Diagnostic technique for secondary immunodeficient disease in pulmonary tuberculosis

FIELD: medicine.

SUBSTANCE: prescribing the specific chemotherapy is preceded by the immunological examination of peripheral blood of the patients suffering pulmonary tuberculosis and the determination of cell, humoral and molecular-genetic parameters of the patient's immune status. The peripheral blood CD4⁺CD25^-Foxp3⁺ regulatory T-cell count more than 2.6%, the peripheral blood CD4⁺CD25⁺Foxp3⁺ regulatory T-cell count more than 5.1%, the peripheral blood CD3⁺CD4^- CD25⁺ regulatory T-cell count more than 0.2%, the peripheral blood CD4⁺CD25⁺Foxp3^- regulatory T-cell/activated T-helper count less than 25.5%, the peripheral blood CD3⁺CD4⁺CD25^- T-helper count less than 35.4%, the peripheral blood γδT-lymphocyte count less than 4.9%, the peripheral blood CD45R0⁺ memory T-cell count less than 8.9%; 1L-2 less than 22.3 pg/ml, IL-4 more than 40.0 pg/ml, IL-10 more than 25.3 pg/ml, TGFβ more than 1109.0 pg/ml in a combination with the carriage of the allele C and genotype GG T-330G of IL2 gene and the allele A and genotype AA C-592A of IL10 gene, of the allele T and genotype TT C-509T of TGFB gene, and the allele T and genotype TT C-590T of IL4 gene, secondary immunodeficient disease is diagnosed.

EFFECT: invention enables the well-timed diagnosing of the immune system defects in the patients with pulmonary tuberculosis and provides the measures of therapeutic modality correction with using the advanced method of immunomodulatory therapy.

6 dwg, 4 tbl, 1 ex

The invention relates to medicine, namely to Phthisiology and pulmonology, clinical immunology, Allergology and pathological physiology and can be used to diagnose secondary immunological failure (WINES) in newly diagnosed patients with widespread destructive pulmonary tuberculosis (TB) before assigning specific chemotherapy.

The basis of pathogenesis of widespread destructive TB is the dysregulation of antigen-specific Th1-dependent immune response [1, 2]. But, still, it remains an open question whether the inclusion of any mechanisms of immune response to Mycobacterium tuberculosis (MW) and at what stage contributes to "immune deviation", pathological progressive course of tuberculosis infection and the formation of multi-component secondary immunological failure in patients with TB. On the other hand, the development of TB is almost always occurs on the background of the functional inefficiency of the immune response. Thus, the WINES can be both a cause and a consequence of the clinical manifestation of tuberculosis.

In the last decade in the study of the peculiarities of the immunopathogenesis of TB special attention is given to regulatory T-cells that have suppressor activity and help to reduce the intensity of halogen with exceptiona the protective antigen-specific immune response, aimed at the eradication of the pathogen [3, 4, 5]. It is assumed that mediated regulatory T-cell inhibition of an antigen differentiation of Th0 lymphocytes and clonal expansion of Th and activated Th1 lymphocytes underlies the formation of T-cell and tuberculin anergy and, as a consequence, WINES TB patients [6, 7]. "The repertoire of regulatory T-cells are very diverse, but the specific mechanisms by which they exert their immunosuppressive potency, remain unidentified. Meanwhile, it is known that T-lymphocytes with immunophenotype CD4⁺CD25⁺containing intracellular transcription factor Foxp3 (Treg)differ from Th1-, Th2-, Th17-, Th9-, Th22-, T_FH-, Th-activated cells along the spectrum of secreted cytokines and are able to supressive functions of all these clones of T helper cells [3, 8]. Conversion, induction of Treg and implementation of suppressor properties mainly mediated by immunoregulatory cytokines - interleukin (IL) 2, IL-4, IL-10, transforming growth factor (TGF) β. Pets also the possibility of direct contact between Treg-cell-target effect of Treg on apoptosis and proliferation of lymphocytes [9, 10].

In the immunopathogenesis of TB are closely interrelated mechanisms of innate and adaptive immunity, intermediaries are considered, including γδ-cells [1, 12]. They play a crucial role not only in the implementation of response "first line of defense" on the pathogen, but also perform regulatory functions. In the structure of the cytokines secreted by regulatory T-cells, the most wide range of suppressor effects have TGFβ, IL-10 and IL-4 [13]. In addition, there is an assumption about the relationship genetically determined Hyper - or gobeproductive cytokine suppressor with the quality of the immune response, severity and duration of infectious diseases [14, 15].

In connection with the above, a comprehensive study of etiopathogenetic factors immunopathological changes and analysis of causal relationships between TB infection is relevant and can serve as a basis for developing new approaches to diagnostics and personalized correction of WINES in newly diagnosed patients with widespread destructive forms of TB.

There is a method of predicting the course of TB. This variant of predicting the course of TB is based on isolation of L-forms of mycobacteria from sputum of TB patients, and identify the causative agents of the disease antilysocyme activity [16]. In that case, if the parameter is the antilysocyme activity was equal to or greater than 4 µg/ml, we can confidently predict exacerbation of TB. One of vagnas the x disadvantage of this method is the complexity and the risk of infection of laboratory workers, as for this method, you must allocate the L-forms of mycobacteria. In addition, in this model analyzed the prognosis of the underlying disease, rather than the formation of accompanying WINES.

There is a method of forecasting the possible variants of immunomodulator therapy in secondary immunodeficiency States, which has wide application in many fields of medicine such as internal medicine, Pediatrics, Phthisiology, surgery and immunology. The invention is based on the control parameters of the immune status and different from any other patents that at each stage of immunomodulator therapy is the restoration of the most damaged immune system, and then again produced a study of the parameters of immune status with sequential selection of drugs immunotherapy aimed at restoring newly discovered violations in the relevant parts of the immune system [17]. Though informative, this invention is quite difficult to perform and does not allow to identify violations of the immune response at the molecular-genetic level.

