RU2613127C1 - Immune dysfunction assessment method for preschool children with recurrent respiratory infection, residing near chemically hazardos production - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: to assess the immunity dysfunction for preschool children with recurrent respiratory infection, living in areas near the chemically hazardous industries, samples of peripheral blood are selected, and allelic versions A₁A₁ and A₁A₂ of TNFα cytokine gene and its quantitative content, as well as indicators of IL-10, IFNγ and IgA cytokine quantitative content are determined in lymphocytes by polymerase chain reaction. When the allelic version is A₁A₁, TNFα contentα is more than 30 pg/ml IL-10 is less than 20 pg/ml, IFNγ is less than 12 pg/ml, IgA is less than 1 g/l, immunity dysfunction against recurrent respiratory infection is predicted. When the allelic version is A₁A₂, TNFα contentα is less than 40 pg/ml IL-10 is more than 26 pg/ml, IFNγ is more than 40 pg/ml, IgA is more than 1.5 g/l, good recurrent respiratory infection flow without immune dysfunction development is predicted.

EFFECT: application of this method allows immunological monitoring of children with different clinical forms of recurrent respiratory infection under the conditions of anthropogenic load.

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Description

The invention relates to biological research and medicine.

It is known that the adverse effects of anthropogenic stress coming from the habitat affect the health of the child population. Therefore, the diagnosis and prediction of immunity dysfunction in preschool children with recurrent respiratory infection, living in areas around chemically hazardous industries, are relevant. Using the example of many regions of the Russian Federation, it has been shown that combined anthropogenic pressures on the children's body are manifested by an increase in the frequency of allergic diseases, a high frequency of recurring respiratory infections and immunopathological syndromes. The problem of frequent respiratory infections in children, directly related to the state of the environment, continues to remain in the focus of attention of pediatricians.

In recent years, an intensive search and development of objective and informative indicators of early changes in the body, due to the influence of anthropogenic environmental factors, has been carried out. Based on experimental data and field observations, it is confirmed that the immune system is one of the most sensitive to environmental influences, since the development of the immune response is based on the intensive proliferation of antigen-specific progenitor cells. Dividing cells are particularly susceptible to various factors and agents. Such environmental susceptibility makes it possible to use indicators of the immune system as indicators of the effects of these factors on the child.

Allergic diseases are multifactorial diseases, the development of which involves many genes. An unfavorable genetic background is realized when interacting with environmental factors and manifests itself in the formation of a pathological phenotype (see, for example, NN Ramazanova et al. Polymorphism of the IL4 (590 C> T) and IL4R (ILе50Val) genes in children with bronchial asthma living in the Republic of Bashkortostan. Kazan Medical Journal. 2007; T. 88; 6: 574-576).

Currently, more than 600 genetic markers are known to cause the development of allergic diseases. The results of many studies have been accumulated, indicating the association of allelic variants of interleukin genes (IL-4, IL-10, IL-13, TNFα), receptors (IgE receptor (MS4A2), IL-4 receptor (IL4R), adrenergic receptor gene (ADRB2) ), xenobiotic detoxification enzyme genes in patients with allergic diseases (see, for example, E.Ya. Grechanin and others. Immunogenetic characteristics of bronchial asthma. ASTMA TA ALERGIA. 2011; 3: 23-26; Yu.V. Ostankova et al. Association of polymorphism of the promoter region of the TNFα gene with the development of atopic bronchial asthma. ogicheskaya Genetics 2005; T.III 3:. 33-37).

Cytokines determine the complex intercellular cooperative relationships of immunocompetent cells and at the same time have their own genetic marker. Interleukins, in turn, determine the different types of Th1 and Th2 immune responses and the formation of various clinical variants of allergic diseases. Cytokines play a significant role in controlling all stages of the development and maintenance of allergic reactions and inflammation, therefore, the analysis of the regulation of their activity is very important for understanding the molecular basis of the pathogenesis of allergies and inflammation. A variety of genetic factors determines the clinical polymorphism and heterogeneity of the pathology. The polymorphism of the studied genotypes can vary significantly depending on ethnicity and regional factor and determines the content of cytokines. (see, for example, E.Yu. Bragin et al. Polymorphism of the biotransformation genes of xenobiotics GSTT1, GSTM1, CYP2E1 and CYP2C19 in patients with atopic bronchial asthma. Bulletin SB RAMS. 2005; 3 (117): 121-125; Annunziato F., Romagnani S. The transient nature of the Th17 phenotype. Eur J Immunol. 2010; 40 (12): 3312-3316).

