KR101592068B1 - Marker and Kit for Diagnosising and Distingushing Aspirin exacerbated respiratory diseases and Aspirin tolerant asthma - Google Patents

Abstract

The present invention relates to an aspirin-inducing respiratory disease (AERD) and an aspirin-resistant asthma (ATA) discriminating marker composition and a diagnostic kit comprising plasma apolipoprotein H.
ELISA was performed to identify and quantify ApoH in plasma between AERD and ATA subjects. Eight protein spots showed a relative intensity difference between collected plasma of AERD ( n = 8) and ATA ( n = 8) subjects. MALDI-TOF / TOF analysis showed a decrease in the levels of alpha-fibrinolytic rsP, ApoH, fibrin beta, and proapolipoprotein in AERD compared with ATA, human complement component C3, 90-kDa thermal shock protein, complement component C4a and kininogen-1 isoform 2 increase. Apo H concentration was significantly increased in plasma of ATA subjects compared to AERD and general control results ( P <0.01). AERD is characterized by changes in protein levels associated with the coagulation and complement pathway. In addition, ApoH is increased in ATA compared to AERD and normal control, indicating that ApoH is associated with other etiologic causes of AERD and ATA.

Description

Technical Field [0001] The present invention relates to an aspirin-sensitive respiratory disease disease-causing aspirin-resistant asthma-specific marker and a diagnostic marker for aspirin-exacerbated respiratory diseases and aspirin-tolerant asthma including plasma apolipoprotein H,

The present invention relates to a biomarker composition for diagnosing aspirin-induced respiratory diseases (AERD) and aspirin-resistant asthma (ATA) including plasma apolipoprotein H, and a diagnostic kit.

Aspirin-sensitized respiratory disease (AERD) refers to the development of signs of bronchoconstriction and nose and eyes in asthmatic patients following administration of aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs) (Stevenson, Sanchez-Borges, and Szczeklik 2001) . Recently, aspirin hypersensitivity has attracted much attention because it is associated with an increase in asthma susceptibility, including life-threatening asthma attacks and possible remodeling of other upper and lower airways (Mascia et al. 2005) .

25% of asthmatic patients requiring emergency mechanical emergency machine ventilation are resistant to aspirin (Picado et al., 1989; Marquette et al., 1992). However, because AERD is not fully aware of the relationship between ASA intake and asthma exacerbations, most asthma patients are not properly diagnosed. In a multicenter study of 500 patients in 10 European countries, 15% of patients did not observe aspirin sensitivity before the aspirin challenge test (Szczeklik, Nizankowska, and Duplaga 2000). In Korea, the sensitivity and specificity of the aspirin sensitivity history for AERD prediction were 64.7% and 92.0%, respectively (Chang et al.). Therefore, identification of aspirin sensitivity is essential to avoid serious complications, especially if not known.

Increased ASA capacity can be assured only by conducting an aspirin stimulation test. Aspirin Oral Induction (OAC) is a gold standard for confirming diagnosis (Szczeklik and Nizankowska 2000). However, OAC is a time consuming procedure and often causes serious complications. Therefore, the development of non-invasive methods for easy diagnosis is needed to prevent unexpected complications of aspirin use in suspect patients.

We have found that the human complement components C3a and C4a are increased in AERD compared to aspirin resistant asthma (ATA) after baseline and aspirin administration (Lee et al. 2006).

In addition, we found changes in apolipoprotein A-1, fertilized albumin and unknown proteins. However, identifying the presence of a very abundant plasma protein was a technical limitation of previous studies that did not have the ability to illuminate proteins with spots overlapping these very abundant proteins in two-dimensional gel electrophoresis (2DE) images.

In the present invention, we removed much of the protein from the plasma prior to performing the 2DE assay and compared the intensity of the protein spots between AERD and ATA.

The present invention provides a biomarker composition for diagnosing discrimination between ATA and AERD, which comprises a plasma apolipoprotein H as a biomarker for discriminating and diagnosing AERD from ATA patients to solve the above problems, And to provide a kit.

In order to achieve the above object, the present invention provides a biomarker for discriminating and diagnosing AERD (aspirin-tolerant asthma) patients from AERD (plasma aspirin-resistant respiratory diseases) and plasma apolipoprotein H (ApoH) The present invention also provides a biomarker composition for distinguishing between ATA and AERD.