A known method for the diagnosis of secondary immunodeficiency, which includes examination of the peripheral blood, which determine the formula of an imbalance in the immune system by selecting three n is the most different from the norm indicators. Selection is performed using the criteria of the diagnostic value of the number of T-, b-lymphocytes and immunoglobulins (Ig) classes A, G, M in the direction of the dynamics of their changes. During the development of this method of diagnosis WINES were surveyed 1304 people from 33 different pathologies, which revealed three parameters, most significantly different from control values and indicates in which direction was changed immunological indices having a characteristic for each of the pathologies. Diagnosis is carried out for a specific pathology in the table. The method provides improved accuracy and reducing the time of diagnosis of secondary immunodeficiency [18]. However, this method of diagnosis of WINES is not specific in relation to TB and gives a very schematic overview of specific defects of the immune system.

Was providen patent search foreign patent database Worldwide www.espacenet.com), which revealed the following inventions. In 2009 at the University of California registered serological method of rapid diagnosis in mammals active tuberculous inflammation, pulmonary and extrapulmonary, based on the determination of the activity of mycobacterial glutamine synthetase. Glutamine-synthetase - enzymatic protein with a molecular mass of 53 kDa. He is plays a Central role in the metabolism of nitrogen, catalyzes the synthesis of L-glutamine to L-glutamate, ammonia and ATP. It was determined that only pathogenic mycobacteria, such as .tuberculosis and .bovis, release large quantities of glutamine synthetase in contrast to non-pathogenic mycobacteria (.smegmatis and .phlei). Thus, the high level of activity in the culture filtrate of glutamine synthetase correlates with pathogenicity of mycobacteria. The method proposed by the authors of the invention is the determination of enzyme activity in the serum of patients with objective diagnosis of active tuberculosis and progression from latent infection to active process, and to monitor the effectiveness of specific anti-TB chemotherapy. The high activity of bacterial glutamine synthetase in the blood serum of the patient within 15 days after the start of treatment demonstrates the ineffectiveness of the drugs used, and therefore, the presence of drug resistance of the infecting strain .tuberculosis [19]. In this model, the evaluation demonstrated the effectiveness of specific chemotherapy of TB, while the presence/absence of patients WINES are not parsed.

Differences in the genome (and, as a consequence, in the antigenic structure) between epidemically significant strain .tuberculosis and vaccine strain .bovis were the basis of the method immunological diagnosis of tuberculosis, proposed in 2004 a group of authors from STATENS SERUM INSTITUTE. It is established that the protein components included in the composition of the infecting strains of mycobacteria, differ from those for vaccine strain. The use of a "cocktail" of proteins mycobacterial origin (composition and immunogenic activity of the components of protein mixtures proposed by the authors of the invention) is significantly (up to 93%) increases the sensitivity and specificity of immunological diagnosis of tuberculosis infection. In particular, it was shown that the addition to the culture medium of peripheral blood mononuclear mixture of mycobacterial antigens origin (genes esat6+CFP10+Rv3 873poolA+Rv2654 14/15) causes increased production of IFN-γ in patients with active tuberculosis, and on the contrary, has no activating effect on cytokine-producing activity of cells in BCG-vaccinated individuals [20]. In this model investigated the genotype of the pathogen, which, of course, can modulate the course of the immune response to the office, but not the role of specific defects in the immune system.

In the course of conducting a patent search did not reveal the source of information, which was described method is effective and informative method of diagnostics of WINES in newly diagnosed patients with widespread destructive TB to assign specific chemotherapy.

A new technical task of creating accurate and informative method of diagnostics of WINES TB patients.

To solve the problem in the method for the diagnosis of secondary immunological failure in newly diagnosed patients with TB to assign specific chemotherapy, conduct immunological study peripheral blood of TB patients and determine the cellular, humoral and molecular-genetic parameters of the immune status of the patient, determine the number γδ-lymphocytes, CD3⁺CD4⁺CD25^-T-helper cells, CD4⁺CD25⁺Foxp3^-regulatory T-cells/T-helper cells activated, CD45R0⁺T cells, memory CD4⁺CD25⁺Foxp3⁺, CD4⁺CD25^-Foxp3⁺and CD3⁺CD4^-CD25⁺regulatory T-cells, secretion in vitro IL-2, IL-4, IL-10, TGFβ and allelic variants of genes cytokines IL2, IL4, IL10, TGFB and relative content in peripheral blood CD4⁺CD25⁺Foxp3⁺regulatory T-cells more than 2.6%, CD4⁺CD25^-Foxp3⁺regulatory T-cells more than 5.1%, CD3⁺CD4^-CD25⁺regulatory T-cells more than 0.2%, CD4⁺CD25⁺Foxp3^-regulatory T-cells/T-helper cells activated in less than 25.5 percent, CD3⁺CD4⁺CD25^-T-helper cells less than 35.4%, and γδ-lymphocytes less than 4.9%of CD45R0⁺T-cell memory is less than the 8.9%; the content of IL-2 in supernatant culture suspensions of mononuclear leukocytes of the peripheral is practical blood less than of 22.3 PG/ml, IL-4 is more than 40,0 PG/ml, IL-10 - more than a 25.3 PG/ml TGFβ - more than 1109,0 PG/ml simultaneously with the carriage of "low producing" the G allele and GG genotype (T-330G) gene IL2 and "high producing" allele a and genotype AA (P-A) IL10 gene, allele T and TT genotype (P-T) TGFB gene and allele T and TT genotype (P-T) gene IL4 diagnose secondary immunological failure.

The method is as follows:

Make the fence peripheral blood from a vein in the morning, on an empty stomach. The material for this study are blood lymphocytes and supernatant culture suspensions of mononuclear cells from peripheral blood of patients with pulmonary tuberculosis.

