WO2016068584A1 - Marker for diagnosing asthma after having bronchiolitis, and use thereof - Google Patents

Marker for diagnosing asthma after having bronchiolitis, and use thereof Download PDF

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Publication number
WO2016068584A1
WO2016068584A1 PCT/KR2015/011391 KR2015011391W WO2016068584A1 WO 2016068584 A1 WO2016068584 A1 WO 2016068584A1 KR 2015011391 W KR2015011391 W KR 2015011391W WO 2016068584 A1 WO2016068584 A1 WO 2016068584A1
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edn
months
wheezing
capillary bronchitis
level
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PCT/KR2015/011391
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French (fr)
Korean (ko)
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김창근
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인제대학교 산학협력단
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Priority to JP2017522176A priority Critical patent/JP6420475B2/en
Priority to CN201580057894.0A priority patent/CN107109476A/en
Publication of WO2016068584A1 publication Critical patent/WO2016068584A1/en

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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD

Definitions

  • the present invention relates to the use of eosinophil-derived neurotoxin (EDN) as a marker for asthma diagnosis after capillary bronchitis.
  • EDN eosinophil-derived neurotoxin
  • Capillary bronchitis is an infectious disease that occurs in the bronchioles, the smallest branch of the respiratory tract.
  • the respiratory tract begins from the nose and branches through the upper bronchus into smaller and smaller bronchus branches that reach the alveoli.
  • the smallest bronchial branch just above the alveoli is called the bronchiole, and the bronchiole serves to deliver air directly to the alveoli, which receives it and supplies oxygen to the blood.
  • Capillary bronchitis in the bronchus is mainly caused by a viral infection, which causes the bronchial inflammatory response, which causes the bronchial mucosa to swell and secrete, resulting in narrowing of bronchioles, which causes impaired oxygen supply to the alveoli. Eventually, systemic hypoxia occurs.
  • Capillary bronchitis is a disease caused by inflammation of the lower respiratory tract due to lower respiratory tract infections, and mainly occurs in infants before 2 years old.
  • the main symptoms of bronchiolitis are coughing and wheezing, and viral infections are the most common causes of respiratory syncytial virus (RSV), and other parainfluenza viruses, influenza viruses, rhinoviruses, adenoviruses, and measles. Viruses and mycoplasmas can also cause this.
  • RSV respiratory syncytial virus
  • RSV respiratory syncytial virus
  • RSV infection symptoms caused by RSV infection vary according to the age of the child, and children usually have mild upper respiratory tract infections, but in infants under 2 years of age, severe lower respiratory tract disease often causes acute capillary disease in infants. 50-90% of bronchitis and pneumonia are caused by RSV.
  • Acute capillary bronchitis caused by RSV is one of the most important causes of respiratory failure in infants.
  • the mortality rate is as low as 0.5-1.5%, but premature infants, neuromuscular disease, chronic respiratory disease, congenital heart disease, or reduced immune function. High-risk patients in the group have a much higher mortality rate. Immunity obtained from natural infections is insufficient, and reinfection often occurs within one to two years after an actual RSV infection.
  • the first symptoms in infants are mainly occlusive or runny nose. Over the next 1-3 days, the infection progresses to the lower respiratory tract and coughing begins with acute laryngitis, bronchitis, capillary bronchitis, pneumonia, or a combination of these symptoms. The symptoms worsen and last about 10 days, then gradually recover. Fever is most often mild, with a maximum body temperature of around 38 ° C and may not have fever, but very young infants infected with RSV may develop life-threatening apnea.
  • Capillary bronchitis caused by RSV causes bronchial hypersensitivity and develops in many cases, such as repeated wheezing or asthma in infancy. Severe severity, atopy findings, and environmental factors may be involved in the cause of these repeated wheezing, but the cause is not yet known.
  • the present inventors examined the degree of symptoms such as asthma, wheezing, dyspnea and cough in infants with capillary bronchitis by examining the correlation with the degree of eosinophil-derived neurotoxin (EDN) expression.
  • EDN eosinophil-derived neurotoxin
  • An object of the present invention is to provide a method for measuring the level of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis to provide information necessary for asthma or wheeze diagnosis after capillary bronchitis will be.
  • EDN eosinophil-derived neurotoxin
  • Another object of the present invention is for diagnosing recurrent wheezing after capillary bronchitis, including an agent for measuring the concentration of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis.
  • EDN eosinophil-derived neurotoxin
  • the present invention to solve the above problems,
  • the present invention provides a method for measuring the level of eosinophil-derived neurotoxin (EDN) 3 to 5 months after capillary bronchitis to provide information necessary for asthma or wheezing diagnosis.
  • EDN eosinophil-derived neurotoxin
  • the level of EDN may be the concentration of EDN in serum or sputum.
  • the capillary bronchitis is respiratory syncytial virus (RSV), metapneumovirus, adenovirus, parainfluenza virus, influenza virus, rhinovirus, adenovirus, measles virus and mycoplasma It may be caused by the infection of harmful bacteria selected from the group consisting of.
  • capillary bronchitis when capillary bronchitis is caused by infection with respiratory syncytial virus (RSV), eosinophil-derived neurotoxin (EDN) at 4 months of RSV capillary bronchitis At levels above 45 ng / ml, relapsed wheeze has a positive predictive value (PPV) of 65%, a negative predictive value (NPV) of 80%, a sensitivity of 70% and specificity ) May have a 72% value.
  • RSV respiratory syncytial virus
  • EDN eosinophil-derived neurotoxin
  • eosinophil-derived neurotoxin EDN
  • NPV negative predictive value
  • specificity specificity
  • PPV positive predictive value
  • NPV negative predictive value
  • the method may be to measure the serum EDN level of infants 1 month to 24 months of age.
  • the EDN level when the EDN level is 100ng / ml or more in the natural course of RSV capillary bronchitis, there is a 75% chance of recurring asthma or wheezing at least two times later. If the EDN level is 80ng / ml or higher in the natural process, there is an 80% chance of recurring asthma or wheezing at least 3 times later, and if the EDN level is 130ng / ml or higher in the natural course of mycoplasma, recurrence is more than 3 times later. There may be a 83% chance of asthma or wheezing.
  • the level of eosinophil-derived neurotoxin (EDN) at 4 months after capillary bronchitis is 70ng / ml If abnormal, regressive wheeze had positive predictive value (PPV) of 73%, negative predictive value (NPV) of 87%, sensitivity of 78% and specificity of 70%.
  • PV positive predictive value
  • NPV negative predictive value
  • the present invention also provides a kit for diagnosing recurrent wheezing after capillary bronchitis, comprising an agent for measuring the concentration of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis. to provide.
  • EDN eosinophil-derived neurotoxin
  • the agent may be a primer, probe or antibody that can specifically bind to EDN.
  • the present invention analyzes the significant correlation between eosinophil-derived neurotoxin (EDN) levels and recurrent asthma or wheeze after capillary bronchitis, thereby asthma or wheeze in infants and children with capillary bronchitis It can be used as a marker for predicting and verifying the prognosis of recurrence, and by preventing the EDN level according to the type of bacteria causing capillary bronchitis, it is a preventive medicine that can continuously manage and manage recurrent asthma or wheezing through a more effective and accurate diagnosis. And therapeutic drug development.
  • EDN eosinophil-derived neurotoxin
  • Diagnosis means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to confirm the recurrence and progression of wheezing and asthma after capillary bronchitis.
  • Diagnostic markers, diagnostic markers or diagnostic markers is a substance capable of diagnosing a diseased cell according to the present invention from normal cells, a polypeptide showing an increase in disease cells compared to normal cells Or organic biomolecules such as nucleic acids (eg mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, polysaccharides, etc.).
  • nucleic acids eg mRNA
  • lipids lipids
  • glycolipids glycoproteins
  • sugars monosaccharides, disaccharides, polysaccharides, etc.
  • Prognosis refers to the prediction of the course and outcome of a disease
  • the present invention includes the prediction of wheezing, recurrence and progression of asthma after capillary bronchitis. Therefore, an indicator that can accurately predict the recurrence and progression of asthma or wheezing is very important, and a factor that can predict the response of treatment while complementing clinical indicators such as tissue differentiation and staging, in the present invention eosinophils Eosinophil-derived neurotoxins (EDNs) have been shown to function as indicators and can be used as recurrent wheezing prognostic factors.
  • EDNs Eosinophil-derived neurotoxins
  • Subject or “patient” means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects who participated in clinical research trials showing no disease clinical findings or subjects who participated in epidemiologic studies or who used as controls.
  • tissue or cell sample means a collection of similar cells obtained from a tissue of a subject or patient.
  • Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any blood component; expectoration; Urine; Cells at any time of pregnancy or development in the subject.
  • Label or “label” means a compound or composition that facilitates detection of a reagent, directly or indirectly.
  • the label may itself be detected (eg, a radioisotope label or fluorescent label) or, in the case of an enzyme label, may catalyze the chemical modification of the detectable substrate compound or composition.
  • an “effective amount” is an appropriate amount that affects a beneficial or desirable clinical or biochemical result.
  • An effective amount can be administered once or more.
  • an effective amount of inhibitor compound is an amount suitable to temporarily alleviate, ameliorate, stabilize, reverse, slow or slow the progression of a disease state. If the recipient animal can tolerate the administration of the composition, or if the administration of the composition to that animal is suitable, the composition indicates "pharmaceutically or physiologically acceptable”. If the amount administered is physiologically important, it can be said that the agent has been administered in a "therapeutically effective amount.” If the presence of the agent resulted in a physiologically detectable change of the recipient patient, the agent is physiologically meaningful.
  • Treatment means an approach to obtain beneficial or desirable clinical results.
  • beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected.
  • Treatment may also mean increasing survival compared to expected survival when untreated. Treatment refers to both therapeutic treatment and prophylactic or preventive measures. Such treatments include not only the disorders to be prevented but also the treatments required for already occurring disorders. "Palliating" a disease may reduce the extent of the disease state and / or undesirable clinical signs and / or slow or lengthen the time course of progression as compared to untreated treatment. It means losing.
  • “About” means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for reference quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths. , Amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by about 3, 2 or 1%.
  • What is intended to be diagnosed in the present invention relates to the recurrence of asthma or wheezing, especially asthma or wheezing that recurs after capillary bronchitis by a pathogenic virus.
  • RSV respiratory syncytial virus
  • adenovirus adenovirus
  • parainfluenza virus influenza virus
  • rhinovirus adenovirus
  • measles virus mycoplasma
  • the present invention is characterized in that it provides a marker and method for predicting whether asthma or wheezing may occur and the severity of the disease after the occurrence of capillary bronchitis for a specific virus that causes capillary bronchitis, and also eosinophils.
  • Eosinophil-derived neurotoxin EDN
  • the product of eosinophil degranulation can be used to diagnose recurrent asthma or wheezing prognosis.
  • a biological sample of a patient with asthma or wheeze after collecting capsular bronchitis infected with respiratory syncytial virus (RSV), mycoplasma and metapneumovirus Thereafter, the level of eosinophil-derived neurotoxin (EDN) contained in the sample was analyzed to analyze the association between asthma or wheezing that recurs after the occurrence of capillary bronchitis.
  • RSV respiratory syncytial virus
  • EDN eosinophil-derived neurotoxin
  • the present invention relates in one aspect to the use of eosinophil-derived neurotoxin (EDN) as a diagnostic marker for recurrent asthma or recurrent wheezing prognosis after capillary bronchitis.
  • EDN eosinophil-derived neurotoxin
  • the prognosis includes both recurrence and progression.
  • Wheezing is a wheezing or crotch breathing sound when the airway becomes narrow and exhales, and it is also common in asthma, an inflammatory airway obstruction disease that causes shortness of breath. Not only symptom such as difficulty, but also used as a concept including a disease containing such a symptom.
  • Asthma is a condition in which the bronchus in the lungs is very sensitive and sometimes the bronchus is narrowed, causing the person to cough as he breathes and cries.
  • the selection and application of meaningful diagnostic markers determines the reliability of the diagnostic results.
  • Significant diagnostic markers mean markers of high reliability such that the results obtained by diagnosis are accurate, have high validity, and show consistent results in repeated measurements.
  • the recurrent asthma or wheezing prognosis diagnostic marker of the present invention shows the same result in repeated experiments in which expression changes with direct or indirect factors with wheezing or asthma recurrence, and the difference in expression level is very large when compared with the control. Highly reliable markers with little chance of producing results.
  • the results diagnosed based on the results obtained by measuring the level of the significant diagnostic marker EDN of the present invention can be reasonably reliable.
  • the level of the EDN of the present invention preferably can measure the EDN level of 3 months to 5 months after capillary bronchitis.
  • the present invention by quantitatively measuring and analyzing the EDN level of each causative bacterium for harmful causative viruses, bacteria, microorganisms, and the like, the possibility of recurrence of asthma or wheezing after capillary bronchitis caused by a particular causative organism can be predicted. .