A known method for assessing cellular immunity disorders under the influence of phenol (RU 2523418 C1, Federal Research Center for Medical and Prophylactic Technologies for Public Health Risk Management, 07.20.2014). A blood sample is taken and examined using a test and control samples with the preliminary introduction of a toxicant phenol in a test sample at a concentration corresponding to its regional background level, and the establishment of criteria for judging the violation of cellular immunity. As a research method, ex vivo tests of the effect of a toxicant on the production of cytokines by cells of the immune system are used, and the criterion indicators are the specific cell sensitivity index for cytokine of tumor necrosis factor TNFα and the specific cell sensitivity index for cytokine Interleukin-10 IL-10, which are determined by way: an experimental and control blood sample is introduced into a vial with a sterile nutrient medium DMEM with heparin and gentamicin, carried out at 37 ° C for 24 hours the batch of these samples, and then centrifugation for 3 min at 1000 rpm and in the selected supernatant of the experimental sample, the level of phenol-induced production of cytokines, CPI _IL-10 and CPF _TNFα is determined, and in the selected supernatant of the control sample, the level of spontaneous production of cytokines is determined, namely, the level of TWS _{IL-10 of the} spontaneous production of the cytokine IL-10 and the level of TWS _{TNFα of the} spontaneous production of the cytokine TNFα, then calculate the IFI index of _TNFα specific cell sensitivity for the cytokine TNFα, and also calculate the index of IFH _IL-10 special the specific sensitivity of the cells for the IL-10 cytokine and when the simultaneous condition of an IFI of _{TNFα of} more than 10 and an IFI of _{IL-10 of} more than 2 is judged, the cellular immunity of the body is affected by phenol.

However, this method has its drawbacks, namely: a) the method is applicable only in a certain initial situation - the availability of information about environmental pollution, that is, the initially known harmful factor - phenol; b) the study did not take into account the age-related features of the differentiation of lymphocytes in preschool children and the ability of the latter to produce IL-10 and TNFα; c) the allelic variants of the IL-10 and TNFα genes, which also determine the production levels of the IL-10 and TNFα cytokines, have not been determined.

A method for diagnosing allergic reactions is also described (RU 2011126990 A1, MASSACHUSETS INSTITUTE OF TECHNOLOGY OF THE, 10.01.2013), including the selection of the minimum amount of blood, determining a set of cytokine characteristics in it, in particular IFN _γ or IL-2, which are a sign of an autoimmune disease. An increase in the level of IFN _γ or IL-2 compared with the normal level of IFN _γ or IL-2 is a criterion for an autoimmune or infectious disease.

However, this method has the disadvantage that IL-2 and IFN _γ are cytokines that are in homeostatic synergism. IL-2 enhances the synthesis of IFN _γ , therefore, with an increase in the level of IL-2, a synergistic increase in the level of IFN _γ inevitably occurs, that is, the method is not sufficiently informative.

Closest to the invention is a method of immunological monitoring of frequently ill preschool children with hypertrophy of adenoids and / or allergies living in territories with different environmental characteristics. Repeated immunological examination is recommended to determine the content of regulatory T lymphocytes with the phenotype CD4 + CD25 + FOXP3 + for early diagnosis of regulatory immunological deficiency (see “Characterization of natural regulatory T lymphocytes and their molecular marker FOXP3 in frequently ill preschool children living under anthropogenic load ", Vetrova A.D., diss. Candidate of medical sciences, M., 2013 - prototype).

However, factors of nonspecific resistance, indicators of cellular and humoral (adaptive) immunity are not analyzed, which does not allow us to fully rely on the reliability of the conclusions.

The invention is aimed at solving a technical problem - the assessment of immunity dysfunction in preschool children living in conditions of increased anthropogenic stress, using a new set of criteria. To date, a standard and unified methodology for assessing the immunological status has been created, which allows to establish the normative parameters of the main components of the immune system in the studied region. However, criteria have not been developed for conducting immunological monitoring in children with various clinical forms of recurrent respiratory infections living under anthropogenic stress, which the invention is dedicated to.

