Method for detecting functional activity of cytokins that suppress t-lymphocytes in neonatals

FIELD: medicine, immunology.

SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.

EFFECT: higher efficiency of detection.

2 ex

The invention relates to medicine, in particular to immunology, and can be used to assess the immune status of the newborn child.

It is known that cytokines are a group of proteins secreted by various cells and regulating hematopoiesis, development of the immune system, immune response and inflammatory processes. Most of the cytokines of polyfunctionally. However, there are several groups of cytokines on their main biological properties - stimulators of hematopoiesis (interleukin-3 (IL-3), erythropoietin, colony-stimulating factors); stimulators of cellular forms of immune response and inflammation (IL-2, IL-9, IL-12, interferons, tumor necrosis); stimulants humoral immune response and allergic reactions (IL-4, IL-5, IL-6) and cytokines that suppress the immune response and inflammation (IL-10, part IL-4, progesterone-induced factor, peptide humoral factors of the placenta). The ratio of the produced cytokines largely determine the nature and effectiveness of the immune response.

We previously identified two functional defect in T-lymphocytes of newborns, which correlated with severe clinical status of children in the neonatal period. One of these defects is increased sensitivity of T cells to apoptosis, the other due to anergy of T cells absence of the response of these cells to activate, modeling the interaction of the immune system with the foreign antigen (Talayev V.Yu., Lebedeva I.E., Rubtzova I.E., and all. The Cord Blood T-cell Proliferation and Apoptosis: Their Connection to Newborn Children Health; Influence of Interleukin-2, -4, -7 and Dexametasone Exerted upon the Data. / Russian Journal of Immunology. - 2001. - V.6. No. 1. - R.29-38). Studies have shown that the anergy of T cells due to the increased level of production factors that suppress proliferation. One of these factors is IL-10, other factors not identified. At the same time, it is shown that the production of these factors in experimental animals (I..Dozmorov and R.A.Miller. Generation of Antigen-Specific Th2 Cells from Unprimed Mice In Vitro: Effects of Dexamethasone and Anti-IL-10 Antibody. // The Journal of Immunology. - 1998. - V.160. - P.2700-2705)and in humans (Talayev V.Yu., Lebedeva I.E., Rubtzova I.E., and all. The Cord Blood T-cell Proliferation and Apoptosis: Their Connection to Newbom Children Health; Influence of Interleukin-2, -4, -7 and Dexametasone Exerted upon the Data. // Russian Journal of Immunology-2001. - V.6. - No. 1 - P.29-38) may be blocked by a synthetic analogue of the glucocorticoid is dexamethasone. This fact has allowed us to develop a way to evaluate the overall biological activity of the humoral factors that suppress T-lymphocytes. The method serves to identify the characteristic of the neonatal period functional defect of cells of the immune system.

Similar methods of assessment of biological activity of suppressor cytokines are not described. The number produced in cultures cytokinemia be determined using enzyme-linked immunosorbent assay (Mossman, T.R. and Fong T.A.T. Specific assays for cytokine production by T cells. // J. Immunol. Meth. - 1989. - V.116. - P.151-158), and the number of cells producing cytokines - by immunofluorescence staining of intracellular cytokines with subsequent analysis by flow laser cytometer (Jung So, Shauer U., Heusser C., Neumann, S., Rieger C. Detection of intracellular cytokines by flow cytometry. // J. Immunol. - 1993. - V.159. - P.197-207). These methods have high sensitivity and specificity, however, a significant drawback of these methods is the high cost of reagents and equipment necessary for the implementation of these methods. Domestic test-systems for detection of cytokines with suppressor properties does not exist. In addition, as mentioned above, is still not established the molecular nature of all cytokines that suppress the activity of T-lymphocytes of newborns. Accordingly, neither in our country nor in other countries there is no test systems and reagents, allowing you to identify all of these humoral factors.