To assign specific chemotherapy, conduct immunological study peripheral blood of TB patients and determine the cellular, humoral and molecular-genetic parameters of the immune status of the patient, determine the number γδ-lymphocytes, CD3⁺CD4⁺CD25^-T-helper cells, CD4⁺CD25⁺Foxp3^-regulatory T-cells/T-helper cells activated, CD45R0⁺T cells, memory CD4⁺CD25⁺Foxp3⁺, CD4⁺CD25^-Foxp3⁺and CD3⁺CD4^-CD25⁺regulatory T-cells, secretion in vitro IL-2, IL-4, IL-10, TGFβ and allelic variants of genes cytokines IL2, IL4, IL10, TGFB and relative content in peripheral blood CD4⁺CD25 Foxp3⁺regulatory T-cells more than 2.6%, CD4⁺CD25^-Foxp3⁺regulatory T-cells more than 5.1%, CD3⁺CD4^-CD25⁺regulatory T-cells more than 0.2%, CD4⁺CD25⁺Foxp3^-regulatory T-cells/T-helper cells activated in less than 25.5 percent, CD3⁺CD4⁺CD25^-T-helper cells less than 35.4%, and γδ-lymphocytes less than 4.9%of CD45R0⁺T-cell memory is less than the 8.9%; the content of IL-2 in supernatant culture suspensions of mononuclear peripheral blood leukocytes less than of 22.3 PG/ml, IL-4 is more than 40,0 PG/ml, IL-10 - more than a 25.3 PG/ml TGFβ - more than 1109,0 PG/ml simultaneously with the carriage of "low producing" the G allele and GG genotype (T-330G) gene IL2 and "high producing" allele a and genotype AA (P-A) IL10 gene, allele T and TT genotype (With-T) TGFB gene and allele T and TT genotype (P-T) gene IL4 diagnose secondary immunological failure.

To identify surface molecules CD3, CD4, CD25, CD45R0, γδTCR and intracellular marker immunosuppressive activity of Foxp3 in peripheral blood lymphocytes used method of running tri-color laser cytometry using monoclonal antibodies (MCAT), labeled with fluorescent tags. The staining procedure surface (CD3, CD4, CD25, CD45R0, γδTCR) and intracellular (Foxp3) markers were performed according to the protocols of the manufacturer (the Becton Dickinson (BD)", USA).

The order of execution of the study:

Determination of CD4⁺CD25⁺Foxp3^-regulatory T-cells/T-helper cells activated CD4⁺CD25⁺Foxp3⁺and CD4⁺CD25^-Foxp3⁺regulatory T-cells in peripheral blood

After separation of mononuclear leukocytes washed twice with phosphate-saline buffer (PH=7,4) and standardized the number of cells in suspension up to 10×10⁶cells/ml For staining of surface markers (CD4, CD25) of peripheral blood lymphocytes to a suspension of mononuclear leukocytes were added to 20 μl of the respective specific MCAT labeled with fluorescent tags: MCAT to CD4 labeled with FITC (cat. No. 345768), and MCAT to CD25, labeled RE-Su (cat. No. 555433) ("Becton Dickinson (BD), USA). Samples were incubated 20 min at room temperature, protect from light. For staining of intracellular marker Foxp3 was in the process of permeabilization lymphocytes. For this in each tube in turn made working solutions of standard buffers: Human Foxp3 Buffer and Human FoxP3 Buffer With set BD Pharmingen Human Foxp3 Buffer Set (cat. No. 560098). Buffers were diluted according to the instructions provided. Leukocytes were incubated for 30 min at room temperature in a dark place. The cells are then washed twice with 2 ml of phosphate-saline buffer (PH=7,4). In resuspending sediment contributed antibodies to vnutricletocnami Foxp3, labeled with PE (cat. No. 560046), in the amount of 20 μl and incubated for 30 min in the dark at room temperature. The cells are then washed twice with 2 ml of phosphate-saline buffer (PH=7,4), to the precipitate was added 400 μl of phosphate-saline buffer, after which the samples were prepared for measurement. The measurements were performed on a FACSCalibur cytometer (Becton Dickinson, USA)equipped with argon laser with a wavelength of 488 nm and standard filters. Analysis of obtained data was performed using software applications BD CellQuest for Mac OS® X.

Analysis of samples of cell suspensions was performed by five parameters: the direct scattering (FSC), which characterizes the size of the cells, the side scattering (SSC), which characterize the cytoplasmic and membrane characteristics of the cells, and three indicators of fluorescence - green (FITC - 530 nm), orange (RE - 585 nm) and Magenta (Re-Su - 610 nm), detected on the FL1-, FL2 and FL3 channels (Fig.1-3).

Figure 3 in the lower right quadrant are cells simultaneously expressing CD4, CD25 - and not containing intracellular marker Foxp3 (CD4⁺CD25⁺Foxp3^-regulatory T-lymphocytes/T-helpers activated)in the upper right quadrant are CD4⁺CD25⁺Foxp3⁺regulatory T-cells in the upper left quadrant - CD4⁺CD25^-Foxp3⁺regulatory T-cells.

The graph FSC versus SSC allocated gate 1 - lymphocyte is (Figure 1). Gate 1 was applied to the events on the chart SSC versus FL-1 (CD4-FITC), gate 2 was defined population of cells bearing CD4 (Figure 2). Gate 2 was applied to the chart FL-2 (Foxp3-PE) versus FL-3 (CD25 - PE Su) (Figure 3). The results were expressed in percent.

The determination of the number of CD3⁺CD4⁺CD25^-T-helper cells and CD3⁺CD4^-CD25⁺regulatory T-cells in peripheral blood

For detection of surface antigens CD3, CD4 and CD25 on peripheral blood lymphocytes used the method of laser three-color flow cytometry using MCAT labeled with fluorescent tags. The staining procedure markers CD3, CD4 and CD25 were performed according to the Protocol of the manufacturer ("Becton Dickinson (BD), USA).