In addition, the present invention provides an ATA and AERD discrimination diagnostic kit, which comprises plasma apolipoprotein H or an antibody capable of complementarily binding thereto.

In the present invention, ELISA was performed to identify and quantify ApoH in plasma between AERD and ATA subjects. Eight protein spots showed a relative intensity difference between collected plasma of AERD ( n = 8) and ATA ( n = 8) subjects. MALDI-TOF / TOF analysis showed a decrease in the levels of alpha-fibrinolytic rsP, ApoH, fibrin beta, and proapolipoprotein in AERD compared with ATA, human complement component C3, 90-kDa thermal shock protein, complement component C4a and kininogen-1 isoform 2 increase. Apo H concentration was significantly increased in plasma of ATA subjects compared to AERD and general control results ( P <0.01). AERD is characterized by changes in protein levels associated with the coagulation and complement pathway. In addition, ApoH is increased in ATA compared to AERD and normal control, indicating that ApoH is associated with other etiologic causes of AERD and ATA. Thus, the present invention can be used to diagnose aspirin-induced respiratory diseases (AERD) and aspirin-resistant asthma (ATA) including plasma apolipoprotein H.

Figure 1 shows two-dimensional electrophoresis analysis of plasma samples from subjects with aspirin-induced respiratory disease (AERD) ( n = 8) and aspirin-resistant asthma (ATA) ( n = 8). Plasma protein (1 mg) was fitted to pH 3-10 fixed pH slope strips (according to the examples described below), separated by 7.5-20% gradient SDS-PAGE, and visualized. Protein spots identified by MALDI-TOF / TOF were numbered. Spot # 13 is shown enlarged in a small box.
FIG. 2 is a table showing the clinical characteristics of the subject of the present study.
Figure 3 is a table showing the eight proteins representing the differences between the ATA group and the AERD group. The relative intensities of spot numbers 1, 2, 3 and 4 were significantly lower in the AERD group than in the ATA group. In contrast, spots 5, 6, 7 and 8 were significantly higher in the AERD group compared to the ATA group.
Figure 4 details the differentially expressed proteins identified by MALDI-TOF / TOF.
Figure 5 compares Apolipoprotein H (Apo H) expression in ATA and AERD subjects. (A) Apo H plasma levels were significantly lower in AERD subjects than in ATA subjects (ATA, 348.98 ± 9.18 μg / ml; AERD; 300.00 ± 8.34 μg / ml; P = 0.0001). (B) ROC analysis for Apo H below baseline in AERD and ATA patients has an area under the curve of 0.717. Apo H level 315.00 ㎍ / ml cut-off value at AERD below baseline had a sensitivity of 71.6% and a specificity of 56.6%.
FIG. 6 shows the relationship between Apo H expression and FEV ₁ reduction after administration of aspirin in asthmatic patients including AERD and ATA. There was a strong inverse correlation between OA (aspirin challenge) and Apo H concentration and FEV ₁ (one second ventilation) in patients with asthma, including AERD and ATA.

The present invention relates to an aspirin-inducing respiratory disease including plasma apolipoprotein H, and a marker for diagnosing distinction between aspirin resistant asthma and a diagnostic kit.

Hereinafter, the present invention will be described in detail with reference to examples.

[ Example  1: subjects]

Plasma from the AERD ( n = 53) and ATA ( n = 53) and control ( n = 24) subjects was analyzed at the biobank of Soonchunhyang University Bucheon Hospital using the protocols approved by the local Ethics Committee of Soonchunhyang University Hospital (Approval No. Schbc-biobank-2011-007). The control group responded negatively to the screening questionnaire for respiratory symptoms and found that the FEV1 value for one second was above the predicted value of 75% and PC ₂₀ for methacholine less than 10 mg / ml caused a 20% reduction in FEV1 ), And general and patient spouses with generalized findings on chest radiography.

All subjects were Koreans and agreed to blood transfusion in the hospital's biobank. All patients were diagnosed by physicians and matched asthma criteria as defined by the International Asthma Association (GINA) guidelines and online appendix.

Patients with asthma did not experience asthma or respiratory tract infections aggravated by OAC (aspirin orally) 6 weeks prior. OAC was performed increasingly with increased aspirin dose as described in the online appendix (Kim et al .; Nizankowska-Mogilnicka et al. 2007).

