Association of interleukins genes polymorphisms with multi-drug resistant tuberculosis in Ukrainian population

INTRODUCTION: Multi-drug resistant tuberculosis (MDR TB) is a significant health problem in some parts of the world. Three major cytokines involved in TB immunopathogenesis include IL-2, IL-4 and IL-10. The susceptibility to MDR TB may be genetically determined. The aim of the study was to assess the association of IL-2, IL-4, IL-10 gene polymorphisms with multi-drug resistant tuberculosis (MDR TB) in Ukrainian population. MATERIAL AND METHODS: We observed 140 patients suffering from infiltrative pulmonary tuberculosis (PT) and 30 apparently healthy subjects. The patients were assigned to two groups whether they suffer or do not suffer from pulmonary MDR TB. Interleukin gene (IL) polymorphisms, particularly T330G polymorphism in the IL-2 gene, C589T polymorphism in the IL-4 gene and G1082A polymorphism in the IL-10 gene were studied through polymerase chain reaction. Circulating levels of IL-2, IL-4 and IL-10 in venous blood were estimated using ELISA. RESULTS: Prior to treatment, patients with PT showed significant increase of IL-2 levels and decrease of IL-4 and IL-10 levels compared to apparently healthy subjects. Circulating IL-4 and IL-10 levels were significantly decreased whilst serum IL-2 level was significantly increased in patients with MDR TB compared to non-MDR TB. Low IL-4 and IL-10 secretion and considerable IL-2 alterations were shown to be significantly associated with mutations of homozygous and heterozygous genotypes affecting C589T polymorphism in the IL-4 gene, G1082A polymorphism in the IL-10 gene and T330G polymorphism in the IL-2 gene in patients with PT. CONCLUSIONS: Heterozygous genotype and mutations homozygous genotypes gene in polymorphisms determining specified cytokines’ production is a PT risk factor and may lead to disease progression into chronic phase. Heterozygous genotype of aforementioned cytokine genetic polymorphisms was significantly the most frequent in patients with MDR TB.INTRODUCTION: Multi-drug resistant tuberculosis (MDR TB) is a significant health problem in some parts of the world. Three major cytokines involved in TB immunopathogenesis include IL-2, IL-4 and IL-10. The susceptibility to MDR TB may be genetically determined. The aim of the study was to assess the association of IL-2, IL-4, IL-10 gene polymorphisms with multi-drug resistant tuberculosis (MDR TB) in Ukrainian population. MATERIAL AND METHODS: We observed 140 patients suffering from infiltrative pulmonary tuberculosis (PT) and 30 apparently healthy subjects. The patients were assigned to two groups whether they suffer or do not suffer from pulmonary MDR TB. Interleukin gene (IL) polymorphisms, particularly T330G polymorphism in the IL-2 gene, C589T polymorphism in the IL-4 gene and G1082A polymorphism in the IL-10 gene were studied through polymerase chain reaction. Circulating levels of IL-2, IL-4 and IL-10 in venous blood were estimated using ELISA. RESULTS: Prior to treatment, patients with PT showed significant increase of IL-2 levels and decrease of IL-4 and IL-10 levels compared to apparently healthy subjects. Circulating IL-4 and IL-10 levels were significantly decreased whilst serum IL-2 level was significantly increased in patients with MDR TB compared to non-MDR TB. Low IL-4 and IL-10 secretion and considerable IL-2 alterations were shown to be significantly associated with mutations of homozygous and heterozygous genotypes affecting C589T polymorphism in the IL-4 gene, G1082A polymorphism in the IL-10 gene and T330G polymorphism in the IL-2 gene in patients with PT. CONCLUSIONS: Heterozygous genotype and mutations homozygous genotypes gene in polymorphisms determining specified cytokines’ production is a PT risk factor and may lead to disease progression into chronic phase. Heterozygous genotype of aforementioned cytokine genetic polymorphisms was significantly the most frequent in patients with MDR TB

Związek wariantów polimorficznych genów kodujących interleukiny z występowaniem wielolekoopornej gruźlicy w populacji Ukrainy