The progress of the work. After selecting mononuclear leukocytes washed twice with phosphate-saline buffer (pH=7,4), whenever this resuspended and centrifuger for 10 min at 1500 rpm and Then the supernatant liquid was decanted and the remaining precipitate resuspendable in phosphate-buffered saline and standardized the number of cells in suspension up to 10×10⁶cells/ml For staining of lymphocytes in cytometrics tubes made of 50 μl of the suspension of mononuclear leukocytes and 20 μl of the conjugated MCAT - CD3 (PerCP-Cy5,5)/CD4 (FITC)/CD25 (PE) ("Becton Dickinson (BD), USA, cat. No. 333170), mixed by vortex and incubated for 15 min at ControlTemplate in the darkness.

The measurements were performed on a FACSCalibur cytometer (Becton Dickinson, USA)equipped with argon laser with a wavelength of 488 nm and standard filters. Analysis of obtained data was performed using software applications BD CellQuest for Mac OS® X.

Analysis of samples of cell suspensions was performed by five parameters: the direct scattering (FSC), which characterizes the size of the cells, the side scattering (SSC), which characterize the cytoplasmic and membrane characteristics of the cells, and three indicators of fluorescence - green (FITC - 530 nm), orange (RE - 585 nm) and Magenta (PerCP-A,5 - 610 nm), detected on the FL1-, FL2-FLS-channels (Figure 4).

For analysis built two intermediate schedule: FSC vs. SSC which was allocated gate 1 - lymphocytes. Gate 1 was applied to the events on the second graph SSC against FL-3 (CD3-PerCP-Su,5), gate 2 was defined population of lymphocytes bearing CD3. Then gate 2 was applied to the chart FL-1 (CD4-FITC) versus FL-2 (CD25-PE) (Figure 4). The results were expressed in percent.

Figure 4 in the upper left quadrant are the lymphocytes, presenting simultaneously CD3 and CD25 molecules and not presenting CD4 molecule. In the lower right quadrant are lymphocytes with immunophenotype CD3⁺CD4⁺CD25^-(T-helpers).

The determination of the number of CD45R⁺lymphocytes (T-cells memory) and γδTCR⁺T cells (γδ-lymphocytes in peripheral blood is

To determine in peripheral blood lymphocytes, presenting markers CD45R0 and γδTCR, used the method of laser three-color flow cytometry using MCAT to lymphocytic receptors Anti-CD45R0 labeled with PE (cat. No. 347967) ("Becton Dickinson (BD), USA), MCAT to lymphocytic receptors Anti-TCR-γ/δ-1, labeled with PE (cat. No. 333141) ("Becton Dickinson (BD), USA). The procedure for staining of lymphocytes bearing CD45R0-molecule, and lymphocytes, presenting γδTCR, were performed according to the Protocol of the manufacturer ("Becton Dickinson (BD), USA).

The progress of the work. The procedure of sample preparation, staining and measurements were carried out similarly described above. Analysis of samples of cell suspensions was performed by three parameters: the direct scattering (FSC), which characterizes the size of the cells, the side scattering (SSC), which characterize the cytoplasmic and membrane characteristics of cells, and one indicator fluorescence - orange (RE - 585 nm), detected in FL2 channel (5, 6). For analysis built intermediate schedule: FSC vs. SSC, which was allocated gate 1 - lymphocytes. Gate 1 was applied to the events on the chart SSC versus FL-2 (Anti-CD45R0-PE) (Figure 5, 6). Figure 5 and 6 in the lower right quadrants are the lymphocytes, presenting CD45R0-molecule (T-memory cells) and γδTCR (γδT-cells). The results were expressed in percent.

The definition of the content of immunos the untranslated cytokines in supernatant culture suspensions of mononuclear peripheral blood leukocytes in vitro

The selection of mononuclear leukocytes from whole blood. Venous heparinized blood (25 U/ml) was kept at 37°C for 30 min to separate plasma from red blood cells. The resulting plasma was layered on a density gradient ficoll-urographine (ρ=1077 g/cm³) ("MP Biomedical, LLC, USA) at a ratio of 1:2 and centrifuged at 1500 rpm for 20 minutes interphase Formed ring of a mixture of mononuclear cells was collected by pipette, washed three times with medium RPMI-1640 (fsri SRC VB "Vector", Russia), supplemented with 100 µg/ml gentamicin and 5%inactivated fetal calf serum (LLC Biolot", Russia), consistently resuspended and centrifuger each time for 10 min at 1500 Rev/min When using a gradient specified density of 90-95% of all isolated mononuclear cells were lymphocytes.

Cooking supernatants. To obtain supernatants selected mononuclear leukocytes resuspendable in complete culture medium consisting of 90% RPMI-1640 (fsri SRC VB "Vector", Russia), 10% inactivated fetal calf serum (LLC Biolot", Russia), 0.3 mg/ml L-glutamine, 100 µg/ml gentamicin. Standardized the number of cells in a suspension of 2.5×10⁶/ml For the induction of secretory function of mononuclear leukocytes in the sample contributed vaccine strain BCG dose of 50 µg/ml In the control and the sample was made a full nutrient medium. Cell suspension (1 ml were incubated at 37°C and 5% CO₂within 24 hours After incubation, the tubes were shaken, centrifuged 10 min at 1500 Rev/min Supernatant collected and used for the quantitative determination of cytokines. The cell sediment was examined for viability. For this purpose, 0.1 ml of the suspension was mixed with an equal volume of 0.5% Trypanosoma blue ("Serva", USA) and fill the counting chamber Goryaeva. The viability of mononuclear leukocytes was assessed by subtracting the number of "dead" cells are colored blue.