  • the 'EDN level measurement' refers to measuring the concentration of eosinophil-derived neurotoxin (EDN) in a biological sample including serum or sputum, and the biological sample can measure the concentration of EDN.
  • Urine, blood, saliva, sputum and the like may be used, but preferably serum or sputum is used.
  • the concentration of the EDN can be appropriately measured by those skilled in the art using a known method, preferably, can be measured using a primer, probe or antibody specific for the EDN.
  • the EDN may be composed of the amino acid sequence of SEQ ID NO: 1, EDN protein is encoded by the nucleotide sequence of SEQ ID NO: 2.
  • the condition of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis can be used to diagnose the condition of recurrence and disease for asthma or wheezing after capillary bronchitis. There is a characteristic.
  • the method also includes measuring eosinophil-derived neurotoxin (EDN) levels from biological samples isolated from patients with capillary bronchitis; And comparing the EDN level with a corresponding EDN level of a normal control sample.
  • EDN eosinophil-derived neurotoxin
  • the patient's capillary bronchitis causative agent was treated with respiratory syncytial virus (RSV), metapneumovirus, adenovirus, parainfluenza virus, influenza virus, rhinovirus, adenovirus, measles virus. Or it is good to go through the step of confirming whether or not due to mycoplasma.
  • RSV respiratory syncytial virus
  • metapneumovirus adenovirus
  • parainfluenza virus influenza virus
  • rhinovirus adenovirus
  • measles virus measles virus
  • capillary bronchitis when capillary bronchitis is caused by infection with respiratory syncytial virus (RSV), 4 months of eosinophil-derived neurotoxin (EDN) of RSV capillary bronchitis At levels above 45 ng / ml, 65% of positive predictive values (PPVs), 80% of negative predictive values (NPVs), 70% of sensitivity and specificity of recurrent wheezing was found to have a value of 72%. Furthermore, in the natural course of RSV capillary bronchitis, if the EDN level is 100ng / ml or more, it is confirmed that there is a 75% chance of recurrent asthma or wheezing at least twice later.
  • RSV respiratory syncytial virus
  • EDN eosinophil-derived neurotoxin
  • capsular bronchitis was caused by metapneumovirus
  • positive predictive value of recurrent wheezing when the level of eosinophil-derived neurotoxin (EDN) was more than 50 ng / ml at 3 months after capsular bronchitis PPV was 70%
  • negative predictive value (NPV) was 82%
  • sensitivity was 75%
  • specificity was 70%.
  • EDN level is 80ng / ml or more, it was confirmed that there is an 80% chance of recurrent asthma or wheezing at least three times later.
  • capsular bronchitis is caused by mycoplasma
  • negative predictive value (NPV) is 77%
  • sensitivity is 78% and specificity is 68%
  • the natural process of mycoplasma In the EDN level of 130ng / ml or more was confirmed that the probability of recurrent asthma or wheezing more than three times 83%.
  • each quantitative value of the measured and analyzed EDN can be used as objective information for predicting and diagnosing the recurrence and severity of asthma or wheezing depending on the specific causative agent of capillary bronchitis.
  • the level of eosinophil-derived neurotoxin (EDN) at 4 months after capillary bronchitis is 70ng / At ml or above, positive predictive value (PPV) is 73%, negative predictive value (NPV) is 87%, sensitivity is 78% and specificity is 70%
  • EDN eosinophil-derived neurotoxin
  • the patient can measure the serum EDN level of infants between 1 month and 24 months of age, and when sputum is used as a sample, the sputum EDN level in children between 7 and 10 years of age. Can be measured.
  • the present invention provides a composition for diagnosing recurrent asthma or recurrent wheezing after capillary bronchitis, including an agent for measuring the concentration of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis. It may be provided, it may be provided a diagnostic kit comprising the composition.
  • EDN eosinophil-derived neurotoxin
  • kits of the present invention can be used to determine the prognosis of recurrent asthma or wheezing after capillary bronchitis by analyzing EDN concentrations at 3 to 5 months of capillary bronchitis.
  • the kit is designed to measure the EDN concentration.
  • Kits of the present invention may optionally include reagents, factors, compositions, and the like, required for EDN concentration analysis.
  • the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases [eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth). ), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)], DNA polymerase cofactors and dNTPs.
  • DNA polymerases eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth).
  • Thermus filiformis Thermis flavus
  • Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)
  • DNA polymerase cofactors e.g, Thermus aquaticus (Taq), Thermus thermophilus (Tth).
  • Thermus filiformis e.g. Thermis flav
  • the kit of the present invention may include an antibody specific for EDN and a reagent for antibody reaction together with a fluorescent marker for easy detection thereof.
  • Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.
  • the diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method.
  • the kit of the present invention may comprise one or more containers including a compartment containing a partitioned carrier means for holding a sample, a means for detecting an EDN concentration.
  • the present invention by measuring the concentration of EDN as a product thereof by using granulation of eosinophils in patients with capillary bronchitis, particularly preferably, by measuring the EDN concentration of capillary bronchitis 3 months to 5th, It was confirmed that the prognosis of asthma can be judged, and it can be used to develop or study recurrent wheezing or diagnosis and treatment of asthma.
  • capillary bronchitis was based on a previously known symptom scoring system (Parkin et al. 1996): respiratory rate, wheezing, retraction, dyspnea And inspiratory-to-expiratory ratio items were divided into 0, 1, and 2 ranges, and the final score was summarized to a maximum of 10. Viral infections were then confirmed by viral antigen in nasopharyngeal inhalation by indirect immunofluorescence. All infants received conventional treatment with ß 2 -agonists during hospitalization but were not prescribed corticosteroids.
  • Mycoplasma Therapeutics [Mycoplasma-Clarithromycin Group] Treatment Group & Matching Placebo Treatment Group
  • sputum was collected by inhalation of 3% hypertonic saline haze prepared using DeVilbiss UltraNeb 99 ultrasonic nebulizer for all experimental and control groups, and sputum was induced at this time, in order to prevent contamination of saliva or manure. Sputum was collected by rinsing the mouth with a 3% hypertonic saline mist and then attempting to drain the sputum every 2 minutes into a sterilized container. It was measured using the immunoassay used.
  • the patient information used in the experiment is as follows.
  • infants with capillary bronchitis caused by the causative agent of infants 1 to 24 months of age were grouped as follows and serum and sputum collected from these patient groups and normal controls were collected and analyzed for correlation.
  • Age range (serum sample analysis target): 1-23 months, 1-23 months, 1-23 months, 1-23 months
  • Age range (targeted for sputum analysis): 7-10 years old, 7-10 years old, 7-10 years old, 7-10 years old
  • Symptom score 1-10, Mean (range): 6.1 (4-9), 5.9 (4-9), n / a
  • Age range (targeted for sputum analysis): 7-10 years old, 7-10 years old, 7-10 years old, 7-10 years old
  • Symptom score 1-10, Mean (range): 6.1 (4-9), 5.9 (4-9), n / a
  • Age range 1-22 months, 1-24 months, 1-21 months
  • Age range (targeted for sputum analysis): 7-10 years old, 7-10 years old, 7-10 years old, 7-10 years old
  • Symptom score 1-10, Mean (range): 6.1 (4-9), 5.9 (4-9), n / a
  • the therapeutic agent treatment group is a disease group
  • the placebo treatment group is a stabilizer group
  • the control group refers to a healthy normal group without disease.
  • the probability of recurrent asthma and wheezing at least three times later is 80%.
  • the probability of asthma and wheezing occurring three or more times is 80%.
  • the probability of recurrent asthma and wheezing three or more times is 83%. That is, when the EDN is 130ng / ml or more, 83% of the three or more asthma and wheezing occurred.
  • the results of ⁇ 1-3> are the results of the analysis of the serum of the experimental group and the control group, and the contents of this analysis are the results of the analysis of the sputum sample. Analysis was performed on the subject.

Abstract

The present invention relates to diagnosis of recurrent wheezing or asthma after having bronchiolitis and to a use of an eosinophil-derived neurotoxin (EDN) marker for diagnosing recurrent wheezing or asthma after having bronchiolitis by analyzing the relationship between an EDN level and recurrent wheezing or asthma after having bronchiolitis.

Description

모세기관지염 후 천식 진단을 위한 마커 및 이의 용도Markers and Their Uses for Diagnosis of Asthma after Capillary Bronchitis
본 발명은 모세기관지염 후 천식 진단을 위한 마커로서 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 용도에 관한 것이다.The present invention relates to the use of eosinophil-derived neurotoxin (EDN) as a marker for asthma diagnosis after capillary bronchitis.
모세기관지염은 호흡기에서 가장 작은 가지인 세기관지에 발생하는 감염성 질환으로 최근에는 모세기관지염이라고 한다. 호흡기는 코에서부터 시작해 상부 기관지를 거쳐 점점 더 작은 기관지로 나뭇가지처럼 분지되어 폐포까지 이르게 되어 있다. 폐포 바로 상부의 가장 작은 기관지 분지를 세기관지라고 하며, 세기관지는 직접 폐포로 공기를 전달하는 역할을 하고 폐포는 이를 받아 혈액으로 산소 공급을 한다.Capillary bronchitis is an infectious disease that occurs in the bronchioles, the smallest branch of the respiratory tract. The respiratory tract begins from the nose and branches through the upper bronchus into smaller and smaller bronchus branches that reach the alveoli. The smallest bronchial branch just above the alveoli is called the bronchiole, and the bronchiole serves to deliver air directly to the alveoli, which receives it and supplies oxygen to the blood.
기관지에 발생하는 모세기관지염은 주로 바이러스 감염에 의해 발생하며, 바이러스는 기관지 염증 반응을 일으켜 기관지 점막이 붓고 분비물이 많아지게 하며 이로 인해 좁은 세기관지 막힘 현상이 발생하여 폐포로의 산소 공급에 장애를 유발하고 결국에는 전신적인 저산소증 상태가 된다.Capillary bronchitis in the bronchus is mainly caused by a viral infection, which causes the bronchial inflammatory response, which causes the bronchial mucosa to swell and secrete, resulting in narrowing of bronchioles, which causes impaired oxygen supply to the alveoli. Eventually, systemic hypoxia occurs.
모세기관지염(bronchiolitis)은 하기도감염에 의해 말초기도의 염증성 폐세로 발생하는 질환으로, 주로 2세 이전의 영아에서 발생한다. 모세기관지염의 주증상은 기침과 천명이며, 바이러스 감염이 원인이 되는데, 호흡기 세포융합 바이러스(respiratory syncytial virus, RSV)가 가장 흔한 원인이고, 기타 파라인플루엔자 바이러스, 인플루엔자 바이러스, 라이노바이러스, 아데노바이러스, 홍역 바이러스, 마이코플라즈마도 원인이 될 수 있다.Capillary bronchitis (bronchiolitis) is a disease caused by inflammation of the lower respiratory tract due to lower respiratory tract infections, and mainly occurs in infants before 2 years old. The main symptoms of bronchiolitis are coughing and wheezing, and viral infections are the most common causes of respiratory syncytial virus (RSV), and other parainfluenza viruses, influenza viruses, rhinoviruses, adenoviruses, and measles. Viruses and mycoplasmas can also cause this.
특히 호흡기 세포융합 바이러스(respiratory syncytial virus, RSV)는 소아에서 하부 기도 질환을 일으키는 가장 흔한 원인 바이러스로 전 세계적으로 매년 10월경부터 이듬해 4월경 사이에 최소 12주 이상 지속되어 유행을 일으킨다. RSV에 의한 감염은 매우 흔하게 발생하여 50% 정도의 소아들이 생후 1세 이내에 감염되고 3세에 이르면 거의 모든 소아들이 경험하는 것으로 보고된 바 있다. In particular, respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract disease in children, and it is epidemic lasting at least 12 weeks worldwide from around October to next April every year. Infection with RSV is very common, and it has been reported that as many as 50% of children are infected within 1 year of age and reach almost 3 years of age.
또한, RSV 감염에 의한 증상은 소아의 연령에 따라 다양하며, 소아에서는 대개 경한 상기도 감염의 증상을 나타내지만, 2세 이하의 영아에서는 심한 하부 기도 질환을 일으키는 경우가 많아 영아에서 발생하는 급성 모세기관지염과 폐렴의 50-90%가 RSV에 의해 발생한다.In addition, symptoms caused by RSV infection vary according to the age of the child, and children usually have mild upper respiratory tract infections, but in infants under 2 years of age, severe lower respiratory tract disease often causes acute capillary disease in infants. 50-90% of bronchitis and pneumonia are caused by RSV.
