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17
Том 17. N 3-4 2013 р.
5. Pabst S., Baumgarten G., Stremmel A. et al. Toll-like receptor (TLR) 4 polymorphisms are associated with a chronic course of sarcoidosis / Clin. Exp. Immunol. - V. 143, № 3. - Р. 420-426.
6. Papadopoulos A. I., Ferwerda B., Antoniadou A. et al. Association of Toll-Like Receptor 4 Asp299Gly and Thr399Ile Polymorphisms with Increased Infection Risk in Patients with Advanced HIV-1 Infection / Clin. Infect. Dis. - 2010. -V. 51, № 2. - Р. 242-247.
7. Peakman M.. Basic and clinical Immunology/ M. Peakman, D. Vergani.- 2 edition. -USA- 2009.- 486 p.
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English version: RELATIONSHIP OF GENE'S POLYMORPHISM OF TOLL-LIKE RECEPTOR 4 WITH CLINICAL AND IMMUNOLOGICAL PARAMETERS IN ATOPIC ASTHMA
Lyakhovskaya N. V.
Bronchial asthma is an example of multifactorial disease that occurs in the interaction of environmental factors and genetic predisposition. The aim of the study was to evaluate the significance of polymorphism Asp299Giy change A to G at position 896 (rs4986790) TLR4 gene and its relationship with the immunoreguiatory factors in the mechanisms of atopic asthma (AAA). Materials and Methods: We examined 45 people AA patients. The diagnosis of the AA and its severity is set in accordance with the approved criteria. Results: TLR4 gene polymorphism 896A / G statistically significant (p = 0.04 ) is found in the group of patients with AA than in heatthy individuals, which allows us to consider TLR 4 gene as a candidate gene in the disease. In patients who carry the gene TLR4 896G allele the disease began in chHdhood (p = 0.03), the spectrum of sensitization were dietary factors (p = 0.02), and there were other manifestations of allergic disease (p = 0.045). A single nucleotide polymorphism of TLR4 gene in patients with atopic asthma is characterized by over-compensated signfficant reduction in IL-10, the absolute number of CD4 + / 25 +/ Foxp3+ - cells and abcence of reia-tionnshifs between these lymphocytes and other immunological parameters. Conclusion: The study of the genetic aspects of atopic asthma optimize diagnostic and treatment protocols of this disease and contribute to the implementation of preventive measures for the development and dissemination of allergic diseases.
Kay words: atopic asthma, Toll like receptor, polymorphism
Bronchial asthma is an example of multifactorial disease that occurs in the interaction of environmental factors and genetic predisposition. In recent years the study of the genetic basis of atopic asthma (AA) is actively pursued, as this approach allows to extend the understanding of the mechanisms of development of this complex pathological phenotype [19]. The study of the genetic aspects of asthma propensity to create new perspectives in the development of diagnostic criteria for individual programs of atopic disease [4,15]. Genetics of innate immunity has become a center of active international studies as a possible pathogenetic link of
atopy [1]. Much attention is focused concept on single nucleotide polymorphism in the genome DNA ( SNP) coding structure of Toll - like receptor 4, that can be a key factor imbalance various lymphocytes lymphocytes in patients with AA [2, 3]. The important role of these patterns recognition receptors is prove in the pathogenesis of several diseases : atopic asthma in children [14], urogenital infections [13], inflammatory periodontal disease [16], gerpevirusny infection [6], diabetes mellitus [17] rheumatoid arthritis [12], chronic sarcoidosis [5].
* To cite this English version: Lyakhovskaya N. V. Relationship of Gene's Polymorphism of To-ike Receptor 4 with arnical and Immunological Parameters in atopic asthma // Problemy e^logi ta medytsyny. - 2013. - Vol 17, № 3-4. - P. 30 -33.
npoSAeMH eKOAorii Ta MejHUHHH
The aim of the study is to evaluate the significance of polymorphism Asp299Gly change A to G at position 1187 (rs4986790) TLR4 gene and its relationship with the immunoregulatory factors in the mechanisms of AA.