Enzyme-linked immunosorbent assay for the quantitative determination of cytokines. For determination of IL-2, IL-4, IL-10 and TGFβ in supernatant culture suspensions used a solid-phase immunoenzymatic "sandwich" method (ELISA). The ELISA procedure was performed according to the instructions offered by domestic (JSC "Vector-best", LLC "Protein component") and foreign ("Bender Medsystems (USA), "high-speed rail Diagnostics (USA)) manufacturers of test systems. The optical density of the cell contents of the tablet were recorded on a spectrophotometer Multiscan EX (Finland) at a wavelength of 450 nm. The concentration of cytokines was calculated based on the calibration curve. The results were expressed in PG/ml.

Study of the polymorphism of cytokine genes

DNA isolation. The isolation of DNA from peripheral blood was performed according to the instructions, Mgr is suggested to set "DNA-Sorb-B" ("Interlabservice Russia).

The principle of the method. DNA adsorbed on the sorbent, then washed with buffer solution and the sorbent is removed. The resulting DNA is used for further definitions.

The progress of the work. Before you begin lyse solution and the solution for washing 1 was warming at 65°C to dissolve crystals. Selected the required number of disposable test tubes. Made in each tube 300 ál lyse solution and 100 μl of venous blood. Samples were thoroughly mixed on a vortex. Carefully resuspendable sorbent on the vortex was added to each tube single tip for 25 ál resuspending sorbent. The samples were mixed on the vortex, put in a rack for 2 min, then stirred and left to stand for 5 minutes Besieged sorbent tubes by centrifugation at 5000 rpm for 30 C. the supernatant was Removed into the flask trap using vacuum odesilatel and a separate tip for each sample.

Added to the samples (300 ál solution for washing 1 and stirred on the vortex until complete resuspendable sorbent. Besieged sorbent by centrifugation at 5000 rpm on microcentrifuge within 30 C. supernatant was Removed by vacuum odesilatele and a separate tip for each sample. Later in the samples were introduced into 500 ál of solution for cleaning 2, was stirred to complete resus is andromania sorbent, were centrifuged for 30 s at 10,000 rpm, the supernatant was collected by vacuum odesilatele and a separate tip for each sample (the washing procedure was repeated twice). Tubes with loosened caps were placed in a thermostat 65°C for 5-10 min for drying the sorbent. Then the test tube was added 50 μl of TE-buffer for DNA elution, was stirred on a vortex and placed in a thermostat 65°C for 5 min, shaking occasionally on the vortex. Centrifuged the tubes on microcentrifuge at 12 (100 rpm for 1 min. the resulting supernatant contained the purified DNA.

Study of the polymorphism of cytokine genes

The study of polymorphic sites of cytokine genes was performed using allelespecific amplification of specific regions of the genome [21].

The principle of the method. Analyzed allelic variants of genes related to SNP polymorphisms, i.e. characterized by the replacement of one nucleotide. For detection is used allelespecific polymerase chain reaction (PCR). The presence or absence of the amplification product can make a conclusion about the nature of the polymorphism.

The progress of the work. Amplification was carried out according to the instructions that came with the set AmpliSens-200-1 (Interlabservice Russia), in tubes of the type "Eppendorf" by PCR, using the structure of the primers and the parameters of temperature cycles described in the literature using amplifier "Terzic MC2" ("DNA-technology", Russia).

Was studied four polymorphic variants of genes of four cytokines: T-330G IL2 gene (rs2069762), C-590T IL4 gene (rs2243250), C-592A IL10 gene (rs1800872), C-509T TGFB gene (rs1800469). All studied polymorphisms are localized in the promotor regions of the respective genes.

The General reaction mixture for amplification volume of 25 μl contained: "bottom" mixture consisting of a mixture of primers ("Synthol", Russia), and a mixture of deoxynucleotides, 2 mm dNTP-mix in equal parts in separate tubes for each pair of primers; "top" mixture at a final concentration of Mg²⁺2, 5, 10 µl of PCR buffer, and 2.5 μl of 50 mm MgSO₄at 6.5 μl of N₂Oh and 1.0 μl of Tag polymerase (per tube). Excavated in microtubes for PCR, 5 µl of the "bottom" of the mixture, and 10 ál of the "top" of the mix. The top was dropping 1 drop of oil for PCR. Made on top of the oil in a 10 µl sample DNA. The amplification program consisted of a pre-denaturation at 96°C for 1 min, followed by 10 cycles, each of which consisted of denaturation at 96°C (15 s), annealing at specific pairs of primers at a temperature of 92°C (50 s), chain elongation at 72°C (40 s). Followed by 20 cycles consisting of denaturation at 96°C (10 s), annealing at 60°C (50 s), and elongation at 72°C (40 s). The program was completed with a final elongation at 72°C for 1 min Analysis of amplification products Provo is or separation of DNA fragments in 2% agarose gel: 2 g of agarose was mixed with 2 ml of 50×Tris-acetate (TAE) buffer (242 g triaminobenzene, with 37.2 g EDTA, to 57.1 ml glacial acetic acid to bring up to 1 l H₂O, pH 8.0) and heated in a microwave oven until complete melting agarose. Cooled the solution to 50-60°C and poured into the chamber for electrophoresis, in which pre-set comb for forming holes. For the formation of gel left it on for 20-30 minutes, then the chamber was filled with 1×TAE buffer (10 ml 50×TAE buffer, and 490 ml of H₂O) so that it slightly covers the gel and cleaned the comb. After PCR 5 ál of amplificate were separated in agarose gel containing 0.5 mg/ml ethidium bromide, at a voltage of 150 B in a few minutes for subsequent visualization under UV light, confirming the presence of the amplification product. As a marker of the amount of DNA used plasmid pUC19 cleaved with the restriction enzyme MspI ("Simanim", Russia).