WSTĘP: Gruźlica wielolekooporna (MDR-TB) stanowi poważny problem zdrowotny w pewnych regionach świata. Interleukiny 2 (IL-2), 4 (IL-4) i 10 (IL-10) odgrywają istotną rolę w immunopatogenezie gruźlicy. Podatność na gruźlicę wielolekooporną może być genetycznie uwarunkowana. Celem badania była ocena związku pomiędzy polimorfizmem genów IL-2, IL-4, IL-10 a występowaniem gruźlicy wielolekoopornej w populacji ukraińskiej. MATERIAŁ I METODY: Do badania włączono 140 chorych na gruźlicę naciekową i 30 osób zdrowych (grupa kontrolna). Wyodrębniono grupę chorych na gruźlicę wielolekooporną (MDR TB) i gruźlicę z zachowaną opornością na leki przeciwgruźlicze (non-MDR TB). Zbadano polimorfizm T330G genu IL-2, C589T genu IL-4 i G1082A genu IL-10 przy zastosowaniu łańcuchowej reakcji polimerazy. Stężenia IL-2, IL-4 and IL-10 w surowicy oznaczono za pomocą testu ELISA. WYNIKI: Przed leczeniem u chorych na gruźlicę stężenia w surowicy IL-2 były wyższe, a IL-4 i IL-10 niższe w porównaniu z grupą kontrolną. Stężenia IL-4 i IL-10 były istotnie niższe, podczas gdy stężenie IL-2 było istotnie wyższe w grupie MDR TB w porównaniu z pozostałymi chorymi (non- MDR TB). Opisane zmiany związane były z homozygotycznymi lub heterozygotycznymi mutacjami polimorficznymi C589T genu IL-4, G1082A genu IL-10 i T330G genu IL-2. WNIOSKI: Mutacje genów badanych cytokin mogą stanowić czynnik ryzyka gruźlicy i prowadzić do progresji i przewlekania się choroby. W grupie MDR TB genotypy heterozygotyczne badanych cytokin występowały najczęściej.WSTĘP: Gruźlica wielolekooporna (MDR-TB) stanowi poważny problem zdrowotny w pewnych regionach świata. Interleukiny 2 (IL-2), 4 (IL-4) i 10 (IL-10) odgrywają istotną rolę w immunopatogenezie gruźlicy. Podatność na gruźlicę wielolekooporną może być genetycznie uwarunkowana. Celem badania była ocena związku pomiędzy polimorfizmem genów IL-2, IL-4, IL-10 a występowaniem gruźlicy wielolekoopornej w populacji ukraińskiej. MATERIAŁ I METODY: Do badania włączono 140 chorych na gruźlicę naciekową i 30 osób zdrowych (grupa kontrolna). Wyodrębniono grupę chorych na gruźlicę wielolekooporną (MDR TB) i gruźlicę z zachowaną opornością na leki przeciwgruźlicze (non-MDR TB). Zbadano polimorfizm T330G genu IL-2, C589T genu IL-4 i G1082A genu IL-10 przy zastosowaniu łańcuchowej reakcji polimerazy. Stężenia IL-2, IL-4 and IL-10 w surowicy oznaczono za pomocą testu ELISA. WYNIKI: Przed leczeniem u chorych na gruźlicę stężenia w surowicy IL-2 były wyższe, a IL-4 i IL-10 niższe w porównaniu z grupą kontrolną. Stężenia IL-4 i IL-10 były istotnie niższe, podczas gdy stężenie IL-2 było istotnie wyższe w grupie MDR TB w porównaniu z pozostałymi chorymi (non- MDR TB). Opisane zmiany związane były z homozygotycznymi lub heterozygotycznymi mutacjami polimorficznymi C589T genu IL-4, G1082A genu IL-10 i T330G genu IL-2. WNIOSKI: Mutacje genów badanych cytokin mogą stanowić czynnik ryzyka gruźlicy i prowadzić do progresji i przewlekania się choroby. W grupie MDR TB genotypy heterozygotyczne badanych cytokin występowały najczęściej

Quantification of anti-aggregation activity of chaperones: a test-system based on dithiothreitol-induced aggregation of bovine serum albumin.