Materials and Methods
We examined 45 people patients with AA. The diagnosis of the AA and its severity is set in accordance with the approved criteria (international recommendations GINA, 2011) on the basis of allergy and pulmonary department of the Poltava Regional Hospital. History data collected using by a special questionnaire. All patients with AA were held general clinical laboratory, instrumental and allergy testing. Sensitization to allergens diagnosed through skin testing ( prick test) with the major aeroallergens (household, pollen, epidermal, fungal) and food allergens ( "Immunology", Vinnitsa). The survey was conducted in the absence of the patient's worsening primary or concomitant chronic, acute lack of intercurrent infections and severe comorbidity, which could affect the results of the study. The control group included 90 healthy individuals with no allergic history with the bases of DNA Research Institute of Genetic and immunological basis for the development of pathology
*P
In DNA samples of 90 people that were part of a control group, the mutant genotype GG was not identified, the frequency of the heterozygous genotype AG was 4.5 % (4 people), the frequency of "wild" AA genotype was 95.6 % (86 people). Patients with AA had next results : GG - was not detected, AG - 15,6% (7 people), the genotype AA -84.4 % (38 people). etween the frequencies of genotypes in the control group and patients with AA noted a significant difference (p = 0.04),
The characteristic clinical feature of this TLR4 SNP was associated with allergic pathology (rhinitis and conjunctivitis) (p = 0.045). The vast majority of patients had gastrointestinal diseases and frequent manifestations of SARS. We identified a number of interesting facts that can play an important role in understanding the characteristics of the immunopathogenesis of the AA in the analysis of data on
and pharmacogenetics " Ukrainian Medical Dental Academy." The study was conducted according to the provision of a written survey and determine the commission on ethics and bioethics issues of this institution. Isolation of genomic DNA was performed by phenol - chloroform extraction. The definition of polymorphism 896A / G TLR4 gene carried by the polymerase chain reaction [13]. Lymphocyte phenotype was analyzed by determining the level of expression of cell surface antigens using the monoclonal antibodies, CD4, CD25 ( production of " Sorbent ", Russia), and intracellular protein Foxp3 («eBioscience», USA) by flow cytometry. At flow cytofluorometry EPIX LX - MCL (Beckman Coulter, USA) using a program System II TM software, serum total IgE, IL -4, 10 (OOO " Ukrmed -Don," Ukraine) in serum was determined by indirect IFA. Mathematical processing of the data was performed using the program «STATISTICA 6.0» (StatSoft Inc). Results. We analyzed the frequency of polymorphism Asp299Gly change of adenine to guanine at position 1187 (rs 4986790) in the TLR4 gene in patients with AA and in the group of population controls ( Table 1).
Table 1
The Frequency Distribution of Genotypes and Alleles gene's TLR4
it's characterizing the TLR 4 gene, as the gene -candidate for the development of AA. The patients who are carriers of the allele G (Table 2) has polisensibilisa-tion: domestic and/or pollen allergies with dietary factors -is observed in all 7 patients ( p = 0.013). 6 persons in this group (p = 0.03) has manifestations of the AA in early childhood, and 4 patients underwent standard procedures " atopic march."
Table 2
The Clinical Features of Atopic Asthma
the state of the immune system in carriers homo - and heterozygotes Toll - like receptor 4. Based on the classic principles of modern immunological science [7] you may notice signs of an imbalance in the immune system in these groups of patients with compensated over AA. This is particularly true for the high activity of CD4 - cells. A characteristic feature (Table 4)
Genotypes, alleles Control group (n=90) Patients with atopic asthma (n=45) P*
AA 95,6 (86) 84,4 (38)
AG 4,5 (4) 15,6 (7) 0,04
GG 0 0
G 97,8 (176) 92,2 (83) 0,06
A 2,2 (4) 7,8 (7)
< 0,05 in comparison with the control group
Attribute Patients with atopic asthma "wild" allele (A) TLR4 carriers, (n=38) Patients with atopic asthma mutant allele (G) TLR4 carriers, (n=7) P*
Polysensibilisation that including food alergens yes no 7 31 7 0 0,013