Aspirin-induced bronchospasm with FEV ₁ decline (%) was calculated as [pre-dose FEV ₁ ] - [post-dose FEV ₁ ] / [pre-dose FEV ₁ ]. The OAC response was divided into two groups: (1) a 15% reduction in FEV ₁ or a nasal reaction (AERD), or (2) a naso-ocular or skin reaction Less than 15% at FEV ₁ (ATA). Plasma was obtained from whole peripheral vein blood of OAC.

[ Example  2 : plasma  Manufacturing and 2-dimensional gel electrophoresis (2 DE )]

1.6 ml of the collected plasma samples collected 200 μl of plasma from each of the eight AERD subjects and the same collected plasma samples were collected from eight ATA subjects. The collected plasma was diluted with four times the equilibration buffer and filtered through a 0.22-μm microcentrifuge and a microcentrifuge filter tube (Agilent Technologies). (MARS) High Performance Liquid Chromatography (HPLC) column (4.6 x 100 mm; Agilent Technologies), the six most abundant plasma proteins (albumin, IgG, antitrypsin, IgA, (Peng et al.). After depletion, the passage fluid fraction was collected by centrifugation on Amicon Ultra-4 tubes (5-kDa molecular weight cut-off, Millipore). The collected samples were stored in aliquots at -70 ° C for subsequent use. 2DE was performed as described above (Lee et al. 2006) as described in the online appendix. The coumarin-stained spots on the gels were quantified on a normalized volume basis using the following equation: relative spot intensity = spot volume / total spot volume total

[ Example  3: Intra - come digestion  And mass analysis mass spectrometric 분석 )]

The differentially expressed protein spots were separated from the gel, cut into small pieces, digested with trypsin (Promega), and subjected to mass spectrometry with MALDI-TOF (Shevchenko et al. 1996; Lee et al. MALDI-TOF mass spectrometry (MS) analysis was performed according to the online appendix (Lee et al. 2006).

[ Example  4 : ELISA On by plasma Apo  H level measurement]

Plasma Apo H levels were measured using a quantitative sandwich enzyme immuno kit (Abnova) according to the manufacturer's instructions. The lower and upper limits of the detection range are 0.625 and 40 ng / mL. Plasma was diluted 2 * 10 ⁵ times with phosphate buffered saline prior to analysis. The value below the lower limit is estimated to be the lower limit for statistical analysis purposes.

[ Example  5: Statistical analysis]

Statistical analysis was performed with SPSS 10.0. Differences between independent groups and samples were compared using the non-parametric Kruskal-Wallis H test for continuous data. When the differences were found to be statistically significant, the Mann-Whitney U test was used to analyze the differences between the two groups.

The receiver operating characteristic (ROC) curve was analyzed. All data are expressed as mean ± standard deviation. Statistical significance is defined as P <0.05.

The characteristics of the patients who participated in this study are as follows. 106 asthmatic patients were classified into two groups (AERD and ATA) according to the results of OAC (Table 1). Eight AERD subjects and eight ATA subjects were selected for 2DE analysis. There was no significant difference in the age, onset age, sex, FVC%, FEV1%, eosinophil%, total IgE, atopic frequency or smoking history between AERD and ATA subjects.

A 2D electrophoresis analysis of the patient plasma sample is as follows. To investigate differences in protein expression in AERD and ATA patients, collected plasma from 8 subjects in each group was analyzed by high resolution 2DE after removal of large amounts of protein. There was no significant difference in number of spots between groups (AERD, 585; ATA, 590) (Fig. 1). Eight proteins that differ between the two groups were identified by applying a cut-off value of at least 3-fold in plasma protein expression as measured by 2DE relative intensity (FIGS. 1 and 2). The relative intensities of spot numbers 1, 2, 3 and 4 were significantly lower in the AERD group than in the ATA group. In contrast, spots 5, 6, 7 and 8 were significantly higher in the AERD group compared to the ATA group (Figs. 1 and 3).

The proteins were confirmed by MALDI-TOF MS (Fig. 3). Spots 1 and 3 correspond to alpha-fibrinogen precursor and fibrin beta, respectively. Spots 2 and 4 were identified as apolipoprotein H and proapolipoprotein, respectively. Spots 5 and 7 were identified as complement C3 and C4a, respectively. The other two proteins were identified as 90-kDa heat shock protein and kininogen-1 isoform 2.