The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin-target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5 = 53 and 58 mM, respectively)

The values of refractive index (<i>n</i>), density (ρ) and dynamic viscosity (η) of solutions of arginine, arginine ethylester, arginine amide and proline at 45°C (0.1 M Na-phosphate buffer, pH 7.0).

<p>The values of refractive index (<i>n</i>), density (ρ) and dynamic viscosity (η) of solutions of arginine, arginine ethylester, arginine amide and proline at 45°C (0.1 M Na-phosphate buffer, pH 7.0).</p

Effect of Pro on DTT-induced aggregation of BSA ([BSA] = 1.0 mg/ml, 2 mM DTT).

<p>(A) The dependences of the light scattering intensity on time obtained at the following concentrations of Pro: (1) 0, (2) 500 and (3) 1000 mM. Points are the experimental data. The solid curves were calculated from Eq. (5). (B) The dependences of the hydrodynamic radius (<i>R</i>_h) of the protein aggregates on time obtained in the absence of Pro (1) and in the presence of 1000 mM Pro (2). (C) The dependence of the <i>K</i>_agg/<i>K</i>_agg,0 ratio on the concentration of Pro. Points are the experimental data. The solid curve was calculated from Eq. (20). Inset shows the dependence of the duration of the lag period (<i>t</i>₀) on the concentration of Pro.</p

Effect of Arg on DTT-induced aggregation of BSA ([BSA] = 1.0 mg/ml, 2 mM DTT).

<p>(A) The dependences of the light scattering intensity on time obtained at the following concentrations of Arg: (1) 0, (2) 50 and (3) 400 mM. Points are the experimental data. The solid curves was calculated from Eq. (5). (B) The dependences of the hydrodynamic radius (<i>R</i>_h) of the protein aggregates on time obtained in the absence of Arg (1) and in the presence of 400 mM Arg (2). (C) The dependence of the <i>K</i>_agg/<i>K</i>_agg,0 ratio on the concentration of Arg. Points are the experimental data. The solid curve was calculated from Eq. (20). Inset shows the dependence of the duration of the lag period (<i>t</i>₀) on the concentration of Arg.</p

The values of parameters of Eq. (20) for suppression of DTT-induced aggregation of BSA by chemical chaperones (<i>R</i>² is the coefficient of determination).

<p>The values of parameters of Eq. (20) for suppression of DTT-induced aggregation of BSA by chemical chaperones (<i>R</i>² is the coefficient of determination).</p

Iinitial rate of DDT-induced aggregation of BSA at 45°C as a function of cross-linked α-crystallin concentration.

<p>The solid line was calculated from Eq. (12) at <i>S</i>₀ = 4.7 subunits of α-crystallin per one BSA molecule. The dotted line corresponds to the dependence of (<i>K</i>_agg/<i>K</i>_agg,0)^1/<i>n</i> on concentration of intact α-crystallin (<i>n</i> = 1.6).</p

Initial rate of DDT-induced aggregation of BSA as a function of α-crystallin concentration (45°C; 2 mM DTT).

<p>The dependence of (<i>K</i>_agg/<i>K</i>_agg,0)^1/<i>n</i> on α-crystallin concentration (lower abscissa axis) or the ratio of the molar concentrations of α-crystallin and BSA (upper abscissa axis <i>x</i> = [α-crystallin]/[BSA]; <i>n</i> = 1.6). Points are the experimental data. The solid line in the interval of <i>x</i> values from 0 to <i>x</i>₁ = 0.17 was calculated from Eq. (12) at <i>S</i>₀ = 0.40 subunits of α-crystallin per one BSA molecule. The solid line in the interval of <i>x</i> values from <i>x</i>₁ = 0.17 to <i>x</i>₂ = 2.6 was calculated from Eq. (13) at <i>Y</i>₀ = 0.94 and <i>x</i>_0.5 = 0.093. The inset shows the dependence of the adsorption capacity (AC) of α-crystallin with respect to the target protein on <i>x</i>.</p