RU2285264C2 - Method for carrying out early stage non-invasive diagnosis of bronchial remodeling processes in chronic obstructive lung disease in male cases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves determining transforming growth factor in male saliva β1 (TGF-β1) using immunoenzyme assay method with a set of standards for determining the TGF-β1. The TGF-β1 value growing 2, 4 and more times as large when compared to a value taken for healthy patient norm, bronchus remodeling is to be diagnosed. The value taken as healthy patient norm is equal to 403.88±102.58 selected as a result of forced expiration volume per 1 s comparison in healthy patients and bronchus biopsy results.

EFFECT: high information capacity, safety and reliability of diagnosis method.

2 cl, 2 tbl

Description

The invention relates to medicine, in particular to the field of pulmonology, and can be used for early diagnosis of obstructive disorders developing against the background of progression of inflammation and the addition of remodeling processes in the lower respiratory tract.

Remodeling processes underlie chronic obstructive pulmonary disease (COPD). COPD is a major socio-medical problem and is rightly considered a disease of the century (along with coronary heart disease) due to its high prevalence, a steady increase in morbidity and mortality, and tremendous economic damage to society.

There is a method for diagnosing the initiation and attachment of processes of morphological rearrangement of the bronchi (remodeling), described in the "Global strategy for the diagnosis, treatment and prevention of chronic obstructive pulmonary disease." Revision of 2003, Moscow: Atmosphere Publishing House, 2003, p. 39. Remodeling can be reliably detected only by biopsy and subsequent morphological evaluation of sections from the walls of the bronchi.

The disadvantage of this method is that for diagnosis, each time it is necessary to take an analysis of a section from the wall of the bronchi (research material), which requires certain technical skills from medical personnel and causes pain, certain inconveniences for the patient, which ultimately causes him fear and a kind of psychological trauma before the study, as a result of which the patient simply refuses this kind of research, goes to it only in extreme cases when the disease is already running.

Currently, in clinical practice, the “gold standard” for diagnosing the processes of irreversible and reversible obstruction of the bronchi remains the method of functional research of the function of external respiration (HF) with a post-bronchodilation test, in particular, the assessment of forced expiratory volume in 1 second of FEV1), described by A. Chuchalin. in the book "Chronic obstructive pulmonary disease", M., CJSC "Publishing House Binom", St. Petersburg, "Nevsky dialect", 1998, p. 512.

The disadvantage of this method is that before the diagnosis it is necessary to cancel all medications per day, the method is time-consuming for the patient, it takes 45 minutes, diagnostically significant changes in FEV1 are detected much later than the morphological processes in the bronchi that underlie remodeling.

A known method for predicting bronchial obstruction in chronic bronchitis is described in the application of the Russian Federation No. 2002111601, A 61 5/00, published November 20, 2003. The method consists in using quantitative laboratory risk criteria for the development of bronchial obstruction, including determining the level IL-1β in serum from the ulnar vein, saliva, bronchoalveolar fluid (BALF) (using bronchial lavage, a flush from the bronchial tree is obtained) and assessment of risk criteria for the level of IL-1β in patients with chronic m bronchitis unchanged FVD (FEV1).

This method predicts bronchial obstruction only in chronic bronchitis, when the narrowing of the bronchi can be due to mucus and sputum, and it is reversible, that is, it indirectly indicates a narrowing of the lumen of the tracheobronchial tree, in addition, the disadvantage of this method is that for diagnosis three analyzes at once, which takes a lot of time for laboratory tests, when taking a blood test, there is a potential risk of infection for both the medical staff and the patient, when taking bronchoalveolar th liquid - the fear of examination of the patient.

The invention is aimed at solving problems: diagnosis of bronchial remodeling in chronic obstructive pulmonary disease (COPD), increasing the safety of the method. the possibility of application in the early stages of morphological processes in the bronchi. increased informativeness and reliability of diagnosis (eliminating the drawbacks of the invasive nature of taking the material for research, the medical staff and the patient have a risk of infection, the patient has fear, pain).

The tasks are solved in that in the claimed method of early non-invasive diagnosis of bronchial remodeling processes in chronic obstructive pulmonary disease (COPD) in men, the patient takes material for research, which is saliva, and according to the invention, the concentration of transforming growth factor β1 is determined in saliva TGF-β1), an enzyme-linked immunosorbent assay (ELISA) using a set of standards for determining TGF-β1 select an indicator considered normal for a healthy patient, the value of TGF-β1 equal to 403.88 ± 102 , 58 pg / ml, with an increase of 2, 4 or more times, bronchial remodeling is diagnosed. In this case, an indicator considered normal for a healthy patient, a TGF-β1 value of 403.88 ± 102.58 pg / ml is selected by comparing the FEV1 indicator and the results of a bronchial biopsy with a healthy patient.