A case-control study was conducted to determine changes in Apo H protein levels in AERD. The Apo H concentrations in the plasma of AERD patients ( n = 53), ATA patients ( n = 53) and control ( n = 23) are shown in FIG. Plasma concentrations of Apo H were significantly lower in AERD patients compared to ATA patients (300.00 ± 8.34 μg / ml vs. 348.98 ± 9.18 μg / ml, respectively; P = 0.0001). Apo H levels were very high in ATA patients (p = 0.002), while Apo H levels in AERD patients were similar to those in the control group, as compared to the plasma Apo H concentration (288.59 ± 7.72 μg / ml) in the control group. In asthmatic patients, there was a significant inverse correlation between Apo H concentration and FEV 1% reduction after OAC (r ² = -0.246, P = 0.005; 6).

ROCs were made for Apo H in AERD and ATA (Fig. 6B). The ROC analysis of ApoH autocorreates the region below the 0.717 curve (Fig. 6B). The 315.00 g / ml cut-off value for Apo H predicts AERD as a sensitivity of 71.6% and a specificity of 56.6%.

Despite the rationality that aspirin contributes to the effect of releasing the 5-lipoxygenase (5-LO) inhibitory pathway by endogenous PGE ₂ , it has mainly focused on other factors affecting the difference between AERD and ATA. To better understand the molecular basis of this pathology, we have adopted a proteomic approach. In previous studies, among the over 500 proteins in the 2DE gel, the complement component in the myristic acid complex, apolipoproteins AI and AII, Less than 10 spots containing serum albumin differ between aspirin resistant asthma (AIA) and ATA (Lee et al. 2006). In the present study, large amounts of plasma proteins, including albumin, IgG, antitrypsin, IgA, trans-perferin and haptoglobin, were removed as initial treatment steps, It is because. Experimental results revealed 8 proteins with a difference of more than 3 times the relative intensity in 2DE gels between the two groups. Of these, five have been identified as new candidate proteins in AERD (the other three (two complement components and one proapolipoprotein have been identified in previous studies) (Lee et al. 2006). These proteins are 90-kDa heat Fibrin beta, kininogen, and apolipoprotein H. These data suggest that changes in the coagulation pathway may contribute to the development of aspirin hypersensitivity in asthma AERD subjects had a significant decrease in euglobulin lysis time during aspirin-induced bronchoconstriction and a decrease in plasma calcium (plasma) time (owalski et al. 1987), indicating that fibrinolysis is associated with plasma hydrolytic activity (plasm a proteolytic activity, which may contribute to the onset of aspirin resistance.

The protein spot for ApoH in the 2DE gel was approximately 50 kDa with pI 6.3. These values are similar to beta-2-glycoprotein I (b2GPI), a 50-kDa phospholipid-binding protein (Wurm 1984; Bendixen et al. 1992).

In this study, beta-2-glycoprotein I (apolipoprotein H) was reduced in the plasma of AERD subjects compared to plasma of ATA subjects. However, ATA patients had a significantly higher Apo H level (p = 0.002) compared to plasma Apo H levels in the control group, whereas Apo H levels in AERD patients were similar to the control group.

These data indicate that functional changes in the coagulation pathway may be present in aspirin-resistant asthma and may be generalized in AERD. Evidence suggests that there is a change in the coagulation pathway in asthma. b2GPI has been known to inhibit prothrombinase activity and factor XII activity in platelets and is a natural anticoagulant that regulates (ADP) -dependent activation in platelets (Matsuura et al.). The interaction between b2GPI dimer and apolipoprotein E reporter 2 in platelets is likely to result in increased collagen binding to platelets as a result of induction of thromboxane synthesis (Pennings et al. 2007). The major role of TXA ₂ in asthma is a potential inducer of bronchospasmodic activity and a role as platelet activation agent (Gresele et al. 1993).

Recently, platelets have been implicated in airway remodeling (Pitchford et al. 2004). Strong evidence at ATA indicates that excessive arachidonate production precedes exposure to aspirin / NSAIDs. Even under the condition reference value, before administration of aspirin, TXB ₂ levels and its metabolites are increased in the urine and airway (Oh et al;.. Kumlin et al 1992). After aspirin administration, urine and bronchoalveolar lavage (BAL) fluids from both AIA and ATA patients were reported to contain reduced levels of TXB ₂ (Szczeklik et al., 1996). Is contributing to the progression of asthma through the generation of thromboxane.