RSV에 의한 급성 모세기관지염은 영아에서 가장 중요한 호흡부전의 원인 중 하나이며 사망률은 0.5-1.5% 정도로 낮지만 미숙아, 신경 근육계질환, 만성호흡기 질환, 선천성 심장질환 등을 가지고 있거나 면역 기능이 저하된 상태에 있는 고위험군 환자들에서는 훨씬 높은 사망률을 나타낸다. 자연적인 감염에 의해 얻어진 면역력은 불충분하여 실제로 RSV 감염을 앓은 이후 1-2년 내에 재감염이 일어나는 경우가 많다.Acute capillary bronchitis caused by RSV is one of the most important causes of respiratory failure in infants. The mortality rate is as low as 0.5-1.5%, but premature infants, neuromuscular disease, chronic respiratory disease, congenital heart disease, or reduced immune function. High-risk patients in the group have a much higher mortality rate. Immunity obtained from natural infections is insufficient, and reinfection often occurs within one to two years after an actual RSV infection.
한편, 영유아기에서 RSV 감염 이후 재발성 천명 발생과 관련이 있다는 것은 여러 역학 연구에서 증명되었다. 알레르기 질환의 유전적 소인과 선천성 기관지 과민성이 모세기관지염 후의 천명과 천식 발생에 관여하는 것으로 알려져 있으며, RSV 감염 자체가 알레르기 염증 반응과 알레르겐 감작 빈도를 높여 천식을 일으킨다는 연구 결과도 있다. 특히 몇몇 연구에서는 첫 1년 이내에 중증의 RSV 모세기관지염을 앓는 소아에서 이후 재발성 천명 및 알레르겐 감작이 일어날 가능성이 높다고 하였다.On the other hand, several epidemiologic studies have been associated with recurrent wheezing after RSV infection in infants and young children. Genetic predisposition and allergic bronchial hypersensitivity of allergic diseases are known to be involved in wheezing and asthma after capillary bronchitis, and RSV infection itself causes asthma by increasing the frequency of allergic inflammatory reactions and allergen sensitization. In particular, some studies have shown that children with severe RSV capillary bronchitis within the first year are more likely to develop recurrent wheezing and allergen sensitization.
영아에서 처음 나타나는 증상은 주로 비폐색, 콧물 등이다. 이후 1-3일에 걸쳐 감염이 하부 기도로 진행하면서 기침이 시작되는데 급성 후두염, 기관지염, 모세기관지염, 폐렴 또는 이들이 복합된 다양한 증상이 나타나게 된다. 이후 증상이 심해지면서 10일 정도 지속되다가 점차 회복된다. 발열은 심하지 않은 경우가 대부분이며 최고 체온이 38℃ 정도이며 열이 없을 수도 있지만, RSV로 감염된 매우 어린 유아에게서는 생명을 위협하는 무호흡증이 발병할 수도 있다.The first symptoms in infants are mainly occlusive or runny nose. Over the next 1-3 days, the infection progresses to the lower respiratory tract and coughing begins with acute laryngitis, bronchitis, capillary bronchitis, pneumonia, or a combination of these symptoms. The symptoms worsen and last about 10 days, then gradually recover. Fever is most often mild, with a maximum body temperature of around 38 ° C and may not have fever, but very young infants infected with RSV may develop life-threatening apnea.
RSV에 의한 모세기관지염은 기관지 과민도를 유발하고 상당수에서 영아기의 반복적 천명 혹은 천식 등으로 발전한다. 이들 반복적 천명의 원인으로 첫번째 감염시 중증 정도, 아토피 소견, 환경요소들이 관여할 수 있으나 아직 그 원인이 확실히 알려져 있지 않다. Capillary bronchitis caused by RSV causes bronchial hypersensitivity and develops in many cases, such as repeated wheezing or asthma in infancy. Severe severity, atopy findings, and environmental factors may be involved in the cause of these repeated wheezing, but the cause is not yet known.
특히, 영유아의 호흡기 감염을 치료하거나 예방하기 위해, RSV에 의한 모세기관지염 후 2-3년간 반복적 천명을 일으키는 경우를 예측할 수 있다면 향후 천명 진단과 치료에 상당히 도움이 될 것이다.In particular, in order to treat or prevent respiratory infections in infants, it would be very helpful for the diagnosis and treatment of wheezing in the future if it can be predicted to cause repeated wheezing for 2-3 years after capsular bronchitis caused by RSV.
이에, 본 발명자들은 모세기관지염에 걸린 영아들을 대상으로 천식, 천명, 호흡곤란, 기침 등의 증상정도를 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN) 발현정도와의 상관관계를 조사함으로써, 특정 시점에서 EDN의 수준을 측정할 경우, 모세기관지염 이후 재발될 수 있는 천명 발작 여부를 우수한 정확도로 예측할 수 있음을 확인함으로써 본 발명을 완성하였다.In this regard, the present inventors examined the degree of symptoms such as asthma, wheezing, dyspnea and cough in infants with capillary bronchitis by examining the correlation with the degree of eosinophil-derived neurotoxin (EDN) expression. When measuring the level of EDN in, the present invention was completed by confirming that it can predict with high accuracy whether wheezing attacks that can recur after capillary bronchitis.
본 발명의 목적은 모세기관지염 이후 천식 또는 천명 진단에 필요한 정보를 제공하기 위해, 모세기관지염 3개월~5개월째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준을 측정하는 방법을 제공하기 위한 것이다.An object of the present invention is to provide a method for measuring the level of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis to provide information necessary for asthma or wheeze diagnosis after capillary bronchitis will be.
본 발명의 다른 목적은 모세기관지염 3개월~5개월째의 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 농도를 측정하는 제제를 포함하는, 모세기관지염 이후 재발성 천식 또는 천명(recurrent wheezing) 진단용 키트를 제공하게 위한 것이다.Another object of the present invention is for diagnosing recurrent wheezing after capillary bronchitis, including an agent for measuring the concentration of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis. To provide a kit.
본 발명은 상기 과제를 해결하기 위해, The present invention to solve the above problems,
본 발명은 모세기관지염 이후 천식 또는 천명 진단에 필요한 정보를 제공하기 위해, 모세기관지염 3개월~5개월째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준을 측정하는 방법을 제공한다.The present invention provides a method for measuring the level of eosinophil-derived neurotoxin (EDN) 3 to 5 months after capillary bronchitis to provide information necessary for asthma or wheezing diagnosis.
본 발명의 일실시예에 있어서, 상기 EDN의 수준은 혈청 또는 객담 내 중 EDN의 농도일 수 있다. In one embodiment of the invention, the level of EDN may be the concentration of EDN in serum or sputum.
본 발명의 일실시예에 있어서, 상기 모세기관지염은 호흡기 세포융합 바이러스(respiratory syncytial virus, RSV), 메타뉴모 바이러스, 아데노바이러스, 파라인플루엔자 바이러스, 인플루엔자 바이러스, 라이노바이러스, 아데노바이러스, 홍역 바이러스 및 마이코플라즈마로 이루어진 군 중에서 선택되는 유해균의 감염으로 유발되는 것일 수 있다.In one embodiment of the present invention, the capillary bronchitis is respiratory syncytial virus (RSV), metapneumovirus, adenovirus, parainfluenza virus, influenza virus, rhinovirus, adenovirus, measles virus and mycoplasma It may be caused by the infection of harmful bacteria selected from the group consisting of.
본 발명의 일실시예에 있어서, 상기 모세기관지염이 호흡기 세포융합 바이러스(respiratory syncytial virus, RSV)에 의한 감염으로 유발된 경우, RSV 모세기관지염 4개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 45ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 65%, 음성 예측값(negative predictive value, NPV)은 80%, 민감도(sensitivity)는 70% 및 특이도(specificity)는 72% 값을 가질 수 있다.In one embodiment of the present invention, when capillary bronchitis is caused by infection with respiratory syncytial virus (RSV), eosinophil-derived neurotoxin (EDN) at 4 months of RSV capillary bronchitis At levels above 45 ng / ml, relapsed wheeze has a positive predictive value (PPV) of 65%, a negative predictive value (NPV) of 80%, a sensitivity of 70% and specificity ) May have a 72% value.
본 발명의 일실시예에 있어서, 상기 모세기관지염이 메타뉴모 바이러스에 의해 유발된 경우, 모세기관지염 3개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 50ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 70%, 음성 예측값(negative predictive value, NPV)은 82%, 민감도(sensitivity)는 75% 및 특이도(specificity)는 70% 값을 가질 수 있다.In one embodiment of the present invention, when the capillary bronchitis is induced by metapneumovirus, recurrent wheezing when the level of eosinophil-derived neurotoxin (EDN) at 3 months after capillary bronchitis is 50ng / ml or more The positive predictive value (PPV) of 70%, negative predictive value (negative predictive value (NPV)) may have a value of 82%, sensitivity (75%) and specificity (specificity) may have a value of 70%.
본 발명의 일실시예에 있어서, 상기 모세기관지염이 마이코플라스마에 의해 유발된 경우, 모세기관지염 5개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 60ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 63%, 음성 예측값(negative predictive value, NPV)은 77%, 민감도(sensitivity)는 78% 및 특이도(specificity)는 68% 값을 가질 수 있다.In one embodiment of the present invention, when the capillary bronchitis is caused by mycoplasma, recurrent wheezing when the level of eosinophil-derived neurotoxin (EDN) is more than 60 ng / ml at 5 months after capillary bronchitis The positive predictive value (PPV) may have a value of 63%, the negative predictive value (negative predictive value (NPV)) may have a value of 77%, sensitivity (78%) and specificity (specificity) of 68%.
본 발명의 일실시예에 있어서, 상기 방법은 생후 1개월~24개월의 영유아의 혈청 내 EDN 수준을 측정하는 것일 수 있다. In one embodiment of the present invention, the method may be to measure the serum EDN level of infants 1 month to 24 months of age.
본 발명의 일실시예에 있어서, RSV 모세기관지염의 자연적인 과정에서 EDN 수준이 100ng/ml 이상인 경우 추후 2회 이상 재발성 천식 또는 천명이 나타날 확률이 75%이고, 메타뉴모 바이러스에 의한 모세기관지염의 자연적인 과정에서 EDN 수준이 80ng/ml 이상인 경우 추후 3회 이상 재발성 천식 또는 천명이 나타날 확률이 80%이고, 마이코플라스마의 자연적인 과정에서 EDN 수준이 130ng/ml 이상인 경우 추후 3회 이상 재발성 천식 또는 천명이 나타날 확률이 83%일 수 있다.In one embodiment of the present invention, when the EDN level is 100ng / ml or more in the natural course of RSV capillary bronchitis, there is a 75% chance of recurring asthma or wheezing at least two times later. If the EDN level is 80ng / ml or higher in the natural process, there is an 80% chance of recurring asthma or wheezing at least 3 times later, and if the EDN level is 130ng / ml or higher in the natural course of mycoplasma, recurrence is more than 3 times later. There may be a 83% chance of asthma or wheezing.
본 발명의 일실시예에 있어서, 객담 시료를 사용하는 경우 상기 모세기관지염이 마이코플라스마에 의해 유발된 경우, 모세기관지염 4개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 70ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 73%, 음성 예측값(negative predictive value, NPV)은 87%, 민감도(sensitivity)는 78% 및 특이도(specificity)는 70% 값을 가질 수 있다.In one embodiment of the present invention, when the sputum sample is used when the capillary bronchitis is induced by mycoplasma, the level of eosinophil-derived neurotoxin (EDN) at 4 months after capillary bronchitis is 70ng / ml If abnormal, regressive wheeze had positive predictive value (PPV) of 73%, negative predictive value (NPV) of 87%, sensitivity of 78% and specificity of 70%. Can have
본 발명의 일실시예에 있어서, 객담 시료를 사용하여 분석하는 경우에는 7세~10세의 아동기의 객담 내 EDN 수준을 측정할 수 있다. In one embodiment of the present invention, when analyzing using a sputum sample, it is possible to measure the EDN level in the sputum of children 7 to 10 years of age.
또한 본 발명은 모세기관지염 3개월~5개월째의 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 농도를 측정하는 제제를 포함하는, 모세기관지염 이후 재발성 천식 또는 천명(recurrent wheezing) 진단용 키트를 제공한다.The present invention also provides a kit for diagnosing recurrent wheezing after capillary bronchitis, comprising an agent for measuring the concentration of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis. to provide.
본 발명의 일실시예에 있어서, 상기 제제는 EDN에 특이적으로 결합할 수 있는 프라이머, 프로브 또는 항체일 수 있다.In one embodiment of the invention, the agent may be a primer, probe or antibody that can specifically bind to EDN.
본 발명은 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN) 수준과 모세기관지염 후 재발성 천식 또는 천명의 유의미한 상관관계를 분석함으로써, EDN을 모세기관지염을 앓았던 영유아 및 아동들에서의 천식 또는 천명의 재발에 대한 예후 예측 및 검증용 마커로 사용할 수 있으며, 모세기관지염의 원인 세균의 종류에 따른 EDN 수준을 객관화 시킴으로써 보다 효과적이고 정확한 진단을 통해 재발성 천식 또는 천명을 치료하며 지속적으로 관리할 수 있는 예방약 및 치료약 개발 등에 유용하게 적용할 수 있다.The present invention analyzes the significant correlation between eosinophil-derived neurotoxin (EDN) levels and recurrent asthma or wheeze after capillary bronchitis, thereby asthma or wheeze in infants and children with capillary bronchitis It can be used as a marker for predicting and verifying the prognosis of recurrence, and by preventing the EDN level according to the type of bacteria causing capillary bronchitis, it is a preventive medicine that can continuously manage and manage recurrent asthma or wheezing through a more effective and accurate diagnosis. And therapeutic drug development.