The determination of the transforming growth factor β1 (TGF-β1) by saliva allows us to save the patient from fear, pain, and the risk of infection of both the medical staff and the patient, and also makes it possible to use the morphological processes in the bronchi in the early stages, increases the information content and reliability of the diagnosis.

The inventive method has a novelty in comparison with the prototype, differs from it in that the concentration of transforming growth factor β1 (TGF-β1) is determined in saliva, the value of which determines the patient’s predisposition to bronchial remodeling or directly remodeling.

The applicant does not know how to confirm the processes of bronchial remodeling by transforming growth factor β1 (TGF-β1), determined by saliva, so he believes that the claimed solution meets the criterion of "inventive step".

The inventive method of laboratory non-invasive confirmation of the processes of bronchial remodeling in chronic obstructive pulmonary disease (COPD) in men by the transforming growth factor β1 (TGF-β1), determined by saliva, can be widely used in medicine, namely in pulmonology, therefore, meets the criterion " industrial applicability. "

In the pathogenesis of the development of remodeling, a transforming growth factor (TGF-β1) takes an active part.

TGF-β1 is described in 1978 (without quantitative indicators). The factor exists in three isoforms. It is produced by a large number of cells (macrophages, stromal cells) in an inactive form. Activation occurs with the help of proteinases: plasmin, cathepsin, formed during inflammation. When TRF-β1 acts on the immune system, inhibitory effects predominate. The factor suppresses hematopoiesis, the synthesis of inflammatory cytokines, the response of lymphocytes to IL-2, 4, 7, the formation of cytotoxic NK cells and T cells. At the same time, it causes the switching of immunoglobulin isotypes to JgA and, together with IL-2, increases JgA synthesis tenfold. thereby contributing to the protection of the mucous membranes. TGF-β1 enhances the synthesis of proteins of the intercellular matrix, collagen, stimulates the proliferation of fibroblasts, activates neutrophils, promotes the formation of connective tissue and blood vessels, directly initiates the processes of histomorphological rearrangement of the bronchial obstructive nature. Consequently, a change in the structure of the bronchi occurs much earlier than a change in the functional indicators of the function of external respiration. In the walls of the respiratory system there is a well-defined apparatus of immune defense. These are accumulations of lymphoid tissue in the mucous membrane of the larynx, trachea and bronchi, as well as quite numerous lymphoid cells scattered in the mucous membrane. dubbed lymphoid tissue associated with the bronchi or BALT. When activated, BALT mucosal epithelial cells enhance the secretion of cytokines, inhibitors of pro-inflammatory agents, molecules of the main histocompatibility complex and intercellular interactions. The list of cytokines secreted by these cells includes interleukins (IL) 1α, 1β, 6, 7, 10, 15, TNF, NGF, TGF-β1, -2, -3-α, IL-7, chemokines. These cytokines, with the exception of TGF-β1, are more involved in the activation of the inflammatory process in the respiratory tract, and only TGF-β1, being a fibrogenic cytokine, stimulates the proliferation of fibroblasts and changes in the structure of the bronchial wall, its remodeling. This morphological rearrangement in the wall of the bronchus leads to the appearance of partially reversible or irreversible airway obstruction, which underlies the pathogenesis of COPD.

Considering the literature data on the production of TGF-β1 by mucosal epithelial cells, cells of broncho-associated lymphoid tissue, as well as materials on the pathogenetic significance of this factor in bronchial remodeling, unlike other cytokines, we undertook a study of the level of TGF-β1 in saliva in men with CB and COPD with the purpose of early detection of a rise in the level of transforming growth factor as a marker and initiator of remodeling.

The proposed method is as follows. A patient with COPD in the morning on an empty stomach takes saliva. In saliva, the concentration of TGF-β1 (pg / ml) is determined. The results are compared with the norm, which is considered the value of TGF-β1, equal to 403.88 ± 102.58 pg / ml, an increase of 2-2.5 times TGF-β1 in the patient’s saliva indicates irreversible obstruction, remodeling of the bronchi.