Criteria of the proposed method for the diagnosis of WINES TB patients selected on the basis of data analysis in-depth clinical and immunological examination of 338 patients with newly diagnosed widespread destructive TB to assign specific chemotherapy and 118 conditionally healthy volunteers who meet the following criteria. The criterion for inclusion in the study was the presence in newly diagnosed patients with infiltrative, disseminated and fibro-cavernous TB. In the study did not include: b is further, not wishing to take part in the study, patients treated at the time of the study the anti-TB therapy, nonsteroidal anti-inflammatory drugs and corticosteroids; patients with severe concomitant diseases (cancer, diabetes, bronchial asthma); patients with immune, including autoimmune and allergic diseases, infected with hepatitis viruses and HIV; patients who were administered immunotherapy. Included in the study, healthy volunteers were compared with TB patients by sex and age, had no history of chronic somatic (including autoimmune) diseases and allergic reactions.

After applying the above criteria were formed three clinical groups of patients with different clinical forms of TB. The clinical form of the disease was established on the basis of data of x-ray examinations of the lungs. In all patients noted common (more than 4 segments) destructive lesions of the lung tissue with involvement in the pathological process mainly both lungs.

Infiltrative pulmonary tuberculosis (ITB) was diagnosed in 209 patients and was characterized by x-ray by the presence of one or more heterogeneous shadows of tuberculatocostata diameter from 5 to 7 cm with pockets of decay and contamination.

Disseminated tuberculosis (TB) was detected in 89 patients, radiographic sign of which was the presence of one or both lungs of small and medium focal changes of heterogeneous structure due to infiltration and decay.

The diagnosis of fibro-cavernous pulmonary tuberculosis (FGTB) was exhibited 40 patients and was characterized by x-ray multiple ring shadows more than 2 cm in diameter, often in subpleural departments, as well as pronounced pneumovirus.

The causative agent of tuberculosis was identified by direct light microscopy sputum smear stained by Zn, as well as by the method of fluorescent microscopy using fluorochromes (auramine). Patients were recorded in 100% of cases. For the species identification of Mycobacterium tuberculosis (MBT) produced sputum on nutritionally dense Lowenstein-Jensen and Finn-2.

All patients to establish the presence/absence of WINES was carried out a comprehensive study of parameters of cellular and humoral immunity, and immunogenetic study.

Analysis of primary data was carried out using the methods of statistical description and testing statistical hypotheses. All quantitative parameters were tested for normality of distribution using criter what I Shapiro-Wilke. For normally distributed samples was calculated srednevyborgskoe features: arithmetic mean (X), standard deviation (σ), the error of the mean (m). For samples, the distribution of which differed from the normal, expected median (M), first and third quartiles (Q₁, Q₃). For indicators of quality signs indicate the absolute number (n) and relative frequency of occurrence (%). If the sign in the studied samples with normal distribution test hypotheses about the equality of means of sample values was performed using one-way ANOVA.

To assess the validity of differences of numerical characteristics of samples that do not obey the normal distribution, used the criterion of Kruskal-Wallis.

For pairwise comparison of parameters in the studied groups criterion was used Mann-Whitney (for independent groups) and the Wilcoxon criterion (dependent groups). The difference of the indices in the two groups were considered statistically significant at a significance level of p≤0,05. The distribution of genotypes in the studied polymorphic loci were checked for compliance with the equilibrium hardy-Weinberg equilibrium using an exact test of Fisher. For comparison of allele frequencies between different groups used the criterion χ²Perso the and amendment of Yates.

Indicators of cellular immunity in TB patients and healthy volunteers established in the course of the study are summarized in table 1. The presented results indicate a marked imbalance in the structure of subpopulations of regulatory and effector T lymphocytes in patients with TB. Demonstrated increase in the number of Foxp3-positive regulatory T cells with suppressor activity and CD3⁺CD4^-CD25⁺regulatory T-lymphocytes with the decline in CD4⁺CD25⁺Foxp3^-regulatory T-cells/T-helper cells activated CD3⁺CD4⁺CD25^-T-helper cells, γδ-lymphocytes and CD45R0⁺T-cell memory in patients with TB compared with similar indices in healthy volunteers. Note that these changes are most pronounced and cases DTB and FCTB, which are always less favorable terms as the clinical course of the disease and the effectiveness of protective immune response. Thus, patients with TB there is a dysregulation of the cellular immune system with predominance of the number of subpopulations of T-lymphocytes, performing suppressor function, which indicates the inefficiency of cellular reactions and immunological failure.

Table 2 presents data characterizing features of cytokineproducing in vitro at ballnacht. In General, the obtained results allow to conclude that if TB infection is an imbalance of cytokineproducing associated with deficiency of IL-2 secretion ("growth factor" T-cells), due to hypersecretion of anti-inflammatory cytokines with immunosuppressive activity: IL-4, IL-10 and TGFβ. While these changes are unidirectional, as in the case of basal secretion of cytokines and the induction of cell culture vaccine strain BCG. This allows to conclude that in addition to increasing the number of cells suppressor of the immune response in TB patients is significantly increased and their functional activity, as regulatory T cells exert their immunosuppressive effects mainly due to the production of cytokines inhibitors and possible sequestration of IL-2 through its binding by IL-2 receptors (molecules CD25) on the cell surface [3].