본 발명에서 사용되는 용어에 대한 정의는 이하와 같다.Definitions of terms used in the present invention are as follows.
"진단"은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 진단은 모세기관지염 이후 천명 및 천식의 재발 및 진행 여부를 확인하는 것이다. "Diagnosis" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to confirm the recurrence and progression of wheezing and asthma after capillary bronchitis.
"진단용 마커, 진단하기 위한 마커 또는 진단 마커(diagnosis marker)란 본 발명에 따른 질환 발병 대상 세포를 정상 세포와 구분하여 진단할 수 있는 물질로, 정상 세포에 비하여 질환 세포에서 증가양상을 보이는 폴리펩타이드 또는 핵산(예: mRNA 등), 지질 , 당지질, 당단백질, 당(단당류, 이당류, 다당류 등)과 같은 유기 생체 분자 등을 포함한다. "Diagnostic markers, diagnostic markers or diagnostic markers (diagnosis markers) is a substance capable of diagnosing a diseased cell according to the present invention from normal cells, a polypeptide showing an increase in disease cells compared to normal cells Or organic biomolecules such as nucleic acids (eg mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, polysaccharides, etc.).
"예후(Prognosis)"란 질병의 경과와 결과의 예측을 의미하는 것으로, 본 발명에서는 모세기관지염 이후 천명, 천식의 재발(Recurrence) 및 진행(progression)을 예측하는 것을 포함한다. 이에, 천식 또는 천명(wheezing)의 재발 및 진행을 정확히 예측할 수 있는 지표가 매우 중요하며 조직의 분화도와 병기와 같은 임상적 지표를 보완하면서 치료의 반응을 예측할 수 있는 인자가 필요한데, 본 발명에서는 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)이 이러한 지표 기능을 하므로 재발성 천명 예후 인자로 이용할 수 있다는 것을 규명하였다. "Prognosis" refers to the prediction of the course and outcome of a disease, the present invention includes the prediction of wheezing, recurrence and progression of asthma after capillary bronchitis. Therefore, an indicator that can accurately predict the recurrence and progression of asthma or wheezing is very important, and a factor that can predict the response of treatment while complementing clinical indicators such as tissue differentiation and staging, in the present invention eosinophils Eosinophil-derived neurotoxins (EDNs) have been shown to function as indicators and can be used as recurrent wheezing prognostic factors.
"대상" 또는 "환자"는 인간, 소, 개, 기니아 피그, 토끼, 닭, 곤충 등을 포함하여 치료가 요구되는 임의의 단일 개체를 의미한다. 또한, 임의의 질병 임상 소견을 보이지 않는 임상 연구 시험에 참여한 임의의 대상 또는 역학 연구에 참여한 대상 또는 대조군으로 사용된 대상이 대상에 포함된다. "Subject" or "patient" means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects who participated in clinical research trials showing no disease clinical findings or subjects who participated in epidemiologic studies or who used as controls.
"조직 또는 세포 샘플"은 대상 또는 환자의 조직으로부터 얻은 유사한 세포의 집합체를 의미한다. 조직 또는 세포 샘플의 공급원은 신선한, 동결된 및/또는 보존된 장기 또는 조직 샘플 또는 생검 또는 흡인물로부터의 고형 조직; 혈액 또는 임의의 혈액 구성분; 객담; 뇨; 대상의 임신 또는 발생의 임의의 시점의 세포일 수 있다. "Tissue or cell sample" means a collection of similar cells obtained from a tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any blood component; expectoration; Urine; Cells at any time of pregnancy or development in the subject.
"표지" 또는 "라벨"는 직접 또는 간접적으로 시약의 검출을 용이하게 하는 화합물 또는 조성물을 의미한다. 표지는 그 자체가 검출될 수 있거나 (예를 들어, 방사성 동위원소 표지 또는 형광 표지), 효소 표지의 경우에, 검출 가능한 기질 화합물 또는 조성물의 화학적 변형을 촉매 할 수 있다."Label" or "label" means a compound or composition that facilitates detection of a reagent, directly or indirectly. The label may itself be detected (eg, a radioisotope label or fluorescent label) or, in the case of an enzyme label, may catalyze the chemical modification of the detectable substrate compound or composition.
"유효량"은, 이롭거나 바람직한 임상적 또는 생화학적 결과에 영향을 주는 적절한 양이다. 유효량은 한번 또는 그 이상 투여될 수 있다. 본 발명의 목적을 위하여, 저해제 화합물의 유효량은 질병 상태의 진행을 일시적으로 완화, 개선, 안정화, 되돌림, 속도를 늦춤 또는 지연시키는데 적절한 양이다. 만약, 수혜동물이 조성물의 투여에 견딜 수 있거나, 조성물의 그 동물에의 투여가 적합한 경우라면, 조성물은 "약학적으로 또는 생리학적으로 허용 가능함"을 나타낸다. 투여된 양이 생리학적으로 중요한 경우에는 상기 제제는 "치료학적으로 유효량"으로 투여되었다고 말할 수 있다. 상기 제제의 존재가 수혜 환자의 생리학적으로 검출 가능한 변화를 초래한 경우라면 상기 제제는 생리학적으로 의미가 있다. An "effective amount" is an appropriate amount that affects a beneficial or desirable clinical or biochemical result. An effective amount can be administered once or more. For the purposes of the present invention, an effective amount of inhibitor compound is an amount suitable to temporarily alleviate, ameliorate, stabilize, reverse, slow or slow the progression of a disease state. If the recipient animal can tolerate the administration of the composition, or if the administration of the composition to that animal is suitable, the composition indicates "pharmaceutically or physiologically acceptable". If the amount administered is physiologically important, it can be said that the agent has been administered in a "therapeutically effective amount." If the presence of the agent resulted in a physiologically detectable change of the recipient patient, the agent is physiologically meaningful.
"치료"는 이롭거나 바람직한 임상적 결과를 수득하기 위한 접근을 의미한다. 본 발명의 목적을 위해서, 이롭거나 바람직한 임상적 결과는 비제한적으로, 증상의 완화, 질병 범위의 감소, 질병 상태의 안정화 (즉, 악화되지 않음), 질병 진행의 지연 또는 속도의 감소, 질병 상태의 개선 또는 일시적 완화 및 경감 (부분적이거나 전체적으로), 검출가능하거나 또는 검출되지 않거나의 여부를 포함한다. 또한, "치료"는 치료를 받지 않았을 때 예상되는 생존율과 비교하여 생존율을 늘이는 것을 의미할 수도 있다. 치료는 치료학적 치료 및 예방적 또는 예방조치 방법 모두를 가리킨다. 상기 치료들은 예방되는 장애뿐만 아니라 이미 발생한 장애에 있어서 요구되는 치료를 포함한다. 질병을 "완화(Palliating)"하는 것은 치료를 하지 않은 경우와 비교하여, 질병상태의 범위 및/또는 바람직하지 않은 임상적 징후가 감소되거나 및/또는 진행의 시간적 추이(time course)가 늦춰지거나 길어지는 것을 의미한다."Treatment" means an approach to obtain beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected. "Treatment" may also mean increasing survival compared to expected survival when untreated. Treatment refers to both therapeutic treatment and prophylactic or preventive measures. Such treatments include not only the disorders to be prevented but also the treatments required for already occurring disorders. "Palliating" a disease may reduce the extent of the disease state and / or undesirable clinical signs and / or slow or lengthen the time course of progression as compared to untreated treatment. It means losing.
"약"이라는 것은 참조 양, 수준, 값, 수, 빈도, 퍼센트, 치수, 크기, 양, 중량 또는 길이에 대해 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 또는 1% 정도로 변하는 양, 수준, 값, 수, 빈도, 퍼센트, 치수, 크기, 양, 중량 또는 길이를 의미한다."About" means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for reference quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths. , Amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by about 3, 2 or 1%.
본 명세서를 통해, 문맥에서 달리 필요하지 않으면, "포함하다" 및 "포함하는"이란 말은 제시된 단계 또는 원소, 또는 단계 또는 원소들의 군을 포함하나, 임의의 다른 단계 또는 원소, 또는 단계 또는 원소들의 군이 배제되지는 않음을 내포하는 것으로 이해하여야 한다.Throughout this specification, the terms “comprises” and “comprising”, unless otherwise indicated in the context, include a given step or element, or group of steps or elements, but any other step or element, or step or element It should be understood that this group is not excluded.
이하, 본 발명에 대하여 구체적으로 설명한다. EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely.
본 발명에서 진단하고자 하는 것은 천식 또는 천명(wheezing)의 재발, 특히 병원성 바이러스에 의한 모세기관지염 후 재발되는 천식 또는 천명에 관한 것이다.What is intended to be diagnosed in the present invention relates to the recurrence of asthma or wheezing, especially asthma or wheezing that recurs after capillary bronchitis by a pathogenic virus.
3세 이전의 영유아에서 천명의 재발은 흔히 보는 문제이며 영아기에는 알레르기 질환에 의한 천명의 빈도는 적은 반면 바이러스에 의한 호흡기 감염이 천식과 천명의 중요한 원인이 된다. 호흡기 감염을 유발할 수 있는 병원성 인자로서 호흡기 세포융합 바이러스(respiratory syncytial virus, RSV), 메타뉴모 바이러스, 아데노바이러스, 파라인플루엔자 바이러스, 인플루엔자 바이러스, 라이노바이러스, 아데노바이러스, 홍역 바이러스, 마이코플라즈마 등이 있다.Relapse of wheeze is a common problem in infants before 3 years of age, and in infants, the incidence of wheezing due to allergic diseases is low, while viral respiratory infections are an important cause of asthma and wheezing. Pathogenic factors that can cause respiratory infections include respiratory syncytial virus (RSV), metapneumovirus, adenovirus, parainfluenza virus, influenza virus, rhinovirus, adenovirus, measles virus and mycoplasma.
특히 본 발명에서는 모세기관지염을 유발하는 특정 바이러스에 대한 모세기관지염 발생 후, 천식 또는 천명이 재발될 수 있는지의 여부와 질환의 중증도를 예측할 수 있는 마커 및 방법을 제공한다는 점에 특징이 있으며, 또한 호산구 탈과립화(eosinophil degranulation)가 재발성 천명과 상관관계가 있음을 최초로 규명하여, 호산구 탈과립화의 산물인 EDN(eosinophil-derived neurotoxin)을 재발성 천식 또는 천명 예후 진단에 활용할 수 있음을 발견하였다.In particular, the present invention is characterized in that it provides a marker and method for predicting whether asthma or wheezing may occur and the severity of the disease after the occurrence of capillary bronchitis for a specific virus that causes capillary bronchitis, and also eosinophils. Eosinophil-derived neurotoxin (EDN), the product of eosinophil degranulation, can be used to diagnose recurrent asthma or wheezing prognosis.
구체적으로 본 발명의 일실시예에서는 세지관지염 유발 원인균 중에서, RSV(respiratory syncytial virus), 마이코플라즈마 및 메타뉴모 바이러스에 감염되어 모세기관지염을 앓은 후 천식 또는 천명이 재발된 환자의 생물학적 시료를 수집한 후, 시료에 함유된 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준을 분석하여 모세기관지염의 발생 이후 재발하는 천식 또는 천명과의 연관 관계를 분석하였다. Specifically, in one embodiment of the causative bronchitis-causing bacterium, a biological sample of a patient with asthma or wheeze after collecting capsular bronchitis infected with respiratory syncytial virus (RSV), mycoplasma and metapneumovirus Thereafter, the level of eosinophil-derived neurotoxin (EDN) contained in the sample was analyzed to analyze the association between asthma or wheezing that recurs after the occurrence of capillary bronchitis.
진단 마커 용도 및 진단키트Diagnostic Marker Applications and Diagnostic Kits
따라서, 본 발명은 일 관점에서 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 모세기관지염 이후 재발성 천식 또는 천명(recurrent wheezing) 예후 진단 마커로서의 용도에 관한 것이다. Accordingly, the present invention relates in one aspect to the use of eosinophil-derived neurotoxin (EDN) as a diagnostic marker for recurrent asthma or recurrent wheezing prognosis after capillary bronchitis.
특히, 모세기관지염 이후 EDN 수준의 확인을 통해 재발성 천식 또는 천명의 예후를 진단하는 것이 매우 중요하다. 이때 상기 예후는 재발(Recurrence) 및 진행(progression)의 단계를 모두 포함한다. In particular, it is very important to diagnose the prognosis of recurrent asthma or wheezing by checking EDN levels after capillary bronchitis. In this case, the prognosis includes both recurrence and progression.