The kinin-kallikrein system is produced by kininogen precursors by enzymatic kallikrein, bradykinin (BK) through protein hydrolysis cleavage of high molecular weight kininogen (Dobo et al.). Increased amounts of tissue kallikrein and kinin are present in the bronchial alveolar space in subjects with asthma. Kinin production directly contributes to the asthma response by increasing edema formation and smooth muscle contraction and the release and / or production of secondary mediators such as pre-formed mediators (e.g., histamine) and leukotrienes and platelet-activating elements (Christiansen, Proud, and Cochrane, 1987).

In the complement system, the lectin pathway activates serine proteases MASP-1 and MASP-2, resulting in oxidative stress in the endothelial cells. MASP-1 is capable of degrading high-molecular weight kininogen (HK), thereby producing BK (Dobo et al.). Aspirin interferes with the activation of mediators by interfering with the formation of the essential enzyme kallikrein in the kallenogen. Aspirin is a complement system in vitro And in vitro has been reported as the activator (Drouin et al 2001;. Gerard and Gerard 2002). Based on this data, clot pathway activation, which leads to enhanced kinin production, may be associated with AERD development, in conjunction with complement activation.

To distinguish the differentially expressed proteins from the 2DE gels, the cut-off level between the two conditions was set to a FEV ₁ 20% reduction induced by aspirin administration. However, the percentage decrease in FEV ₁ induced by aspirin administration is a continuous value. Therefore, we shall select the subjects with a 5% FEV ₁ percent or less decrease in the difference can be represented having more than 25% FEV ₁ percent decrease in AERD subjects, ATA subjects between these two phenotypes. From the viewpoint of the inventors, it is not appropriate to analyze only 8 cases in each group, but it was judged that it is enough to examine the difference between the two groups in the initial screening study. However, plasma collection from 8 subjects in each group may be the limit of this study. A Korean study (Kim HS et al. 1998) showed that allelic variants of Apo H: APOH * 1 (10%), APOH * 2 (91.3%) and APOH * 3 It can cause Apo H of level and function. However, no relevant studies have been performed on asthma and aspirin hypersensitivity. The DNA genotype may be a solution to the association of gene mutations and other levels of Apo H in AERD and ATA subjects.

The present invention is the first invention showing differences in plasma expression of proteins involved in the clotting pathway between AERD and ATA patients. The inventors identified four protein-alpha-fibrinogen precursors, fibrin beta, kininogen and ApoH, which are significantly more or less expressed in AERD patients. Plasma levels of apolipoprotein H

Control study that resulted in significantly elevated Apo H levels in plasma of ATA patients compared to AERD and control patients. Apo H levels in asthma were significantly associated with a decrease in FEV ₁ after aspirin administration. These results indicate that Apo H and possibly other clotting factors and complement proteins contribute to AERD as well as ATA incidence.

The above-described embodiments are illustrative and those skilled in the art will be able to make various modifications and alterations to the disclosed embodiments without departing from the spirit of the present invention. The scope of the present invention is not limited by these variations and modifications.

Claims (4)

i) plasma apolipoprotein H; And ii) a detection agent for a protein that is any one selected from the group consisting of an alpha-fibrinogen precursor, fibrin beta, kininogen, and combinations thereof, wherein the aspirin resistance Wherein the biomarker composition distinguishes between asthma (ATA, aspirin tolerant asthma) and aspirin-exacerbated respiratory disease (AERD). The method according to claim 1,
Wherein said plasma apolipoprotein H, alpha fibrinogen precursor or fibrin beta is expressed in aspirin-resistant asthma and said kininogen distinguishes between aspirin-resistant asthma and aspirin-hypersensitive respiratory system disease, wherein the expression level increases in an aspirin-sensitive respiratory system disease &Lt; / RTI &gt; The method according to claim 1,
Wherein the kininogen is a kininogen-1 isoform 2 protein. i) plasma apolipoprotein H; And ii) an antibody that binds complementarily to a protein that is any one selected from the group consisting of alpha-fibrinogen precursor, fibrin beta, kininogen, and combinations thereof. (AERD, aspirin-tolerant asthma) and aspirin-exacerbated respiratory disease (AERD).

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