The purpose of the invention is the identification of the functional defect in the immune system, characteristic of the neonatal period and is due to increased production factors with immunosuppressive activity.

The method is based on the ability of a synthetic analogue of the hormone of the adrenal cortex - dexamethasone to suppress the production of a wide range of cytokines by lymphocytes che is oweka in culture. As a result, dexamethasone inhibits the proliferation of activated T-lymphocytes. At the same time, recombinant human IL-7 is able to fully compensate created dexamethasone deficit of cytokines that stimulate the multiplication of T-lymphocytes. As a result, the introduction of dexamethasone in the culture of mononuclear cells in cord blood T-lymphocytes, activated with antibodies to CD3 molecule in the presence of IL-7, does not lead to suppression of proliferation in cultures with normal production of cytokines or significantly increases cell proliferation in cultures in which the action of dexamethasone was observed enhanced products suppress proliferation factors. The proposed method allows to estimate the total biological activity of the humoral factors produced by cells of the blood of newborns and suppress the activity of T-lymphocytes.

The method is as follows.

Fence umbilical cord blood is carried out after the cutting of the umbilical cord from the placenta of a segment of the umbilical cord in a sterile vial with a volume of 50 ml containing 1 ml of heparin on the environment DMEM or physiological solution. The concentration of the solution of heparin 100 u/ml Volume of selected blood samples of 5-10 ml of sample collection blood can be stored up to 8 hours at +4°C.

Mononuclear cells from blood samples emit a traditional is pushed way over the layer ficoll-urografin, ficoll pack or histopaque with a density of 1.077 g/ml in sterile conditions. Selected mononuclear cells are washed twice with the medium for cell cultures, DMEM precipitating cells by centrifugation at 1500 rpm./min for 10 minutes Before the second centrifugation, the number of cells counted in the camera Goryaeva. After centrifugation to prepare a cell suspension containing 10⁶cells in 1 ml of medium for cell cultures, DMEM with 10% fetal calf serum 584 mg/ml L-glutamine and 50 mg/ml gentamicin.

Suspension cells contribute 15 holes 96 hole sterile round-bottom tablets for immunological reactions or 96-well flat-bottomed tablets for cell cultures. In the first 3 holes do not contribute additional reagents. These wells serve as a negative control (K).

In the next 3 holes by using the pipettor contribute 10 μl of a solution of purified monoclonal antibodies IR-90 to the CD3 molecule. A solution of monoclonal antibodies, and solutions of all other reagents, cooked in a medium of the same composition that was used to prepare cell suspension. The solution should not contain preservatives (sodium azide, thimerosal, and others). The antibody concentration in the applied solution of 20 mcg/ml. Data 3 wells are used to assess the proliferative response of T-cell activation via the CD3 molecule (ACT). Activation of T cells via the CD3 molecule will delirum in polyclonal embodiment, the activation of the cells by the antigen in an immune response.

In the next 3 holes together with 10 μl of the solution of antibodies IR-90 make a 10 μl solution of sodium salt of fastfat dexamethasone. The concentration of the solution of dexamethasone - 8 µg/ml These 3 holes are the control actions of dexamethasone on activated T-cells (ACT D).

In the next 3 holes contribute 10 μl/ml of antibodies IR-90 and solution of recombinant human IL-7. Solution concentration of IL-7 is 200 ng/ml These holes serve to control the proliferation of activated T cells in the presence of IL-7 (ACT IL-7). According to the literature and research of the authors of IL-7 is the best factor in the growth and survival of T-lymphocytes of newborns, is able to fully compensate for a deficiency of growth factors in culture (Webb LM, Foxwell BM, Feldmann M. Putative role for interieukin-7 in the maintenance of the recirculating naive CD4+T-cell pool. // Immunology. - 1999. - V. - 98(3). - P.400-405).