During the research we were also interested in the question of predisposition surveyed TB to the formation of immunological failure complicating the course of TB infection. To solve this problem, a study was conducted polymorphic cells of cytokine genes (T-330G IL2 gene (rs2069762)-T IL4 gene (rs2243250), C-592A IL10 gene (rs1800872)-T TGFB gene (rs1800469)) and analyzed the correlation of allelic polymorphism of immunoregulatory genes with titoki what produkcia in vitro. In tables 3 and 4 summarizes the data obtained during the study of the peculiarities of the functional polymorphism of cytokine genes. It is established that patients with TB as basal and BCG-induced hipocresia IL-2 and hypersecretion of IL-4, IL-10 and TGFβ due carrier "low producing" the G allele and GG genotype (T-330G) gene IL2 and "high producing" allele A and genotype AA (P-A) IL10 gene, allele T and TT genotype (P-T) TGFB gene and allele T and TT genotype (P-T) IL4 gene. Thus, patients with TB immunological failure is including deterministic carrier polymorphic variants immunoregulatory genes IL2, IL4, IL10 and TGFB, as excess cytokines with suppressor activity, undoubtedly, leads to the induction of, conversion and generation at the periphery of regulatory T-cells, and the lack of IL-2 contributes to the inhibition of clonal expansion of effector T-helper cells.

Examples for the implementation of the method

Example 1. Patient F., 47 years old.

Diagnosis: subacute disseminated pulmonary tuberculosis in the phase of infiltration and decay, MBT (+). Diagnosis of tuberculosis was established on the basis of history, clinical picture of the disease, radiographic examination of the lungs, data, microscopic and bacteriological examination of the sputum.

For admission to inpatient treatment in Tomsk about actnow clinical tuberculosis hospital 28.11.2011 in parallel with the described further examinations of the patient F. was produced blood sampling for determination of immunological and molecular-genetic parameters according to the proposed method. The survey yielded the following results. Relative abundance of CD4⁺CD25⁺Foxp3⁺regulatory T-cells in a patient F. was 10.2%, CD4⁺CD25Foxp3⁺regulatory T-cells to 8.3%, CD3⁺CD4^-CD25⁺regulatory T-cells by 2.2%, CD4⁺CD25⁺Foxp3^-regulatory T-cells/T-helper cells activated to 15.1%, CD3⁺CD4⁺CD25^-T-helpers - 18%, γδ-lymphocytes - 1,5%, CD45R0⁺T-cell memory is 3.1%; the content of IL-2 in supernatant culture suspensions of mononuclear peripheral blood leukocytes of the patient F. was 12.0 PG/ml, IL-4 - 66,4 PG/ml, IL-10 - of 47.8 PG/ml TGFβ - 1756,7 PG/ml When carrying out the molecular genetic studies in a patient with F. we have found the carriage of the G allele and GG genotype (T330G) IL2 gene, allele a and genotype AA (P-A) IL10 gene, allele T and TT genotype (P-T) gene TGFB and allele T and TT genotype (P-T) IL4 gene. Thus, in a patient F. was diagnosed with secondary immunological failure.

Further examination of the patient revealed the following facts.

Objective status. General condition closer to the guarantee of the WMD, t=37.8°C, position, active, clear consciousness. Pale skin, wet. Peripheral lymph nodes are not enlarged. Musculoskeletal system without visible deformation, movement joints in full.

When viewed thorax cylindrical. Respiratory rate is 14 per minute, the inhalation phase lengthened. Palpation of the chest pain points are missing. Percussion percussion of the lung, over the same symmetric parts. Auscultation is defined by weakened breathing with extended breath, moist low rales on the front and back surfaces of the lungs.

During the inspection the heart of visible pathological changes. HR 82 per minute, corresponds to the pulse. Muffled heart sounds, rhythmic. HELL of 145/90 mm Hg, the same on both hands.

Palpation of the abdomen is soft, painless. Liver and spleen are not enlarged. Gallbladder symptoms negative. Symptom tapping negative on both sides. Dysuric phenomena and peripheral edema no.

Radiographically from 28.11.2011: the projection of the upper lobes of both lungs in the background enriched pulmonary pattern of multiple foci of low intensity, the drain of the nature of shadows, with patches of enlightenment.

In the analysis of sputum from 29.11.2011: office detected by microscopy and sowing method.

General analysis of blood from 28.11.2011: g is moglobin - 102 g/l, the number of erythrocytes - of 2.21×10¹²/l, color index (CPU) of 0.5, the total number of leukocytes (OCP) - 15,5×10⁹/l, eosinophils - 4%, stab neutrophils - 12%, segmented neutrophils - 67%, lymphocytes - 13%, monocytes 7%, and ESR - 44 mm/h

General analysis of urine from 28.11.2011: without features.

Biochemical analysis of blood from 29.11.2011: total protein 59 g/l, ALT - 17 IU/l, ACT - 24 IU/l, thymol test - 4,8 ed, cholesterol - 6.0 mmol/l, total bilirubin is 11.9 µmol/l, glucose - 5.6 mmol/L. Mantoux test - 20 mm

About established and proved diagnosis (subacute disseminated pulmonary tuberculosis in the phase of infiltration and decay, MBT ( + )), the patient was assigned to anti-TB chemotherapy according to standard chemotherapy regimen IIB, including anti-TB drugs back (2nd) series. Patient F. received the specified treatment for 2.5 weeks, but almost positive dynamics were observed. In this regard, the patient was assigned to the consultation of the doctor-immunologist to assign pathogenetically justified immunotherapy. Based on the data obtained in the course of immunological and immunogenetic survey, allowed to diagnose a patient F. genetically deterministic WINES, the doctor-immunologist were assigned to the treatment, including primenenie-activin, p/C, 1.0 ml, once a day for 10 days, licopid, per os, 10 mg, every other day for 20 days, Roncoleukin,/drip 2.0 ml 200 ml of saline once a day for 10 days. While treatment with anti-TB drugs according to the standard chemotherapy regimen IIB was continued. Repeated consultation of the doctor-immunologist was appointed in 1.5 months.