The proposed method for assessing immune dysfunction in preschool children with recurrent respiratory infection living in areas around chemically hazardous industries consists in taking peripheral blood samples and conducting an enzyme-linked immunosorbent assay.

The difference between the method is as follows. In the lymphocytes of the obtained samples, allelic variants A ₁ A ₁ and A ₁ A ₂ of the TNFα cytokine gene and its content, as well as the content of IL-10, IFN _γ , IgA cytokines, are determined by the method of allelespecific polymerase chain reaction (PCR).

With the allelic variant A ₁ A ₁ and TNFα content> 30 pg / ml, IL-10 <20 pg / ml, IFN _γ <12 pg / ml, IgA <1 g / l, the development of immune dysfunction is predicted against the background of a recurrent viral respiratory infection .

With the allelic variant A ₁ A ₂ , TNFα content <40 pg / ml, IL-10> 26 pg / ml, IFNγ> 40 pg / ml, IgA> 1.5 g / l, a favorable course of recurrent respiratory viral infection without the development of dysfunction is predicted immunity.

The technical result of the invention is to increase the reliability of the evaluation of the functions of immunity in children with recurrent respiratory infection.

The invention is based on a large amount of research on the health of preschool children with recurrent respiratory infections living in conditions of increased anthropogenic stress, namely in the areas around chemically hazardous industries. The reactions of the child's body to pollutants, even at sub-threshold concentrations, differ significantly from those in adults. Moreover, the most “environmentally sensitive” are indicators of the state of health of children of early and preschool age. As the concentration of pollutants in the environment increases, the number of children in the population that react to their presence naturally increases. Ecologically unfavorable territories are characterized by a high frequency of recurrent respiratory infections, allergic diseases, chronic somatic diseases, somatoform disorders of the autonomic nervous system, and children lagging behind in physical development.

A total of 187 children were examined, each of which underwent allele-specific PCR and 748 quantitative determinations of the levels of cytokines in peripheral blood. As a result of these studies, the population of the formed sample of the examined patients and the parameters of the immune system homeostasis determining the type of Th1 or Th2 immune response — the allelic variants A1, A2 of the TNFα gene, the content of cytokines — TNFα, IL-10, IFN _γ and IgA, was determined, which made it possible evaluate immunopathogenesis reactions in the diagnosis of allergies or inflammation. Moreover, the established type of immune response - allergy or inflammation - determines the further tactics of treatment and prevention of complications of recurrent respiratory infections in children.

So, it was found that monoalleles of the TNFα gene (6p21.3-p21.1) control different levels of production of this cytokine: the TNFαA1 allele provides a relatively low production of TNFα; TNFαA2 allele - high production of TNFα. It was found that the distribution of allelic variants of the TNFα gene in the analyzed sample corresponds to the European population. This allowed the use of pan-European controls for the content of this cytokine as a comparison parameter for determining the content of TNFα (see table).

It was also established that in the examined children carriers of the allelic variant TNFαA1A1 the content of the cytokine TNFα is lower than in children carriers of the allelic variant TNFαA1A2 (p <0.05). The level of cytokines, IL-10, IFN _γ and immunoglobulin A (IgA), which are one of the main components of the formation of adaptive immunity in preschool children with recurrent respiratory infection, carriers of the allelic variant TNFαA1A2 is significantly higher compared to the group of children carriers of the TNFαA1 A1 allele ( see drawing).

The results of clinical observation of the examined children have the same tendency as laboratory indicators: the incidence of respiratory infections in the group of children carriers of the TNFαA1A2 allele decreases with age compared with the group of children carriers of the TNFαA1A1 allelic variant. The detected resistance to respiratory infections in preschool children carriers of the TNFαA1A2 alele is apparently associated with the predominance of Th 1 type immune responses.

The method is as follows. The material for the study was peripheral blood samples. Blood samples were taken from the child by venipuncture, which was carried out in the supine position of the subject, using the saphenous veins of the elbow bend in compliance with the rules of asepsis and antiseptics with maximum extension of the elbow bend by placing a roller under it. The tourniquet was placed above the venipuncture site several minutes before its implementation.

Molecular genetic research was carried out at the Federal State Budget Scientific Institution Medical Genetic Research Center.