천명(wheezing)은 기도가 좁아져서 숨을 내쉴 때 쌕쌕거리거나 가랑가랑하는 호흡음이 나타나는 것으로, 호흡 곤란을 일으키는 염증성 기도 폐쇄 질환인 천식에서도 흔히 나타나는 바, 본 발명에서의 천명은 이러한 기침, 호흡곤란 등의 증상 뿐만 아니라, 이러한 증상을 포함하는 질환 포함 개념으로 사용되고 있다.Wheezing is a wheezing or crotch breathing sound when the airway becomes narrow and exhales, and it is also common in asthma, an inflammatory airway obstruction disease that causes shortness of breath. Not only symptom such as difficulty, but also used as a concept including a disease containing such a symptom.
천식은 폐 속에 있는 기관지가 아주 예민해진 상태로, 때때로 기관지가 좁아져서 숨이 차고 가랑가랑하는 숨소리가 들리면서 기침을 심하게 하는 증상을 나타내는 질환이다. Asthma is a condition in which the bronchus in the lungs is very sensitive and sometimes the bronchus is narrowed, causing the person to cough as he breathes and cries.
한편, 유의성 있는 진단 마커의 선택과 적용은 진단 결과의 신뢰도를 결정짓는다. 유의성 있는 진단 마커란, 진단하여 얻은 결과가 정확하여 타당도(validity)가 높고 반복 측정시에도 일관된 결과를 나타내도록 신뢰도(reliability)가 높은 마커를 의미한다. 본 발명의 재발성 천식 또는 천명 예후 진단 마커는, 천명 또는 천식의 재발과 함께 직접적 또는 간접적 요인으로 발현이 변화하는 반복된 실험에도 동일한 결과를 나타내며, 발현 수준의 차이가 대조군과 비교할 때 매우 커서 잘못된 결과를 내릴 확률이 거의 없는 신뢰도가 높은 마커들이다.On the other hand, the selection and application of meaningful diagnostic markers determines the reliability of the diagnostic results. Significant diagnostic markers mean markers of high reliability such that the results obtained by diagnosis are accurate, have high validity, and show consistent results in repeated measurements. The recurrent asthma or wheezing prognosis diagnostic marker of the present invention shows the same result in repeated experiments in which expression changes with direct or indirect factors with wheezing or asthma recurrence, and the difference in expression level is very large when compared with the control. Highly reliable markers with little chance of producing results.
그러므로 본 발명의 유의성 있는 진단 마커 EDN의 수준을 측정하여 얻은 결과를 토대로 진단된 결과는 타당하게 신뢰할 수 있다.Therefore, the results diagnosed based on the results obtained by measuring the level of the significant diagnostic marker EDN of the present invention can be reasonably reliable.
특히, 본 발명의 EDN의 수준은, 바람직하게는 모세기관지염 이후 3개월~5개월째의 EDN 수준을 측정할 수 있다.In particular, the level of the EDN of the present invention, preferably can measure the EDN level of 3 months to 5 months after capillary bronchitis.
나아가 본 발명에서는 모세기관지염을 유발하는 유해 원인 바이러스, 세균, 미생물 등에 대해 각 유발 원인균에 대한 EDN 수준을 정량적으로 측정 및 분석함으로써 특정 원인균에 의한 모세기관지염 이후의 천식 또는 천명의 재발 가능성을 예측할 수 있다.Furthermore, in the present invention, by quantitatively measuring and analyzing the EDN level of each causative bacterium for harmful causative viruses, bacteria, microorganisms, and the like, the possibility of recurrence of asthma or wheezing after capillary bronchitis caused by a particular causative organism can be predicted. .
상기 'EDN 수준 측정'이란, 혈청 또는 객담을 포함하는 생물학적 시료 등에서 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 농도를 측정하는 것을 말하며, 생물학적 시료로는 EDN의 농도를 측정할 수 있는 것으로 뇨, 혈액, 타액, 객담 등을 사용할 수 있으나, 바람직하게는 혈청 또는 객담을 사용한다. 상기 EDN의 농도는 공지의 방법을 이용하여 당업자가 적절히 측정할 수 있는데, 바람직하게는 EDN에 특이적인 프라이머, 프로브 또는 항체를 사용하여 측정할 수 있다. The 'EDN level measurement' refers to measuring the concentration of eosinophil-derived neurotoxin (EDN) in a biological sample including serum or sputum, and the biological sample can measure the concentration of EDN. Urine, blood, saliva, sputum and the like may be used, but preferably serum or sputum is used. The concentration of the EDN can be appropriately measured by those skilled in the art using a known method, preferably, can be measured using a primer, probe or antibody specific for the EDN.
본 발명에서 상기 EDN은 서열번호 1의 아미노산 서열로 이루어진 것일 수 있고, EDN 단백질은 서열번호 2의 염기서열로 암호화되어 있다.In the present invention, the EDN may be composed of the amino acid sequence of SEQ ID NO: 1, EDN protein is encoded by the nucleotide sequence of SEQ ID NO: 2.
본 발명에서는 모세기관지염 3개월~5개월째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준을 측정한 결과를 이용하여 모세기관지염 이후의 천식 또는 천명에 대한 재발 및 질병의 상태를 진단할 수 있는 특징이 있다.In the present invention, the condition of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis can be used to diagnose the condition of recurrence and disease for asthma or wheezing after capillary bronchitis. There is a characteristic.
또한, 상기 방법은 모세기관지염의 환자로부터 분리된 생물학적 시료로부터 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN) 수준을 측정하는 단계; 및 상기 EDN 수준을 정상 대조군 시료의 해당 EDN 수준과 비교하는 단계를 포함할 수 있다. The method also includes measuring eosinophil-derived neurotoxin (EDN) levels from biological samples isolated from patients with capillary bronchitis; And comparing the EDN level with a corresponding EDN level of a normal control sample.
이 때, EDN의 수준측정은 모세기관지염 3개월~5개월째 측정하는 것이 바람직하다.At this time, it is preferable to measure the level of EDN 3 months to 5 months capillary bronchitis.
또한 EDN의 수준을 측정하기 전에, 환자의 모세기관지염 원인 균이 호흡기 세포융합 바이러스(respiratory syncytial virus, RSV), 메타뉴모 바이러스, 아데노바이러스, 파라인플루엔자 바이러스, 인플루엔자 바이러스, 라이노바이러스, 아데노바이러스, 홍역 바이러스 또는 마이코플라즈마에 의한 것인지 확인하는 단계를 미리 거치는 것이 좋다.Also, before measuring the level of EDN, the patient's capillary bronchitis causative agent was treated with respiratory syncytial virus (RSV), metapneumovirus, adenovirus, parainfluenza virus, influenza virus, rhinovirus, adenovirus, measles virus. Or it is good to go through the step of confirming whether or not due to mycoplasma.
본 발명의 일실시예에 따르면, 모세기관지염이 호흡기 세포융합 바이러스(respiratory syncytial virus, RSV)에 의한 감염으로 유발된 경우, RSV 모세기관지염 4개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 45ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 65%, 음성 예측값(negative predictive value, NPV)은 80%, 민감도(sensitivity)는 70% 및 특이도(specificity)는 72% 값을 가진다는 사실을 확인하였고, 나아가 RSV 모세기관지염의 자연적인 과정에서 EDN 수준이 100ng/ml 이상인 경우 추후 2회 이상 재발성 천식 또는 천명이 나타날 확률이 75%이라는 것을 확인할 수 있었다.According to one embodiment of the present invention, when capillary bronchitis is caused by infection with respiratory syncytial virus (RSV), 4 months of eosinophil-derived neurotoxin (EDN) of RSV capillary bronchitis At levels above 45 ng / ml, 65% of positive predictive values (PPVs), 80% of negative predictive values (NPVs), 70% of sensitivity and specificity of recurrent wheezing Was found to have a value of 72%. Furthermore, in the natural course of RSV capillary bronchitis, if the EDN level is 100ng / ml or more, it is confirmed that there is a 75% chance of recurrent asthma or wheezing at least twice later.
또한, 모세기관지염이 메타뉴모 바이러스에 의해 유발된 경우, 모세기관지염 3개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 50ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 70%, 음성 예측값(negative predictive value, NPV)은 82%, 민감도(sensitivity)는 75% 및 특이도(specificity)는 70% 값을 가진다는 것을 확인할 수 있었고, 나아가 메타뉴모 바이러스에 의한 모세기관지염의 자연적인 과정에서 EDN 수준이 80ng/ml 이상인 경우 추후 3회 이상 재발성 천식 또는 천명이 나타날 확률이 80%임을 확인할 수 있었다.In addition, if capsular bronchitis was caused by metapneumovirus, positive predictive value of recurrent wheezing when the level of eosinophil-derived neurotoxin (EDN) was more than 50 ng / ml at 3 months after capsular bronchitis PPV) was 70%, negative predictive value (NPV) was 82%, sensitivity was 75% and specificity was 70%. In the natural course of capillary bronchitis, if the EDN level is 80ng / ml or more, it was confirmed that there is an 80% chance of recurrent asthma or wheezing at least three times later.
또한, 모세기관지염이 마이코플라스마에 의해 유발된 경우, 모세기관지염 5개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 60ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 63%, 음성 예측값(negative predictive value, NPV)은 77%, 민감도(sensitivity)는 78% 및 특이도(specificity)는 68% 값을 가진다는 것을 알 수 있었으며, 나아가 마이코플라스마의 자연적인 과정에서 EDN 수준이 130ng/ml 이상인 경우 추후 3회 이상 재발성 천식 또는 천명이 나타날 확률이 83%라는 것을 확인할 수 있었다.In addition, if capsular bronchitis is caused by mycoplasma, positive predictive value (PPV) of recurrent wheeze when the level of eosinophil-derived neurotoxin (EDN) is greater than 60 ng / ml at 5 months after capsular bronchitis ) Is 63%, negative predictive value (NPV) is 77%, sensitivity is 78% and specificity is 68%, and the natural process of mycoplasma In the EDN level of 130ng / ml or more was confirmed that the probability of recurrent asthma or wheezing more than three times 83%.
따라서 이렇게 측정되어 분석된 EDN에 대한 각각의 정량적 수치는 모세기관지염 유발 특정 원인균에 따라 천식 또는 천명의 재발 가능성 및 중증도를 예측 및 진단하기 위한 객관적인 정보로 사용할 수 있다.Therefore, each quantitative value of the measured and analyzed EDN can be used as objective information for predicting and diagnosing the recurrence and severity of asthma or wheezing depending on the specific causative agent of capillary bronchitis.
상기 기술된 수치 범위는 혈청을 시료로 하여 분석한 결과인 반면, 객담을 시료로 하는 경우에 대해서도 정량적인 결과를 토대로 한 객관적 진단 범위를 제공할 수 있다.While the above-described numerical range is the result of analyzing the serum as a sample, it is possible to provide an objective diagnosis range based on quantitative results even in the case of sputum as the sample.
즉, 본 발명의 일실시예에 따르면 객담 시료를 사용하는 경우 상기 모세기관지염이 마이코플라스마에 의해 유발된 경우, 모세기관지염 4개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 70ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 73%, 음성 예측값(negative predictive value, NPV)은 87%, 민감도(sensitivity)는 78% 및 특이도(specificity)는 70% 값을 가진다는 것을 알 수 있었고, 나아가 마이코플라스마의 자연적인 과정에서 EDN 수준이 130ng/ml 이상인 경우 추후 3회 이상 재발성 천식 또는 천명이 나타날 확률이 83%인 것으로 확인할 수 있었다. That is, according to one embodiment of the present invention, when using the sputum sample, when the capillary bronchitis is induced by mycoplasma, the level of eosinophil-derived neurotoxin (EDN) at 4 months after capillary bronchitis is 70ng / At ml or above, positive predictive value (PPV) is 73%, negative predictive value (NPV) is 87%, sensitivity is 78% and specificity is 70% In addition, in the natural course of mycoplasma, when the EDN level is 130ng / ml or more, there was an 83% chance of recurrent asthma or wheezing at least three times later.
본 발명에서 제공하는 진단 방법에 있어서 상기 환자는 생후 1개월~24개월의 영유아의 혈청 내 EDN 수준을 측정할 수 있고, 객담을 시료로 하는 경우에는 7세~10세의 아동기의 객담 내 EDN 수준을 측정할 수 있다.In the diagnostic method provided by the present invention, the patient can measure the serum EDN level of infants between 1 month and 24 months of age, and when sputum is used as a sample, the sputum EDN level in children between 7 and 10 years of age. Can be measured.
나아가 본 발명은 모세기관지염 3개월~5개월째의 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 농도를 측정하는 제제를 포함하는, 모세기관지염 이후 재발성 천식 또는 천명(recurrent wheezing) 진단용 조성물을 제공할 수 있고, 상기 조성물을 포함하는 진단용 키트를 제공할 수 있다.Furthermore, the present invention provides a composition for diagnosing recurrent asthma or recurrent wheezing after capillary bronchitis, including an agent for measuring the concentration of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis. It may be provided, it may be provided a diagnostic kit comprising the composition.