During a specified period of time in a patient DF was observed pronounced positive dynamics of clinical course of the disease. The patient was testing the immunological status, except for the molecular genetic studies, because the carrier polymorphic variants immunoregulatory genes contributing to the susceptibility to the development of WINE is an etiological factor that cannot be influenced by the purpose of immunomodulators. Data immunoassay patient F. from 10.02.2012,: relative abundance of CD4⁺CD25⁺Foxp3⁺regulatory T-cells in a patient F. was 4.1%, CD4⁺CD25^-Foxp3⁺regulatory T-cells with 5.22%, CD3⁺CD4^-CD25⁺regulatory T-cells - 1,07%, CD4⁺CD25⁺Foxp3^-regulatory T-cells/T-helper cells activated - 20,31%, CD3⁺CD4⁺CD25^-T-helpers - 28,4%, γδ-lymphocytes - 2,8%, CD45R0 +T-cell memory is 5.1%; the content of IL-2 in supernatant culture suspensions of mononuclear peripheral blood leukocytes of the patient F. was 26.4 PG/ml, IL-4 - 48,7 PG/ml, IL-10 - 33,6 PG/ml TGFβ - 1210,5 PG/ml As shown by the data obtained, the patient F. there is a trend towards normalization of parameters of immune status after diagnosed WINES and the course of immunotherapy.

The example illustrates the effectiveness, informative and high accuracy of proposed method. Thus, the developed model diagnostics secondary immunological failure in patients with pulmonary tuberculosis, based on the assessment of quantitative indicators of immune and immune and genetic status of the patient, allows to diagnose the defects of the immune system in patients with pulmonary tuberculosis in identifying the disease and to apply remedial measures of therapeutic effects with the use of modern methods of immunomodulatory therapy.

Application

Figure 1 Histogram of the distribution of mononuclear leukocytes in intensity forward (FSC) and side (SSC) light scattering. Estimated total content of lymphocytes in a mixed suspension of mononuclear leukocytes

Figure 2 Histogram of the distribution of CD4⁺lymphocytes labeled with FITC, intensity SSC-is waterscience and fluorescence on the first channel FL1

Figure 3. The histogram of the regulatory (Treg, T regulatory) CD25⁺Foxp3⁺T-lymphocytes in a population of CD4-positive lymphocytes in the intensity of fluorescence in the FL2-, FL3 channels (labeled D and RE-Su). Estimated CD4⁺CD25⁺Foxp3⁺cells (upper right quadrant), CD4⁺CD25^-Foxp3⁺cells (upper left quadrant), CD4⁺CD25⁺Foxp3^-cells (lower right quadrant).

Figure 4. A histogram of the distribution of CD4⁺CD25⁺cells in the population of CD3-positive lymphocytes in the blood by the intensity of fluorescence in the FL1-, FL2 channels. Considers the content of CD3⁺CD4⁺CD25^+hicells (upper right quadrant) and CD3⁺CD4⁺CD25^-cells (lower right quadrant)

Figure 5. A histogram of the distribution of lymphocytes labeled with Anti-CD45R0-PE intensity SSC and fluorescence on the second channel FL2. Estimated content of lymphocytes expressing the molecule CD45R0

6. A histogram of the distribution of lymphocytes labeled with Anti-γδ-TCR-PE intensity SSC and fluorescence on the second channel FL2. Estimated content of lymphocytes, presenting γδ-TCR

Table 1. Subpopulation structure of regulatory T cells in peripheral blood of patients with tuberculosis of the lungs

Table 2. The indicators of production of immunoregulatory cytokines in vitro in patients with pulmonary tuberculosis

al. The content of cytokines in supernatant culture suspensions of mononuclear blood leukocytes in spontaneous (basal) secretion (PG/ml) in patients with pulmonary tuberculosis depending on the allelic variants of cytokine genes

Table 4. The content of cytokines in supernatant culture suspensions of mononuclear blood leukocytes by inducing vaccine strain BCG (PG/ml) in patients with pulmonary tuberculosis depending on the allelic variants of cytokine genes

Sources of information

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The method of diagnostics is secondary immunologic deficiency pulmonary TB, characterized in that prior to the appointment of specific chemotherapy conduct immunological study peripheral blood of patients with pulmonary tuberculosis and determine the cellular, humoral and molecular-genetic parameters of the immune status of the patient, determine the number γδ-lymphocytes, CD3⁺CD4⁺CD25^-T-helper cells, CD4⁺CD25⁺Foxp3^-regulatory T-cells/T-helper cells activated, CD45R0⁺T cells, memory CD4⁺CD25⁺Foxp3⁺, CD4⁺CD25^-Foxp3⁺and CD3⁺CD4^-CD25⁺regulatory T-cells, the secretion of in vitro IL-2, IL-4, IL-10, TGFβ and allelic variants of genes cytokines IL2, IL4, IL10, TGFB and relative content in peripheral blood CD4⁺CD25⁺Foxp3⁺regulatory T-cells more than 2.6%, CD4⁺CD25^-Foxp3⁺regulatory T-cells more than 5.1%, CD3⁺CD4^-CD25⁺regulatory T-cells more than 0.2%, CD4⁺CD25⁺Foxp3^-regulatory T-cells/T-helper cells activated in less than 25.5 percent, CD3⁺CD4⁺CD25^-T-helper cells less than 35.4%, and γδ-lymphocytes less than 4.9%of CD45R0⁺T-cell memory is less than the 8.9%; the content of IL-2 in supernatant culture suspensions of mononuclear peripheral blood leukocytes less than of 22.3 PG/ml, IL-4 is more than 40,0 PG/ml, IL-10 - more than a 25.3 PG/ml TGFβ - more che is 1109,0 PG/ml simultaneously with the carriage of the G allele and GG genotype of the T-330G IL2 gene and allele a and genotype AA WITH-A gene IL10, allele T and TT genotype WITH-T TGFB gene and allele T and TT genotype WITH-T gene IL4 diagnose secondary immunological failure.