In the next 3 holes contribute 10 μl of the solution of antibodies IR-90, a solution of IL-7 and the solution of sodium salt fastfat dexamethasone (ACT IL-7 D). The concentration of the solution of dexamethasone - 8 µg/ml

On one 96-well tablet can be sown culture of 6 blood samples. Cropped tablets incubated at 37° C in an atmosphere with 5% CO₂(CO₂-the incubator or in a desiccator with a candle) within 72 hours. In the last 24 hours incubation in the seeded wells contribute to the solution of tritium-labeled methyltin the Dean 0.5 µci per well. After incubation tablets frozen, thawed and transferred the contents of the wells on Steklovolokno filters using the harvester. The filters after thorough drying transferred to acquired scintillation vials with 5 ml of acquired scintillation fluid. As acquired scintillation fluid can be used LGL-8 or solution diphenyloxazole and diphenyloxazole benzene toluene for spectroscopy. The radioactivity of the filters was determined on the acquired scintillation counter (beta-1 SBS-2, Beckman). The result values proliferation of cells in each well is expressed in counts per minute Values corresponding to the holes TO be, ACT, ACT ON, ACT IL-7, and the ACT of IL-7 ON average.

The analysis results are as follows.

The reaction of besttransport in response to activation of T cells via the CD3 molecule estimate, calculating the index of stimulation of proliferation - the ratio of average values of cell proliferation in the wells with antibodies to CD3 to the value of proliferation in the control wells (ACT/). The index value of stimulating the proliferation of less than 2 indicates a low proliferative response of T-cell activation.

Products suppress proliferation factors assessed by calculating the index action of dexamethasone is the ratio of average values of cell proliferation in the hole with activated cells in the presence of IL-7 to the value of the cast new is erali in the hole with activated cells in the presence of IL-7 and dexamethasone by the formula:

The index action of dexamethasone = ACT IL-7: the ACT of IL-7 Days

The value of the index action of dexamethasone over 1.2 shows increased production in the culture of cytokines that suppress the proliferation of T-lymphocytes.

Example 1. The patient M. the Newborn with a normal period of intrauterine development (40 weeks), almost healthy. Mononuclear cells from umbilical cord blood of a newborn bred in an environment of DMEM with 10% fetal calf serum, L-glutamine and gentamicina to a concentration of 10⁶cells in ml and 200 μl were seeded in 15 holes 96-well plate to cell cultures without additional reagents (3 holes), with monoclonal antibodies IR-90 (3 holes-ACT), with monoclonal antibodies and dexamethasone (3 holes - ACT D), with monoclonal antibodies and IL-7 (3 holes - ACT IL-7) and with monoclonal antibodies, IL-7 and dexamethasone (3 holes - ACT IL-7 D). Seeded cell cultures were incubated for 72 hours in CO₂-incubator, tritium-labeled methylthymidine made in the last 24 hours incubation. The incorporation of the label was evaluated using a counter Beckman LS-230. The results of the assessment of proliferation:

K - 1004 pulse per min

ACT - 5347 pulses per min

ACT D - 480 pulses per min

ACT IL-7 - 5531 pulse per min

ACT IL-7 D - 4961 pulse per min

Calculate the stimulation index of proliferation 5347/1004=5,33. The proliferative response of T-limp is Titov activation satisfactory.

Calculates the index action of dexamethasone on the production of suppressing proliferation factors 4961/5531=0,9. Enhanced production of cytokines that suppress the proliferation of T cells was not found.

Example 2. The patient Century Child full-term, severe clinical condition. Clinical diagnosis of Respiratory distress syndrome, meconium aspiration, complicated bilateral pneumonia, Perinatal encephalopathy hypoxic Genesis, STC-1, hypochromic anemia moderate, hemangioma of the chest to the right”.