The diagnostic effectiveness of the proposed method was studied in 59 men 40-62 years old, of which 18 with patients with chronic kidney disease, and 31 were diagnosed with COPD. The control group consisted of 10 healthy men aged 35-60 years. The diagnosis of CV and COPD was made according to the anamnesis, complaints, data from an objective study, the results of laboratory, functional, and radiological studies.

Saliva was collected in the morning on an empty stomach, 10 minutes after rinsing the oral cavity with distilled water.

TGF-β1 (pg / ml) was determined by enzyme-linked immunosorbent assay (ELISA) using a set of standards (manufactured by BioSO and RCE Belgium).

Standards and samples are first diluted with buffer acidified with HCl and then neutralized with NaOH. After that, neutralized standards and samples are added to the wells coated with antibodies. After the first incubation, loose material is removed by washing with diluted washing solution. Then the mouse Mav anti-TGF-β1 antibodies, biotinylated anti-mouse IgG antibodies and streptavidin - POD enzyme complex are incubated. An immuno-enzyme sandwich complex is formed. The loose conjugate is removed by washing. Then a substrate solution is added. The color reaction stops with stop solution and is read at 450 nm. The staining intensity is proportional to the concentration of TGF-β1 in the sample.

In parallel with the analysis of saliva for the concentration of TGF-β1 (to determine the value of TGF-β1, which is considered the norm for a healthy patient), FEV1 and bronchial biopsy analysis were determined (table 1, table 2). Four groups were presented for the study: control - healthy, patients with chronic bronchitis (CB). patients with chronic obstructive pulmonary disease of the 1st degree (COPD 1 tbsp.). patients with chronic obstructive pulmonary disease of the 2nd degree (COPD 2 tbsp.).

The indicator considered to be the norm for a healthy patient, the TGF-β1 value of 403.88 ± 102.58 pg / ml was selected taking into account the analysis of the data in table 1 and table 2.

In the control group of healthy patients, the content of TGF-β1 in saliva was 403.88 ± 102.58 (pg / ml).

Change in TGF-β1 in saliva and FEV 1 in healthy and sick

Table 1 Diagnosis TGF-β1 (pg / ml) p≤ * FEV1-% R ** Healthy 403.88 ± 102.58 0.05 95.1 ± 1.05 - HB 464.15 ± 107.94 0.10 91.2 ± 1.02 -0.59 COPD 1 tbsp. 1129.22 ± 165.84 0.05 79.3 ± 0.79 -0.64 COPD 2 tbsp. 1149 ± 189.13 0.05 62.3 ± 0.62 -0.71 * p - probability
** r is the correlation coefficient of TGF-β1 and FEV 1.

As can be seen from table 1, in patients with COPD stages 1 and 2, the value of TGF-β exceeds that in patients with chronic obstructive pulmonary disease, and even more so in healthy patients by 2.4 or more times. The correlation coefficient of TGF-β1 and FEV1 for chronic kidney disease was -0.59, for COPD 1 tbsp. -0.64, with COPD 2 tbsp. -0.71. Thus, the higher the value of TGF-β1 in saliva, the lower the indices of FEV1.

Example No. 1

Patient V., 56 years old, works as a grinder at a large industrial enterprise, smokes 31 years of 15-20 cigarettes a day, suffers from chronic bronchitis for more than 3 years, notes cough with mucous sputum in the morning, shortness of breath with intense physical exertion. During the examination: the correct physique, the skin is clean, the lymph nodes are not enlarged. In the lungs - with percussion, pulmonary sound with a boxy tint, with auscultation - hard breathing, single dry rales. BH - 18. Heart sounds are clear, rhythmic, heart rate - 66 in 1 minute. HELL - 135/90 mm RT. Art. The abdomen is soft, painless. The liver does not protrude from under the costal arch. Physiological administration is normal.

PSV-426, HPF: FEV1 = 2.69 L (73%), FVC = 2.72 L (74%), FV1 / FVC = 100%. The TGF-β1 value in saliva was 1125.92 pg / ml. Final diagnosis: Chronic obstructive pulmonary disease, stage 1.

Thus, the presence of high concentrations of TGF-β1 in saliva corresponds to bronchial remodeling, which is confirmed by these changes in FEV1.