상기 키트는 모세기관지염 3개월~5개월째의 EDN 농도를 분석하여 모세기관지염 이후의 재발성 천식 또는 천명의 예후를 판단하는데 사용할 수 있다. 이를 위해, 상기 키트는 EDN 농도를 측정할 수 있도록 설계된다. 본 발명의 키트는 선택적으로, EDN 농도 분석에 필요한 시약, 인자, 조성물 등을 포함할 수 있다. The kit can be used to determine the prognosis of recurrent asthma or wheezing after capillary bronchitis by analyzing EDN concentrations at 3 to 5 months of capillary bronchitis. For this purpose, the kit is designed to measure the EDN concentration. Kits of the present invention may optionally include reagents, factors, compositions, and the like, required for EDN concentration analysis.
구체적으로 본 발명의 키트가 만일 PCR 증폭 과정에 적용되는 경우, 본 발명의 키트는 선택적으로, PCR 증폭에 필요한 시약, 예컨대, 완충액, DNA 중합효소 [예컨대, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis 또는 Pyrococcus furiosus (Pfu)로부터 수득한 열 안정성 DNA 중합효소], DNA 중합 효소 조인자 및 dNTPs를 포함할 수 있다.Specifically, if the kit of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases [eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth). ), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)], DNA polymerase cofactors and dNTPs.
또한 본 발명의 키트가 면역효소요법을 이용하는 경우, 본 발명의 키트는 EDN에 특이적인 항체 및 이의 용이한 검출을 위한 형광표지자와 함께 항체 반응을 위한 시약 등을 포함할 수 있다. In addition, when the kit of the present invention uses immunoenzymatic therapy, the kit of the present invention may include an antibody specific for EDN and a reagent for antibody reaction together with a fluorescent marker for easy detection thereof.
본 발명의 키트는 상기한 시약 성분을 포함하는 다수의 별도 패키징 또는 컴파트먼트로 제작될 수 있다. 바람직하게, 상기 진단 키트는 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성성분 조성물, 용액 또는 장치를 더 포함하여 구성될 수 있다. 일 구체예로서, 본 발명의 키트는 샘플을 담는 구획된 캐리어 수단, EDN 농도를 검출하는 수단이 함유된 용기를 포함하는 하나 이상의 용기를 포함할 수 있다. Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above. Preferably, the diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method. In one embodiment, the kit of the present invention may comprise one or more containers including a compartment containing a partitioned carrier means for holding a sample, a means for detecting an EDN concentration.
이처럼, 본 발명에서는 모세기관지염 환자에게서 호산구의 과립화 현상이 일어나는 것을 이용하여 이의 산물인 EDN의 농도를 측정함으로써, 특히 바람직하게는 모세기관지염 3개월~5째의 EDN 농도를 측정함으로써 재발성 천명 또는 천식의 예후를 판단할 수 있음을 확인하였고, 이를 활용하여, 재발성 천명 또는 천식 진단 및 치료제를 개발 또는 연구할 수 있음을 시사한다. As such, in the present invention, by measuring the concentration of EDN as a product thereof by using granulation of eosinophils in patients with capillary bronchitis, particularly preferably, by measuring the EDN concentration of capillary bronchitis 3 months to 5th, It was confirmed that the prognosis of asthma can be judged, and it can be used to develop or study recurrent wheezing or diagnosis and treatment of asthma.
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Kim et al(2010)의 문헌을 참조하여 실험을 수행하였다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples. The experiment was performed referring to the literature of Kim et al (2010).
실험 방법Experiment method
1~24개월의 영유아들 중에서 RSV(respiratory syncytial virus), 마이코플라즈마 및 메타뉴모 바이러스에 대해 첫 발작을 가진 100명의 각 환자들을 대상으로 하되, 임의적, 더이중 맹검(double-blind), 플라시보(위약)-조절, 병력군(paralle group)으로 나누었으며, 1~24개월의 영유아들로부터는 분석을 위한 시료로서 혈청을 사용하였다.Covering 100 patients with first seizures of respiratory syncytial virus (RSV), mycoplasma and metapneumovirus among infants between 1 and 24 months of age, randomized, double-blind, placebo (placebo) ) -Controlled, paralle group. Serum was used as a sample for analysis from infants between 1 and 24 months.
또한, 분석 시료로서 객담을 사용하는 경우에는 7~10세의 아동들 중에서 RSV(respiratory syncytial virus), 마이코플라즈마 및 메타뉴모 바이러스에 대해 첫 발작을 가진 100명의 각 환자들을 대상으로 상기 영유아들과 동일한 군으로 그룹핑하여 분석을 수행하였다. In addition, when sputum was used as analytical sample, 100 patients with the first seizure of respiratory syncytial virus (RSV), mycoplasma and metapneumovirus among children 7-10 years old were the same as the infants. Analysis was performed by grouping into groups.
천식 증상 또는 항-천식 치료의 병력을 가진 모세기관지염 환자들은 제외하였고 모세기관지염 이전에 천명이 있는 아이들 역시 제외시켰다. 36주 임신 전에 태어나고 만성적 질환을 앓고 있는 경우도 제외시켰다. 백신 접종을 잘 한 1~24개월의 50명의 정상 아이를 대조군으로 등록하였다. 이들은 알러지 질환, 천식 또는 공기를 통한 어떠한 감염도 없었다.Patients with bronchiolitis who had asthma symptoms or a history of anti-asthma treatment were excluded and children who had wheeze prior to bronchiolitis were also excluded. Those who were born before 36 weeks of pregnancy and had chronic diseases were excluded. Fifty normal children from 1 to 24 months of vaccination were enrolled as controls. They had no allergic disease, asthma or any infection through the air.
한편, 모세기관지염의 중증도 평가는 이전에 공지된 증상 스코어링 시스템을 기초로 하여 이루어졌다(Parkin et al. 1996): 호흡율(respiratory rate), 천명(wheezing), 퇴축(retraction), 호흡곤란(dyspnea) 및 흡기대 호기 비율(inspiratory-to-expiratory ratio) 항목들을 0, 1, 2 범위로 나누고, 최종 스코어는 최대값 10으로 요약하였다. 그리고, 바이러스 감염을 간접적 면역형광법에 의해 비인두 흡입에서 바이러스성 항원에 의해 확인하였다. 모든 영유아들은 입원동안 ß2-아고니스트를 이용한 통상적인 치료를 받았지만 코르티코스테로이드 처방은 받지 않았다.On the other hand, the severity evaluation of capillary bronchitis was based on a previously known symptom scoring system (Parkin et al. 1996): respiratory rate, wheezing, retraction, dyspnea And inspiratory-to-expiratory ratio items were divided into 0, 1, and 2 ranges, and the final score was summarized to a maximum of 10. Viral infections were then confirmed by viral antigen in nasopharyngeal inhalation by indirect immunofluorescence. All infants received conventional treatment with ß 2 -agonists during hospitalization but were not prescribed corticosteroids.
12주 동안 하기와 같은 그룹을 대상으로 하루에 한번 저녁에 4mg을 투여하였다. Merck&Co., Inc.로부터 플라시보(위약)로서 불활성, 경구 유사 그래뉼을 제공받았다. 블라인딩(blinding)의 활성 및 위약 그래뉼을 약제과로부터 컴퓨터-생성된 임의적 할당 스케쥴에 따라 제공받았다. 모든 실험 및 환자들을 전체 실험 기간동안 블라인드 상태로 유지하였다. ß2-아고니스트 이외 처방된 추가의 천식 치료는 이루어지지 않았다.For 12 weeks, the following groups were administered 4 mg once a day in the evening. Inert, oral-like granules were provided as a placebo (placebo) from Merck & Co., Inc. The activity of blinding and placebo granules were received from the Department of Medicine according to a computer-generated random assignment schedule. All experiments and patients were kept blind for the entire experimental period. No additional asthma treatment prescribed except ß 2 -agonists.
병력, 물리적 검토 및 일상적인 혈액 테스트가 전체 환자들에 대해 수행되었고 증상 스코어를 기록하였다. 팔로우(추적)-업 데이터를 12개월 동안 매 1개월마다 수집하였고(즉, 0, 1, 2, 3, 4,,,,12 월), 천명 또는 천식에 대한 2주마다의 전화 또는 의사 방문을 통해 천식 약물 치료에 대한 조사를 행하였다.History, physical review, and routine blood tests were performed on all patients and symptom scores were recorded. Follow-up data was collected every 12 months for 12 months (ie, 0, 1, 2, 3, 4 ,,, December), and 2 week's telephone or doctor visits for wheezing or asthma Was investigated for asthma medication.
이 때, 실험군 및 대조군 환자의 부모들의 동의 및 병원 윤리 위원회의 승인을 받았다. At this time, the consent of the parents of the experimental and control patients and the approval of the hospital ethics committee.
[분석대상 그룹][Group to analyze]
(1) montelukast oral granules[RSV-Montelukast(RSV-MONT) 그룹]처리군 & 매칭 플라시보[RSV-Placebos(RSV-PLC)그룹] 처리군(1) montelukast oral granules [RSV-Montelukast (RSV-MONT) group] treatment group & matching placebo [RSV-Placebos (RSV-PLC) group] treatment group
(2) 메타뉴모바이러스 치료제 처리군[메타뉴모바이러스-Montelukast oral granules) 그룹] & 매칭 플라시보 처리군(2) Metapneumovirus treatment group [Metanumovirus-Montelukast oral granules group] & matching placebo treatment group
(3) 마이코플라스마 치료제[마이코플라스마-클라리스로마이신 그룹]처리군 & 매칭 플라시보 처리군(3) Mycoplasma Therapeutics [Mycoplasma-Clarithromycin Group] Treatment Group & Matching Placebo Treatment Group
혈청 및 객담의 수집과 분석Collection and analysis of serum and sputum
먼저, 각 실험군 및 대조군으로부터 혈청을 수집하고 혈정 내 EDN 수준을 프로토콜(MBL, Woburn, MA)의 설명서에 따라 ELISA에 의해 측정하였고, 결과들을 ng/ml로 표시하였다. 이러한 ELISA <0.62ng/ml의 최소검출 한계, >300ng/ml의 최대 검출 한계 및 ECP와 교차반응 없이 인간 EDN을 검출하였다. 모든 분석은 2회씩 수행하였고, 평균값으로 통계학적 분석에 이용되었다. First, serum was collected from each experimental group and control group and EDN levels in blood serum were measured by ELISA according to the protocol (MBL, Woburn, MA) instructions, and the results were expressed in ng / ml. Human ELDN was detected without this ELISA minimum detection limit of <0.62 ng / ml, maximum detection limit of> 300 ng / ml and without cross-reaction with ECP. All analyzes were performed twice and were used for statistical analysis as mean values.
또한, 객담의 수집은 모든 실험군 및 대조군을 대상으로 DeVilbiss UltraNeb 99 ultrasonic nebulizer을 이용하여 제조한 3% 고장액 식염수 연무를 흡입시켜 객담을 유도하였고, 이때 타액이나 후비류의 오염을 방지하기 위해 생리식염수로 입을 헹군 후 코를 푼 다음 3% 고장액 식염수 연무를 흡입하게 한 다음, 소독된 용기에 매 2분마다 객담 배출을 시도하게 하여 객담을 수집하였고, 객담 내 EDN의 측정은 EDN에 대한 항체를 사용한 면역측정법을 이용하여 측정하였다.In addition, sputum was collected by inhalation of 3% hypertonic saline haze prepared using DeVilbiss UltraNeb 99 ultrasonic nebulizer for all experimental and control groups, and sputum was induced at this time, in order to prevent contamination of saliva or manure. Sputum was collected by rinsing the mouth with a 3% hypertonic saline mist and then attempting to drain the sputum every 2 minutes into a sterilized container. It was measured using the immunoassay used.
통계학적 분석Statistical analysis
모든 데이터들은 처리 코드에 따라 그룹핑되어 모세기관지염 후 호산구 탈과립(eosinophil degranulation)이 천명과 관련이 있는지 여부를 1차 결과로 결정지었다. 천명(wheezing) 또는 천식 재발의 마커로서, 각 측정 순간의 EDN수준을 조사하였다. All data were grouped according to treatment code to determine the primary outcome whether eosinophil degranulation was associated with wheezing after capillary bronchitis. As markers of wheezing or asthma recurrence, the EDN levels at each measurement instant were examined.
3그룹에서 차이를 나타내는 데이터들의 스크리닝은 Kruskall-Wallis test를 이용하여 수행하였고, 유의미한 차이점이 확인되었을 때, Mann-Whitney U test를 이용하여 개별적 그룹들을 비교하고 호산구 탈과립화의 마커 및 다른 파라미터 측정에 사용하였다. Spearman 상관계수를 계산하여 값들 사이의 상관관계를 평가하였다. P-value가 0.05 이하일 때 통계학적으로 유의미한 것으로 간주하였다. Screening of data representing differences in the three groups was performed using the Kruskall-Wallis test, and when significant differences were identified, the individual groups were compared using the Mann-Whitney U test to determine markers of eosinophil degranulation and other parameters. Used. The Spearman correlation coefficient was calculated to evaluate the correlation between the values. When the P-value was less than 0.05, it was considered statistically significant.