Mononuclear cells from umbilical cord blood of a newborn bred in an environment of DMEM with 10% fetal calf serum, L-glutamine and gentamicina to a concentration of 10⁶cells in ml and 200 μl were seeded in 15 holes 96-well plate to cell cultures without additional reagents (3 holes), with monoclonal antibodies IR-90 (3 holes - ACT), with monoclonal antibodies and dexamethasone (3 holes - ACT D), with monoclonal antibodies and IL-7 (3 holes - ACT IL-7) and with monoclonal antibodies, IL-7 and dexamethasone (3 holes - ACT IL-7 D). Seeded cell cultures were incubated for 72 hours in CO₂-incubator, tritium-labeled methylthymidine made in the last 24 hours incubation. The incorporation of the label was evaluated using a counter Beckman LS-230. The results of the assessment of proliferation:

K - 383 pulse per min

ACT 362 of pulses per min

ACT D - 468 pulses per min

ACT IL-7 - 1044 pulse per min

ACT IL-7 D - 3804 pulse per min

The stimulation index of proliferation 362/383=0,95. The proliferative response of T-cell activation is absent.

The index action of dexamethasone on the production of suppressing proliferation factors 3804/1044=3,64. Found enhanced production of cytokines that suppress the proliferation of T-cells. It is concluded that a depressed response of T-cell activation, and at least one of the reasons for the poor response of T-lymphocytes is enhanced production of lymphocytes, humoral factors suppressing the proliferation of lymphocytes.

Thus, the proposed method allows to evaluate both the known functional parameter of the immune system response of T-lymphocytes newborn activation, new immunological indicator - total biological activity of the humoral factors produced by cells of the blood of newborns and suppress the activity of T-lymphocytes. The proliferative response of T-cell activation by antibodies to CD3 (reaction besttransport with antibodies to CD3) is a well-known method for assessing the functional state of the human immune system. The informative value of this method in relation to newborn shows a number of authors, including the authors of this invention (Talev V.Y., Lebedev Iailed determination of the functional state of the immune system in young children. // Methodical recommendations. The Ministry of Health of the Russian Federation. - 1999. - 12 S.). The original method for the determination of total biological activity of humoral factors with immunosuppressive activity allows to identify the characteristic of the neonatal period a defect in immune system cells. The correlation of the severity of the clinical condition detected with this method, a defect in the immune system (Talev V.Y., Lebedev IE, Rubtsova IE, Nikonov F, E. Gracheva, Babakina O.N., Talaia E.B., Lukyanova NV Proliferation and apoptosis of T lymphocytes in cord blood: relationship with clinical status of the newborn, the influence of interleukin-2, -4, -7 and dexamethasone on these parameters. // Immunology. - 2001. No. 3. - P.29-35), suggests the importance of this parameter. Identifying newborns weakness proliferative response (IP proliferation less than 2) and increased production suppressor factors (the index action of dexamethasone over 1.2) requires physicians to neonatologists greater thoroughness in carrying out activities aimed at preventing child-transmission of pathogenic and conditionally pathogenic microorganisms, and upon detection of infection - introduction to the complex of measures for the treatment of infections substitution immune disorders with drugs of intravenous immunoglobulins (if no action is pokazani).

The method is applicable in the conditions of specialized immunological laboratories, research institutes and diagnostic centers. Given the low consumption of expensive drugs (recombinant human interleukin-7), the method is not expensive.

The method developed and tested in the laboratory of cellular immunology Nizhny Novgorod scientific research Institute of epidemiology and Microbiology. Acad. I.N. Blokhina and applied in this Institute to assess the condition of immune system of babies born in the maternity hospital №1 and 3 of Nizhny Novgorod.

How to determine the production of cytokines that suppress the proliferation of T-lymphocytes of newborns, which consists in the fact that they are carrying out the reaction besttransport determine the proliferation of T-lymphocytes activated by antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexamethasone (ACT IL-7 D), calculate the index action of dexamethasone as regards the ACT of IL-7 to the ACT of IL-7 D and the value of the index action of dexamethasone over 1.2 shows an increased production of cytokines that suppress the proliferation of T-lymphocytes of newborns.