HELL - blood pressure;

PSV - peak expiratory flow rate;

BH - the number of respiratory movements in 1 min .;

Heart rate - the number of heart contractions in 1 min:

FVD - functions of external respiration;

FZHEL - forced vital capacity of the lungs

Example No. 2

Patient D., 57 years old, works as a technician at a large industrial enterprise, smokes 40 years of 30 cigarettes a day, suffers from chronic bronchitis for 2 years, annually exacerbating more than 3 times a year. Cough with mucous sputum bothers daily. shortness of breath with moderate physical exertion. Objectively: the condition is satisfactory, the skin is clean, the lymph nodes are not enlarged. In the lungs, harsh breathing, single, dry rales, BH - 20 in 1 minute. Heart sounds are rhythmic, clear. Heart rate - 78 in 1 minute. HELL - 140/90 mm RT. Art. The abdomen is soft, painless. PSV - 386, HPF: FEV1 = 2.48 L (66%), FVC = 2.57 L (68%), FV1 / FVC = 100%. The TGF-β1 value in saliva was 1397.28 pg / ml. The patient underwent diagnostic bronchoscopy with a biopsy. Biopsy protocol: signs of chronic fibrous inflammation of the bronchi: an increase in the number of flat cells, fragments of a multi-row cylindrical epithelium: the basement membrane is fibrosed with round-cell infiltration. All layers of the wall with fibroplastic infiltration: with areas of fibrosis. Separately located sections of fibrin with accumulations of granulocytes. Final diagnosis: Chronic obstructive pulmonary disease, stage 2.

Thus, the presence of high concentrations of TGF-β1 in saliva corresponds to bronchial remodeling, which is confirmed by biopsy with bronchoscopy and a change in FEV1.

Example No. 3

Patient K., 59 years old, works as a grinder at an industrial enterprise, smokes 45 years of 25 cigarettes a day, suffers from chronic bronchitis for 5 years, annually exacerbation more than 2-3 times a year. Daily cough with mucous sputum, shortness of breath during physical exertion. Objectively: the condition is satisfactory, the skin is clean, the lymph nodes are not enlarged. In the lungs, breathing is harsh, single, dry rales. BH - 18 in 1 minute. Heart sounds are rhythmic, clear, heart rate - 66 in 1 minute. HELL - 135/90 mm RT. Art. The abdomen is soft, painless. PSV - 375, HPF: FEV1 = 2.78 L (82%), FVC = 2.85 L (90%), FV1 / FVC = 91%. The TGF-β1 value in saliva was 1227.13 pg / ml. The patient underwent diagnostic bronchoscopy with a biopsy. Biopsy Protocol: The wall of the bronchus is represented by fragments of a flattened epithelium with pseudo-squamous metaplasia of the mucous membrane. The basement membrane is fibrosed with round cell infiltration; fragments with patches of fibrosis. Final diagnosis: Chronic obstructive pulmonary disease, stage 2.

In this clinical case, the patient has clinical signs of COPD; a significant increase in TGF-β1 in saliva; pronounced fibrotic changes in the wall of the bronchi; which indicates the already occurred remodeling of the bronchial tree. At the same time, there are no disturbances in ventilation by high-pressure filters. Therefore, an increase in TGF-β1 in saliva indicates histomorphological changes in the bronchial wall and reacts much earlier than FEV1 in COPD. Changes in the fibrogenic cytokine TGF-β1 and bronchial remodeling data are presented in table 2.

Changes in TGF-β1 data and bronchial biopsy in healthy and sick

The inventive method in comparison with the prototype diagnoses remodeling, improves the safety of diagnosis, provides the possibility of application at the early stages of morphological processes in the bronchi, increases the information content and reliability of the diagnosis (excluding the disadvantages of the invasive nature of taking the material for research, the medical staff and the patient are at risk of infection, the patient has fear pains).

Claims (2)

1. A method for early non-invasive diagnosis of bronchial remodeling processes in chronic obstructive pulmonary disease (COPD) in men, which consists in the fact that the patient is taken material for research, which is saliva, characterized in that the concentration of transforming growth factor β1 is determined in saliva ( TGF-β1) by enzyme-linked immunosorbent assay (ELISA) using a set of standards for determining TGF-β1, choose an indicator considered normal for a healthy patient, the value of TGF-β1 equal to (403.88 ± 102.58) pg / ml, with an increase indicator I am 2.4 or more times diagnosed with bronchial remodeling. 2. A method for early non-invasive diagnosis of bronchial remodeling processes in chronic obstructive pulmonary disease (COPD) according to claim 1, characterized in that the indicator considered normal for a healthy patient, the TGF-β1 value is (403.88 ± 102.58) pg / ml, choose, comparing with the norm for a healthy patient, the FEV1 indicator and the results of a bronchial biopsy.