실시예 1 : EDN과 재발성 천식/천명과의 상관관계 확인Example 1 Check the Correlation between EDN and Recurrent Asthma / Wheezing
실험에 사용된 환자 정보는 다음과 같다. 특히 영유아 생후 1~24개월에 해당하는 발병 원인균에 의한 모세기관지염 발병 영유아들을 아래와 같이 그룹핑하여 이들 환자군 및 정상 대조군으로부터 상기 기술된 바와 같이 혈청 및 객담을 수집하여 상관관계를 분석하였다.The patient information used in the experiment is as follows. In particular, infants with capillary bronchitis caused by the causative agent of infants 1 to 24 months of age were grouped as follows and serum and sputum collected from these patient groups and normal controls were collected and analyzed for correlation.
<1> (RSV-MONT, RSV-PLC, 대조군 각각):<1> (RSV-MONT, RSV-PLC, control group respectively):
- n : 71,79,28n: 71,79,28
- 연령범위(혈청시료 분석대상): 1-23개월, 1-23개월, 1-23개월Age range (serum sample analysis target): 1-23 months, 1-23 months, 1-23 months
- 연령범위(객담시료 분석대상): 7~10세, 7~10세, 7~10세Age range (targeted for sputum analysis): 7-10 years old, 7-10 years old, 7-10 years old
- 성별 M:F : 54:25, 46:25, 12:16-Gender M: F: 54:25, 46:25, 12:16
- 아토피성 유전(atopic heredity) % : 18.5, 16.2, 0.0% Atopic heredity: 18.5, 16.2, 0.0
- 혈중 호산구 mm3, 평균(범위) : 190(11-809), 168(39-900), 91(49-334)Blood eosinophils mm3, mean (range): 190 (11-809), 168 (39-900), 91 (49-334)
- 증상 스코어, 1-10, 평균(범위) : 6.1(4-9), 5.9(4-9), n/aSymptom score, 1-10, Mean (range): 6.1 (4-9), 5.9 (4-9), n / a
<2> (메타뉴모 바이러스-치료제 처리군, 메타뉴모 바이러스-플라시보 군, 대조군 각각):<2> (metaneumo virus-therapeutic treatment group, metapneumo virus-placebo group, control group, respectively):
- n : 70,80,27n: 70,80,27
- 연령범위: 1-23개월, 1-23개월, 1-23개월Age range: 1-23 months, 1-23 months, 1-23 months
- 연령범위(객담시료 분석대상): 7~10세, 7~10세, 7~10세Age range (targeted for sputum analysis): 7-10 years old, 7-10 years old, 7-10 years old
- 성별 M:F : 55:27, 44:26, 11:18Gender M: F: 55:27, 44:26, 11:18
- 아토피성 유전(atopic heredity) % : 18.0, 15.2, 0.0% Atopic heredity: 18.0, 15.2, 0.0
- 혈중 호산구 mm3, 평균(범위) : 190(11-809), 168(39-900), 91(49-334)Blood eosinophils mm3, mean (range): 190 (11-809), 168 (39-900), 91 (49-334)
- 증상 스코어, 1-10, 평균(범위) : 6.1(4-9), 5.9(4-9), n/aSymptom score, 1-10, Mean (range): 6.1 (4-9), 5.9 (4-9), n / a
<3> (마이코플라스마(M. pneumoniae)-치료제 처리군, 마이코플라스마-플라시보 군, 대조군 각각):<3> ( M. pneumoniae -therapeutic treatment group, mycoplasma-placebo group, control group, respectively):
- n : 68,78,27n: 68,78,27
- 연령범위: 1-22개월, 1-24개월, 1-21개월Age range: 1-22 months, 1-24 months, 1-21 months
- 연령범위(객담시료 분석대상): 7~10세, 7~10세, 7~10세Age range (targeted for sputum analysis): 7-10 years old, 7-10 years old, 7-10 years old
- 성별 M:F : 53:24, 40:26, 13:14-Gender M: F: 53:24, 40:26, 13:14
- 아토피성 유전(atopic heredity) % : 18.0, 15.2, 0.0% Atopic heredity: 18.0, 15.2, 0.0
- 혈중 호산구 mm3, 평균(범위) : 190(11-809), 168(39-900), 91(49-334)Blood eosinophils mm3, mean (range): 190 (11-809), 168 (39-900), 91 (49-334)
- 증상 스코어, 1-10, 평균(범위) : 6.1(4-9), 5.9(4-9), n/aSymptom score, 1-10, Mean (range): 6.1 (4-9), 5.9 (4-9), n / a
여기서 상기 치료제 처리군은 질병군이고 플라시보 처리군은 안정기 군이며, 대조군은 질병에 걸리지 않은 건강한 정상군을 말한다. Here, the therapeutic agent treatment group is a disease group, the placebo treatment group is a stabilizer group, and the control group refers to a healthy normal group without disease.
또한 구체적으로, 기준선, 1개월, 2개월, 3개월에서 12개월까지 매월 혈청 EDN 수준 및 천식과 천명 사이의 상관관계를 조사하였다.Specifically, we examined the correlation between serum EDN levels and asthma and wheeze at baseline, 1 month, 2 months, 3 months to 12 months.
<결과 1-1><Result 1-1>
혈청 EDN 분석을 통해 RSV-PLC 그룹(r=0.70, P<0.0001) 및 RSV-MONT 그룹(r=0.52, P<0.001) 에서는 12개월 천명 발작의 전체 횟수와 4개월(치료 마지막)에서의 혈청 EDN 수준이 유의적으로 상관관계가 있었고, 반면, 12개월 째 전체 천명 발작 수와 4개월 미만인 2개월, 3개월 및 4개월을 초과한 5~12개월째의 EDN 수준 사이에는 유의미한 상관관계가 나타나지 않았다.Serum EDN analysis revealed the total number of 12-month wheezing attacks and serum at 4 months (last treatment) in the RSV-PLC group (r = 0.70, P <0.0001) and RSV-MONT group (r = 0.52, P <0.001). There was a significant correlation between EDN levels, whereas there was no significant correlation between the total number of wheezing seizures at 12 months and the EDN levels at 5-12 months beyond 2 months, 3 months and 4 months below 4 months. Did.
또한, 상승된 EDN 수준의 컷 오프(cut-off)로서 45ng/ml(median+2SD)을 이용하여, 12 개월 동안 누적되는 재발 천명에 대한 4 개월째 EDN 수준의 양성 예측값(positive predictive value, PPV)은 65%였고, 음성 예측값(negative predictive value, NPV)은 80%, 민감도(sensitivity)는 70% 그리고 특이도(specificity)는 72%였다. In addition, positive predictive value (PPV) at 4 months of EDN levels for 12 months of cumulative recurrent wheezing, using 45ng / ml (median + 2SD) as cut-off of elevated EDN levels. ) Was 65%, negative predictive value (NPV) was 80%, sensitivity 70% and specificity 72%.
또한, RSV-PLC 그룹(RSV 모세기관지염의 자연적인 과정)에서는 EDN이 100ng/ml 이상인 경우 추후 2회 이상 재발성 천명이 나타날 확률은 75%인 것으로 나타났다. 즉, EDN이 100ng/ml 이상인 경우 2번 이상 천명이 발생한 확률이 75%임을 알 수 있었다.In addition, in the RSV-PLC group (a natural course of RSV capillary bronchitis), if the EDN is 100ng / ml or more, there is a 75% chance of recurring wheezing at least twice later. In other words, when the EDN is 100ng / ml or more, the probability of occurrence of two or more wheezes was 75%.
흥미롭게도, RSV-PLC 그룹(즉, RSV 모세기관지염의 자연적인 과정)에서의 최초의 전체 호산구는 12개월째(spearman r=0.4044, P=0.0059) 천명 발작의 누적적 수와 관련이 있었던 반면, RSV-MONT 그룹(spearman r=0.4-0.05470, P=0.6553)에서 이러한 2 값 사이에는 상관관계가 없었다.Interestingly, the first total eosinophils in the RSV-PLC group (ie, the natural course of RSV capillary bronchitis) were associated with a cumulative number of wheezing seizures at 12 months (spearman r = 0.4044, P = 0.0059), There was no correlation between these 2 values in the RSV-MONT group (spearman r = 0.4-0.05470, P = 0.6553).
그 결과, 혈청 EDN 분석을 통해 RSV-PLC 그룹(r=0.70, P<0.0001) 및 RSV-MONT 그룹(r=0.52, P<0.001) 에서는 12개월 천명 발작의 전체 횟수와 4개월(치료 마지막)에서의 혈청 EDN 수준이 유의적으로 상관관계가 있었고, 반면, 12개월 째 전체 천명 발작 수와 4개월 미만인 2개월, 3개월 및 4개월을 초과한 5~12개월째의 EDN 수준 사이에는 유의미한 상관관계가 나타나지 않았다.As a result, the total number of 12-month wheezing attacks and 4 months (last treatment) in the RSV-PLC group (r = 0.70, P <0.0001) and RSV-MONT group (r = 0.52, P <0.001) were determined by serum EDN analysis. There was a significant correlation between serum EDN levels in, whereas there was a significant correlation between the total number of wheezing seizures at 12 months and EDN levels at 2-12 months, 3 months and 4 months below 4 months. The relationship did not appear.
또한, 상승된 EDN 수준의 컷 오프(cut-off)로서 45ng/ml(median+2SD)을 이용하여, 12 개월 동안 누적되는 재발 천명에 대한 4 개월째 EDN 수준의 양성 예측값(positive predictive value, PPV)은 65%였고, 음성 예측값(negative predictive value, NPV)은 80%, 민감도(sensitivity)는 70% 그리고 특이도(specificity)는 72%였다. In addition, positive predictive value (PPV) at 4 months of EDN levels for 12 months of cumulative recurrent wheezing, using 45ng / ml (median + 2SD) as cut-off of elevated EDN levels. ) Was 65%, negative predictive value (NPV) was 80%, sensitivity 70% and specificity 72%.
또한, RSV-PLC 그룹(RSV 모세기관지염의 자연적인 과정)에서는 EDN이 100ng/ml 이상인 경우 추후 2회 이상 재발성 천명이 나타날 확률은 75%인 것으로 나타났다. 즉, EDN이 100ng/ml 이상인 경우 2번 이상 천명이 발생한 확률이 75%임을 알 수 있었다.In addition, in the RSV-PLC group (a natural course of RSV capillary bronchitis), if the EDN is 100ng / ml or more, there is a 75% chance of recurring wheezing at least twice later. In other words, when the EDN is 100ng / ml or more, the probability of occurrence of two or more wheezes was 75%.
흥미롭게도, RSV-PLC 그룹(즉, RSV 모세기관지염의 자연적인 과정)에서의 최초의 전체 호산구는 12개월째(spearman r=0.4044, P=0.0059) 천명 발작의 누적적 수와 관련이 있었던 반면, RSV-MONT 그룹(spearman r=0.4-0.05470, P=0.6553)에서 이러한 2 값 사이에는 상관관계가 없었다.Interestingly, the first total eosinophils in the RSV-PLC group (ie, the natural course of RSV capillary bronchitis) were associated with a cumulative number of wheezing seizures at 12 months (spearman r = 0.4044, P = 0.0059), There was no correlation between these 2 values in the RSV-MONT group (spearman r = 0.4-0.05470, P = 0.6553).
<결과 1-2><Result 1-2>
혈청 EDN 분석을 통해 메타뉴모 바이러스 치료제 처리군(r=0.50, P<0.0001, 메타뉴모 바이러스 플라시보 처리군(r=0.68, P<0.0001) 에서는 12개월 천식 및 천명 발작의 전체 횟수와 3개월(치료 마지막)에서의 혈청 EDN 수준이 유의적으로 상관관계가 있었고, 반면, 12개월 째 전체 천명 발작 수와 3개월 미만인 2개월, 1개월 및 3개월을 초과한 4~12개월째의 EDN 수준 사이에는 유의미한 상관관계가 나타나지 않았다.Serum EDN analysis showed that the total number of 12-month asthma and wheezing seizures and 3 months (treatment) in the metapneumovirus treatment group (r = 0.50, P <0.0001, metapneumovirus placebo treatment group (r = 0.68, P <0.0001) There was a significant correlation between serum EDN levels at the end), whereas there was a difference between the total number of wheezing seizures at 12 months and EDN levels at 2 months, 1 month, and 3 months over 3 months. There was no significant correlation.
또한, 상승된 EDN 수준의 컷 오프(cut-off)로서 50ng/ml(median+2SD)을 이용하여, 12 개월 동안 누적되는 재발 천명에 대한 3 개월째 EDN 수준의 양성 예측값(positive predictive value, PPV)은 70%였고, 음성 예측값(negative predictive value, NPV)은 82%, 민감도(sensitivity)는 75% 그리고 특이도(specificity)는 70%였다. In addition, positive predictive value (PPV) at the third month of EDN levels for 12 months of cumulative relapse wheezing, using 50ng / ml (median + 2SD) as the cut-off of elevated EDN levels. ) Was 70%, negative predictive value (NPV) 82%, sensitivity 75% and specificity 70%.