We analyzed the polymorphism of TNFα genes using the test systems of NTP Litekh LLC (Moscow) using the allele-specific polymerase chain reaction (PCR) (G-308A) in real-time mode. Quantitative determination of the levels of cytokines TNFα, IL-10 and IFN _{γ was} carried out using enzyme-linked immunosorbent assay systems “Cytokine” (St. Petersburg). IgA was determined using the enzyme immunoassay system "Vector-Best" (Russia).

Further, the allelic variants A ₁ A ₁ and A ₁ A ₂ of the TNFα cytokine gene and its quantitative content, as well as the quantitative content of IL-10, IFN _γ , IgA cytokines, were determined in the lymphocytes of the obtained samples. Then, according to the established criteria, with the allelic variant A ₁ A ₁ , TNFα content> 30 pg / ml, IL-10 <20 pg / ml, IFN _γ <12 pg / ml, IgA <1 mg / ml, immune dysfunction was predicted against a recurrent course respiratory infection. In contrast, with the allelic variant A ₁ A ₂ , TNFα cytokine levels <40 pg / ml, IL-10> 26 pg / ml, IFN _γ > 40 pg / ml, IgA> 1.5 mg / ml, a favorable course of recurrent respiratory viral infection without the development of immune dysfunction.

A total of 187 children were examined: each of them performed 187 molecular genetic studies and 748 quantitative determinations of the levels of cytokines in peripheral blood. The reliability of the forecast was P <0.05.

The achievement of the technical result is illustrated by examples.

Clinical example 1. A child from 3 pregnancies, 3 urgent physiological births, full-term, mature. The course of the neonatal period is without features. In the first 3 years of life, it grew and developed in accordance with age parameters. He was sent for the first time to an allergist-immunologist in connection with registration in a kindergarten. An immunological examination was carried out: allelic version of the TNFα gene A ₁ A ₁ , TNFα concentration - 48 pg / ml, IL 10-18 pg / ml, IFN _γ - 9 pg / ml, IgA -0.6 g / l. Prognosis: immunity dysfunction.

A child under 5 years of age was re-observed after 6-8 months. The development of clinical signs of immune dysfunction was noted: episodes of a respiratory infection> 8 times a year (above the age norm), grade II adenoid hypertrophy, degree II-III tonsil hypertrophy. Forecast confirmed.

Clinical example 2. The child is 3 years old, from the first pregnancy, the first urgent physiological birth, full-term, immature. The course of the neonatal period within the age parameters. Up to 2 years old, grew and developed in accordance with age. For social reasons, a preschool institution began to attend from the age of 2.5. Against the background of visiting a kindergarten for 6 months, the child had 4 episodes of acute respiratory disease. Directed to an allergist-immunologist. An immunological examination was carried out: allelic version of the TNFα gene A ₁ A ₂ , concentration of TNFα - 31 pg / ml, IL 10-38 pg / ml, IFN _γ - 62 pg / ml, IgA - 2.1 g / l.

Prognosis: No dysfunction of the immune system.

When observed for 1.5 years, clinical signs of immunological deficiency were not detected. The frequency of episodes of acute respiratory infection corresponded to age parameters - 4-5 times a year during the period of seasonal increases in the incidence of respiratory infections.

Forecast confirmed.

Thus, the patented method is advisable to apply when conducting immunological monitoring in children with various clinical forms of recurrent respiratory infections living under anthropogenic stress, which will allow more accurately assess the function of immunity in preschool children.

Claims (5)

A method for assessing immune dysfunction in preschool children with recurrent respiratory infection living in areas around chemically hazardous industries, which consists in collecting peripheral blood samples and conducting an enzyme-linked immunosorbent assay, characterized in that in the lymphocytes of the obtained samples, allelic variants A ₁ A ₁ and A ₁ A ₂ of the TNFα cytokine gene and its quantitative content, as well as quantitative content of IL-10, IFN _γ and IgA cytokines, are determined by conducting an allele-specific polymerase chain reaction with the allelic variant A ₁ A ₁ , TNFα contents of more than 30 pg / ml, IL-10 less than 20 pg / ml, IFN _γ less than 12 pg / ml, IgA less than 1 g / l, immune dysfunction is predicted against the background of a recurrent respiratory infection, and with the allelic variant A ₁ A ₂ , TNFα content less than 40 pg / ml, IL-10 more than 26 pg / ml, IFN _γ more than 40 pg / ml, IgA more than 1.5 g / l predict a favorable course of recurrent respiratory infection without the development of dysfunction immunity.

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