  Endoplasmic reticulum Sensitization  asthma, Chronic Obstructive Pulmonary Disease Association of lung cancer

Multiple logistic regression analysis was performed to determine the association between Pseudomonas aeruginosa-derived extracellular vesicle sensitization, asthma, chronic obstructive pulmonary disease and lung cancer.

As a result, as shown in Table 7 below, the association between sensitization to Pseudomonas aeruginosa-derived extracellular vesicles and asthma was evaluated. When sensitized to endoplasmic reticulum, the risk of asthma was 6.2 times higher than that of non-sensitized persons. The analysis of the association between sensitization to Pseudomonas aeruginosa-derived extracellular vesicles and chronic obstructive pulmonary disease showed a 4.5-fold increase in the incidence of endoplasmic reticulum. In addition, the analysis of the association between sensitization to Pseudomonas aeruginosa-derived extracellular vesicles and lung cancer showed a 17.5-fold increase in lung cancer incidence when sensitized to endoplasmic reticulum.

Asthma COPD Lung cancer odds ratio (95% CI) p- value † odds ratio (95% CI) p- value † odds ratio (95% CI) p- value † Aging 1.0 (1.0-1.1) 0.003 1.2 (1.1-1.2) <0.001 1.2 (1.1-1.2) <0.001 Female gender 0.8 (0.4-1.7) 0.594 1.0 (0.3-3.0) 0.997 0.5 (0.2-1.3) 0.168 Cigarette smoking 0.3 (0.2-0.7) 0.005 4.3 (1.6-11.8) 0.005 2.0 (0.8-4.7) 0.133 Anti-Pseudomonas EV IgG (+) * 6.2 (2.1-18.0) 0.001 4.5 (1.2-16.6) 0.024 17.5 (4.7-64.5) <0.001

High serum IgG against Pseudomonas EVs (extracellular vesicles)

† Linear logistic regression analysis vs. control subjects

6-5. Enterobacter Aerogenes ( Enterobacter aerogenes Origin Extracellular  Endoplasmic reticulum and asthma, Chronic Obstructive Pulmonary Disease Association of lung cancer

Multiple logistic regression analysis was performed to determine the association between Enterobacter aerogenes-derived extracellular vesicle sensitization and asthma, chronic obstructive pulmonary disease, and lung cancer.

As a result, as shown in Table 8 below, as a result of evaluating the association between Enterobacter aerogenes-derived extracellular vesicles and asthma, the risk of developing asthma when sensitized to the endoplasmic reticulum is compared to those who are not sensitized. 2.4-fold increase. Analysis of the association between Enterobacter aerogenes-derived extracellular vesicles and chronic obstructive pulmonary disease showed a 2.8-fold increase in the incidence of endoplasmic reticulum. In addition, as a result of analyzing the association between Enterobacter aerogenes-derived extracellular vesicles and lung cancer, lung sensitization was 8.3 times higher than the sensitized endoplasmic reticulum.

Asthma COPD Lung cancer odds ratio (95% CI) p- value † odds ratio (95% CI) p- value † odds ratio (95% CI) p- value † Aging 1.0 (1.0-1.0) 0.005 1.2 (1.1-1.2) <0.001 1.2 (1.1-1.2) <0.001 Female gender 0.8 (0.4-1.7) 0.564 1.0 (0.3-3.1) 0.954 0.6 (0.3-1.6) 0.358 Cigarette smoking 0.3 (0.1-0.7) 0.004 4.6 (1.7-12.7) 0.003 2.3 (1.0-5.4) 0.058 Anti-Enterovacter EV IgG (+) * 2.4 (0.8-7.2) 0.122 2.8 (0.8-10.1) 0.112 8.3 (2.3-30.0) 0.001

High serum IgG against Enterovacter EVs (extracellular vesicles)

† Linear logistic regression analysis vs. control subjects

Example  7. Origin of the above four bacteria Extracellular  On the endoplasmic reticulum Sensitization  Asthma, Chronic Association between obstructive pulmonary disease and lung cancer

Four bacteria of Example 6, namely Staphylococcus aureus , Acinetobacter Baumani ( Acinetobacter) baumannii ), Pseudomonas aeruginosa ), Enterobacter The purpose of this study was to determine the relationship between the number of aerogenes ) -derived extracellular vesicles and their sensitivity to disease.

As a result, as shown in FIG. 7, when IgG antibody was measured, there was no increase in IgG antibody in three or more bacterial-derived vesicles among the four bacterial-derived extracellular vesicles, but asthma, chronic obstructive lung disease, And only in lung cancer patients. When IgG1 antibody was increased in all four bacterial-derived endoplasmic reticulum, as shown in FIG. 8, it was not observed in normal and asthmatic patients, but only in patients with chronic obstructive pulmonary disease and lung cancer. In addition, it is shown in Fig. 9 that the specific IgG4 increased in two or three endoplasmic reticulum among the four bacterial-derived endoplasmic reticulum was observed in lung cancer patients.

The description of the present invention set forth above is for illustrative purposes, and one of ordinary skill in the art may understand that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (8)

A method for diagnosing a respiratory inflammatory disease, comprising the following steps: (A) reacting a patient sample with extracellular vesicles or proteins derived from indoor dust bacteria; (B) reacting the reactant with an anti-human IgG antibody to determine the amount of IgG antibody in the sample; And (C) determining the respiratory inflammatory disease when the amount of IgG antibody in the sample is more than doubled compared to the normal person. The method of claim 1, Diagnostic method The extracellular vesicles, and proteins, characterized in staphylococci (Staphylococcus), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas) and Enterobacter that is (Enterobacter) derived from one strain or more kinds selected from the group consisting of, . The method of claim 2, The Staphylococcus aureus is Staphylococcus aureus , and the Acinetobacter is Acinetobacter. baumannii ), and the pseudomonas is Pseudomonas aeruginosa), and the Enterobacter diagnostic method, characterized in that the Enterobacter aero to Ness (Enterobacter aerogenes). The method of claim 1, The patient sample is characterized in that selected from the group consisting of blood and sputum, diagnostic method. The method of claim 1, The IgG antibody is characterized in that the IgG1 or IgG4, diagnostic method. The method of claim 1, The extracellular vesicles are characterized in that the average diameter of 10-300 nm, diagnostic methods. The method of claim 1, The respiratory inflammatory disease is characterized in that selected from the group consisting of asthma, chronic obstructive pulmonary disease, lung cancer. For the diagnosis of respiratory inflammatory diseases of extracellular vesicles derived from indoor dust bacteria.

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