Comorbidities (rhinitis, conjunctivitis) yes no 6 32 5 2 0,045
"Atopic march" in the history of deases yes no 7 31 6 1 0,029
Frequent SARS ye no 17 21 5 2 0,344
Проблеми екологц та медицини
Tabl.4
Strength of the Correlation due Immunological Parameters depending on the genotype 896A/G gene's TLR4
The pair correlation * Patients with atopic asthma "wild" allele (A) TLR4 carriers, (n=38) Patients with atopic asthma mutant allele (G) TLR4 carriers, (n=7)
1 CD4+ and CD4+/25+ 0,47 --
2 CD4+ and CD4+/25+/Foxp3+ 0,54 --
3 CD4+ and Leucocytes 0,73 0,93
4 CD4 and Eosinophils 0,49 --
5 CD4+ and Lymphocytes 0,87 0,99
6 CD4+/25 and Lymphocytes 0,54 --
7 CD4+/25+ and Leucocytes -- 0,81
8 CD4+/25+/Foxp3+ and Leucocytes 0,46 --
9 CD4+/25+/Foxp3+ and Lymphocytes 0,41 --
10 CD4+/25+/Foxp3+ and IL10 0,52 --
11 Leucocytes and Eosinophils 0,59 --
12 Leucocytes and Lymphocytes 0,75 0,92
13 Eosinophils and Lymphocytes 0,60 --
14 IgE and IL4 0,63 0,96
15 IL10 and IL4 -- -0,77
*- All of the pairs are statistically significant (p < 0,05)
AA compensated flow in patients with heterozygous changes TLR4 is a decrease in the number (from 17 to 6), statistically significant relationships and a significant increase in the strength of correlation of these links, as an example - a direct correlation between the levels of
IgE and IL4, which reflects the typical for AA relationship immunoregulatory and effector pathogenesis. There was a significant change in the level of CD4 + / 25 + / Foxp3 + and IL10 in patients with polymorphism 896A / G gene TLR4 (Table 3).
Table 3
Immunological parameters Dependency Genotypes 896A/G гену TLR4
№ Index Homozygous (AA), (n=38) Heterozygous (AG), (n=7)
1. CD 4+' G/l 0,67 ± 0,07 0,78 ± 0,17
2. CD 4+/25+, G/l 0,16 ± 0,02 0,27 ± 0,08
3. CD 4+/25+/Foxp3+, G/l 0,07 ± 0,01 * 0,03 ± 0,01*
4. Leucocytes, G/l 6,55 ± 0,4 8,7 ± 1,89
5. Eosinophils, G/l 0,06 ± 0,01 0,05 ± 0,02
6. Lymphocytes, G/l 1,47 ± 0,13 2,05 ± 0,45
7. IgE, IU / ml 163,8 ± 14,4 170,8 ± 45,8
8 IL10, pg/l 0,45 ± 0,02 * 0,35 ± 0,03*
9 IL 4, pg/l 63,09 ± 9,28 38,6 ± 8,18
*p < 0,05 in comparison with the homozygous
Interleukin-10 is one of the two main mediators of effector T - reg cells [18, 8]. Changes in the level of IL -10 can be regarded as a systemic immune response, both natural and induced T - lymphocytes regulators -this is confirmed by a number of authors [11]. The significant reduction in the level of CD4 + / 25 + / Foxp3 + in conjunction with its main mediator - IL10 heterozygous carriers of TLR4 gene variant may be an important factor in the pathogenesis of immunogenetic AA. T - reg cells control reactions not only adaptive, but innate immunity, including TLR [9]. It is known that due to the presence of Toll - like receptor on the surface of these regulatory cells may be activated by the same factors which activate the effector cells of innate immunity [10]. It is proved that the increased activity of T - regulatory cells after repeated stimulation of pathogens due not only to the formation of memory B cells, but also the expansion of these regulatory cells through Toll - like receptors [9]. On this basis, it is logical to assume that the genetic changes TLR4 may lead to a breach in the performance of the other component of this mechanism, in our case - the suppression of the link : T - reg - cells and IL10.
Conclusions:
1. TLR4 gene polymorphism 896 A / G statistically significant (p = 0.04) is found in the group of patients ABA than in healthy individuals can be considered TLR 4 gene as a candidate gene in the development of this disease.
2. In patients who carry the gene TLR4 896G allele the disease began in childhood (p = 0.03), the spectrum of sensitization were dietary factors (p = 0.02), and there were other manifestations of allergic disease (p = 0.045).
3. A single nucleotide polymorphism of TLR4 gene in patients with atopic asthma is characterized by over-compensated significant reduction in IL-10, the absolute number of CD4 + / 25 + / Foxp3 + - cells and absence of relationship between these lymphocytes other immu-nological parameters.
4. The study of genetic aspects of atopic asthma optimize diagnostic and treatment protocols of this disease and contribute to the implementation of preventive measures for the development and dissemination of allergic diseases...
npoSAeMH eKOAorii Ta MejHUHHH
^iTepaiypa
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