또한, 메타뉴모 바이러스에 의한 모세기관지염의 자연적인 과정에서 EDN이 80ng/ml 이상인 경우 추후 3회 이상 재발성 천식 및 천명이 나타날 확률은 80%인 것으로 나타났다. 즉, EDN이 80ng/ml 이상인 경우 3번 이상 천식 및 천명이 발생한 확률이 80%임을 알 수 있었다.In addition, in the natural course of capillary bronchitis caused by metapneumovirus, if the EDN is 80ng / ml or more, the probability of recurrent asthma and wheezing at least three times later is 80%. In other words, when the EDN is 80ng / ml or more, it was found that the probability of asthma and wheezing occurring three or more times is 80%.
<결과 1-3><Result 1-3>
혈청 EDN 분석을 통해 마이코플라스마 치료제 처리군(r=0.50, P<0.0001), 마이코플라스마 플라시보 처리군(r=0.68, P<0.0001) 에서는 12개월 천식 및 천명 발작의 전체 횟수와 5개월(치료 마지막)에서의 혈청 EDN 수준이 유의적으로 상관관계가 있었고, 반면, 12개월 째 전체 천명 발작 수와 5개월 미만인 1~4개월 및 5개월을 초과한 6~12개월째의 EDN 수준 사이에는 유의미한 상관관계가 나타나지 않았다.Serum EDN analysis showed that the total number of asthma and wheezing attacks and the 5 months (last treatment) in the mycoplasma treatment group (r = 0.50, P <0.0001) and the mycoplasma placebo treatment group (r = 0.68, P <0.0001). There was a significant correlation between serum EDN levels in), whereas there was a significant correlation between total wheezing seizures at 12 months and EDN levels at 1-4 months less than 5 months and 6-12 months above 5 months. The relationship did not appear.
또한, 상승된 EDN 수준의 컷 오프(cut-off)로서 60ng/ml(median+2SD)을 이용하여, 12 개월 동안 누적되는 재발 천명에 대한 5 개월째 EDN 수준의 양성 예측값(positive predictive value, PPV)은 63%였고, 음성 예측값(negative predictive value, NPV)은 77%, 민감도(sensitivity)는 78% 그리고 특이도(specificity)는 68%였다. In addition, positive predictive value (PPV) at 5 months of EDN levels for 12 months of cumulative relapse wheezing, using 60ng / ml (median + 2SD) as cut-off of elevated EDN levels. ) Was 63%, negative predictive value (NPV) was 77%, sensitivity was 78% and specificity 68%.
또한, 마이코플라스마에 의한 모세기관지염의 자연적인 과정에서 EDN이 130ng/ml 이상인 경우 추후 3회 이상 재발성 천식 및 천명이 나타날 확률은 83%인 것으로 나타났다. 즉, EDN이 130ng/ml 이상인 경우 3번 이상 천식 및 천명이 발생한 확률이 83%임을 알 수 있었다.In addition, in the natural course of capillary bronchitis caused by mycoplasma, when the EDN is 130ng / ml or more, the probability of recurrent asthma and wheezing three or more times is 83%. That is, when the EDN is 130ng / ml or more, 83% of the three or more asthma and wheezing occurred.
<결과 1-4><Result 1-4>
상기 결과 <1-3>의 내용은 실험군과 대조군의 혈청을 대상으로 분석한 결과라면 본 내용은 객담 시료를 대상으로 분석한 결과를 나타낸 것으로, 마이코플라스마에 의한 감염으로 모세기관지염이 유발된 아동들을 대상으로 분석을 수행하였다.The results of <1-3> are the results of the analysis of the serum of the experimental group and the control group, and the contents of this analysis are the results of the analysis of the sputum sample. Analysis was performed on the subject.
상기 기술한 바와 같이, 7세~10세의 아동들을 대상으로 분석한 결과, 상승된 EDN 수준의 컷 오프(cut-off)로서 70ng/ml(median+2SD)을 이용하여, 12 개월 동안 누적되는 재발 천명에 대한 4개월째 EDN 수준의 양성 예측값(positive predictive value, PPV)은 73%였고, 음성 예측값(negative predictive value, NPV)은 87%, 민감도(sensitivity)는 78% 그리고 특이도(specificity)는 70%였다. As described above, the analysis of children aged 7 to 10 years, using 70ng / ml (median + 2SD) as a cut-off of the elevated EDN level, accumulated for 12 months The positive predictive value (PPV) of EDN levels at 73 months for recurrent wheezing was 73%, negative predictive value (NPV) was 87%, sensitivity was 78% and specificity Was 70%.
마이코플라스마에 의한 모세기관지염의 자연적인 과정에서 EDN이 80ng/ml 이상인 경우 추후 3회 이상 재발성 천식 및 천명이 나타날 확률은 88%인 것으로 나타났다. 즉, EDN이 80ng/ml 이상인 경우 3번 이상 천식 및 천명이 발생한 확률이 88%임을 알 수 있었다.In the natural course of capillary bronchitis caused by mycoplasma, if the EDN is 80ng / ml or more, the probability of recurrent asthma and wheezing more than three times is 88%. That is, when the EDN is 80ng / ml or more, it was found that the probability of asthma and wheezing occurring more than three times was 88%.
달리 정의되지 않는 한, 본원에서 사용된 모든 기술적 및 과학적 용어는 본 발명이 속하는 분야의 당업자가 통상적으로 이해하는 것과 동일한 의미를 갖는다. 본원에 기술된 것들과 유사하거나 등가인 임의의 방법 및 재료가 본 발명을 테스트하기 위한 실행에서 사용될 수 있다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention.

Claims (13)

  1. 모세기관지염 이후 천식 또는 천명 진단에 필요한 정보를 제공하기 위해, 모세기관지염 3개월~5개월째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준을 측정하는 방법.A method of measuring the level of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis to provide information necessary for asthma or wheezing after capillary bronchitis.
  2. 제1항에 있어서, The method of claim 1,
    상기 EDN의 수준은 혈청 또는 객담 내 중 EDN의 농도인 것을 특징으로 하는 방법.The level of EDN is a concentration of EDN in serum or sputum.
  3. 제1항에 있어서, The method of claim 1,
    상기 모세기관지염은 호흡기 세포융합 바이러스(respiratory syncytial virus, RSV), 메타뉴모 바이러스, 아데노바이러스, 파라인플루엔자 바이러스, 인플루엔자 바이러스, 라이노바이러스, 아데노바이러스, 홍역 바이러스 및 마이코플라즈마로 이루어진 군 중에서 선택되는 유해균의 감염으로 유발되는 것을 특징으로 하는 방법.The capillary bronchitis is an infection of a harmful bacterium selected from the group consisting of respiratory syncytial virus (RSV), metapneumovirus, adenovirus, parainfluenza virus, influenza virus, rhinovirus, adenovirus, measles virus and mycoplasma. Method characterized in that caused by.
  4. 제1항에 있어서,The method of claim 1,
    상기 모세기관지염이 호흡기 세포융합 바이러스(respiratory syncytial virus, RSV)에 의한 감염으로 유발된 경우, RSV 모세기관지염 4개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 45ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 65%, 음성 예측값(negative predictive value, NPV)은 80%, 민감도(sensitivity)는 70% 및 특이도(specificity)는 72% 값을 가지는 것을 특징으로 하는 방법.When capillary bronchitis is caused by infection with respiratory syncytial virus (RSV), the relapse occurs when the level of eosinophil-derived neurotoxin (EDN) is higher than 45 ng / ml at 4 months of RSV capillary bronchitis. Positive predictive value (PPV) of 65%, negative predictive value (NPV) of 80%, sensitivity of 70% and specificity of 72% How to.
  5. 제1항에 있어서,The method of claim 1,
    상기 모세기관지염이 메타뉴모 바이러스에 의해 유발된 경우, 모세기관지염 3개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 50ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 70%, 음성 예측값(negative predictive value, NPV)은 82%, 민감도(sensitivity)는 75% 및 특이도(specificity)는 70% 값을 가지는 것을 특징으로 하는 방법. If the capillary bronchitis is caused by the metapneumovirus, positive predictive value (PPV) of recurrent wheeze when the level of eosinophil-derived neurotoxin (EDN) is greater than 50 ng / ml at 3 months after capillary bronchitis ) 70%, negative predictive value (NPV) 82%, sensitivity (75%) and specificity (specificity) has a value of 70%.
  6. 제1항에 있어서,The method of claim 1,
    상기 모세기관지염이 마이코플라스마에 의해 유발된 경우, 모세기관지염 5개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 60ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 63%, 음성 예측값(negative predictive value, NPV)은 77%, 민감도(sensitivity)는 78% 및 특이도(specificity)는 68% 값을 가지는 것을 특징으로 하는 방법When capillary bronchitis is caused by mycoplasma, positive predictive value (PPV) of recurrent wheezing when the level of eosinophil-derived neurotoxin (EDN) is greater than 60 ng / ml at 5 months after capillary bronchitis Is 63%, negative predictive value (NPV) is 77%, sensitivity is 78% and specificity is 68%
  7. 제4항 내지 제6항에 있어서,The method according to claim 4 to 6,
    상기 방법은 생후 1개월~24개월의 영유아의 혈청 내 EDN 수준을 측정하는 것을 특징으로 하는 방법. The method is characterized by measuring the EDN level in the serum of infants 1 month to 24 months of age.
  8. 제1항에 있어서,The method of claim 1,
    RSV 모세기관지염의 자연적인 과정에서 EDN 수준이 100ng/ml 이상인 경우 추후 2회 이상 재발성 천식 또는 천명이 나타날 확률이 75%이고, In the natural course of RSV capillary bronchitis, with an EDN level of 100 ng / ml or more, there is a 75% chance of developing recurrent asthma or wheezing at least twice later.
    메타뉴모 바이러스에 의한 모세기관지염의 자연적인 과정에서 EDN 수준이 80ng/ml 이상인 경우 추후 3회 이상 재발성 천식 또는 천명이 나타날 확률이 80%이고, In the natural course of capillary bronchitis caused by metapneumovirus, if the EDN level is 80ng / ml or more, there is an 80% chance of recurring asthma or wheezing at least three times later.
    마이코플라스마의 자연적인 과정에서 EDN 수준이 130ng/ml 이상인 경우 추후 3회 이상 재발성 천식 또는 천명이 나타날 확률이 83%인 것을 특징으로 하는 방법.If the EDN level is more than 130ng / ml during the natural course of mycoplasma, there is a 83% chance of recurring asthma or wheezing three or more times later.
  9. 제2항에 있어서,The method of claim 2,
    객담 시료를 사용하는 경우 상기 모세기관지염이 마이코플라스마에 의해 유발된 경우, 모세기관지염 4개월 째 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 수준이 70ng/ml 이상일 때 재발성 천명의 양성 예측값(positive predictive value, PPV)은 73%, 음성 예측값(negative predictive value, NPV)은 87%, 민감도(sensitivity)는 78% 및 특이도(specificity)는 70% 값을 가지는 것을 특징으로 하는 방법.When sputum samples are used, when capillary bronchitis is caused by mycoplasma, positive predictive value of recurrent wheezing (when the level of eosinophil-derived neurotoxin (EDN) is above 70 ng / ml at 4 months after capillary bronchitis a positive predictive value (PPV) of 73%, a negative predictive value (NPV) of 87%, a sensitivity of 78%, and a specificity (specificity) of 70%.
  10. 제2항에 있어서,The method of claim 2,
    객담 시료를 사용하여 분석하는 경우에는 7세~10세의 아동기의 객담 내 EDN 수준을 측정하는 것을 특징으로 하는 방법. In the case of analysis using a sputum sample, the method characterized by measuring the EDN level in the sputum of children 7 to 10 years old.
  11. 모세기관지염 3개월~5개월째의 호산구-유래 신경독(eosinophil-derived neurotoxin, EDN)의 농도를 측정하는 제제를 포함하는, 모세기관지염 이후 재발성 천식 또는 천명(recurrent wheezing) 진단용 키트.Kit for diagnosing recurrent wheezing after capillary bronchitis, comprising an agent for measuring the concentration of eosinophil-derived neurotoxin (EDN) at 3 to 5 months of capillary bronchitis.
  12. 제11항에 있어서, The method of claim 11,
    상기 EDN의 농도는 혈청 또는 객담 내 EDN의 농도인 것을 특징으로 하는 키트.The concentration of EDN is a kit, characterized in that the concentration of EDN in serum or sputum.
  13. 제11항에 있어서, The method of claim 11,
    상기 제제는 EDN에 특이적으로 결합할 수 있는 프라이머, 프로브 또는 항체인 것을 특징으로 하는 키트.The agent is a kit, characterized in that the primer, probe or antibody that can specifically bind to EDN.
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