RU2050020C1 - Method for modeling secondary immunodeficiency state of t- cell type - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves administering agglutinating pertussis serum to experimental animals at a single intraperitoneal dose of 0.10 ml. EFFECT: simplified method; improved reproducibility. 6 tbl

Description

 The invention relates to medicine, mainly to immunology, and can be used to simulate a thymus-dependent type of immune system deficiency in order to study the pathogenetic mechanisms of the development of secondary immunodeficiency, including secondary immunodeficiency syndrome, the development of new methods of immunocorrection, principles of drug therapy and prediction of treatment.

 In recent years, the number of diseases based on the violation of various parts of the immune system has increased. These include autoimmune diseases, allergoses, post-vaccination complications, sluggish chronic infections and other diseases, the development of which is due to inhibition of T- or B-systems of immunity. This problem is of particular importance in the case of secondary immunodeficiency syndrome (AIDS), the study of pathogenesis, methods of treatment, and the prognosis of the course of this disease. Until now, there are no sufficiently effective methods of immunosuppressive and immunostimulating therapy of such diseases, due to the lack of interpretation of their occurrence and the participation of various biologically active substances of the macroorganism in these processes and reactions. Therefore, the development of simple and effective methods for modeling secondary immunodeficiency states of a thymus-dependent and thymus-independent nature is an urgent problem of applied and fundamental immunology.

 Conducted research on patent and scientific medical literature revealed various methods for modeling secondary immunodeficiency states. So, in the works of A.V. Maksimov "An experimental study of autoimmune hemolytic anemia and the effect of immunomodulators on its course" (1987); B. N. Sofronova, T. G. Shemerovskaya "Immunological tolerance induced in adult immunoreactive organisms" g. Immunology, 1990, N 4, p. 4-6; as well as in ed. St. N 1005157 "A method for modeling the hyperfunction of the immune system", Lube V.M. et al. and auth. N 1205169 "Method for modeling an immunodeficiency state", Sizyakina L.P. et al. Describes methods that suppress immune system reactions using chemical factors of medicinal immunosuppressants, such as cyclophosphomide, batriden, heparin, antilymphocytic globulin, and physical factors of low-frequency ultrasound or hard x-ray radiation. The following disadvantages are characteristic of these methods: the general undifferentiated antimetabolic effect on the body, the need for multiple use of large doses of drugs to obtain the expected effect, and the difficulty in accurately selecting effective, but not fatal doses and immunosuppressant regimens, combining them with pronounced antigenic effects.

 The prototype of the present invention is a method for modeling a secondary immunodeficiency state, ed. N 1476517, Sizyakina L.P. Moskalenko E.P. Klembitskaya I.V. The method is as follows. Experimental animals (white outbred male mice weighing 10-12 g) are injected with gamma globulin against SKO (subcommissural organ) of the brain, obtained as follows: in cattle, the area of the quadruple of the brain is allocated and the subcommissure organ located under the posterior commissure of the brain is excised. Then, 1 g of brain RMSE is crushed with 1 ml of sterile saline solution until a uniform consistency is obtained and mixed with 2 ml of Freund's complete adjuvant. Dogs are immunized with this emulsion in the pads of the hind legs and in the back skin at the rate of 0.2 ml / kg body weight. 14 days after immunization, dogs were bled, hyperimmune serum was obtained, and a gamma globulin fraction was isolated from it by alcohol precipitation. The resulting gamma globulin against brain RMS at a dose of 0.1 mg was intraperitoneally administered once to outbred white male mice weighing 10-12 g. To control the changes in the immune system in animals, the primary immune response to sheep red blood cells was studied. The following disadvantages are characteristic of this method: the duration of obtaining gamma globulin against SKO, the non-standardity of the preparation obtained, as a result of which, when modeling secondary immunodeficiency, a number of adverse reactions occur, a large variability of the results that distort the reproducible model.

 The aim of the present invention is to simplify the method and increase the reproducibility of the model through the use of a standard preparation.

 The method is as follows. To implement the method using pertussis agglutinating serum, manufactured by the Moscow Institute of Epidemiology and Microbiology of the USSR Academy of Medical Sciences N.F. Gamalei. The model of the secondary immunodeficiency state is reproduced on male mice by introducing into the body an intraperitoneal pertussis agglutinating serum in a dose of 0.1 ml once. To control the changes in the immune system, experimental animals studied the primary immune response to sheep erythrocytes 3 (thymus-dependent antigen) and typhoid Vi antigen (thymus-independent antigen). Research data indicate the development of a secondary immunodeficiency state in the absence of an immune response to sheep erythrocytes and the presence of immune responses to typhoid Vi antigen.

This is confirmed by the following data: 2 18 white mongrel male mice weighing 14 g were administered pertussis agglutinating serum of the 184 series, obtained from the Moscow Institute of Epidemiology and Microbiology of the USSR Academy of Medical Sciences N. F. Gamalei at a dose of 0.1 ml intraperitoneally once. To monitor the state of the immune system 24 hours after drug administration, one animal was immunized with ram erythrocytes (thymus-dependent antigen) in a dose of 1 x 10 ⁸ in a volume of 0.5 ml. The second animal was immunized with typhoid Vi antigen at a dose of 5 μg / mouse in the same volume. 5 days after the inoculation of test antigens, the level of antibodies in the blood serum of animals was determined: in the first mouse to sheep erythrocytes (EB) in the hemmagglutination reaction, in the second mouse to typhoid Vi antigen in the reaction of passive hemmagglutination.

 The research data presented in table 1 indicate the development of a secondary immunodeficiency state according to the T-cell type, i.e. suppression of the immune response to the thymus-dependent antigen (sheep erythrocytes), without touching the formation of an immune response to the thymus-independent antigen (typhoid Vi antigen).

 The application of the method allows to simplify the reproduction of the model of secondary immunodeficiency by T-cell type and to avoid variability of the results.

 Modern ideas about the method of maintaining homeostasis indicate that those microorganisms are most important, the reaction to which can be directed to cells with immunosuppressive activity (T-suppressors), which results in the development of systemic auto-aggression. Concerning the pertussis vaccine, the fact of antigenic similarity of microbial substances with the substance of the brain of mammals is interesting (Brusina L.I. Sapozhnikova A.M. Cross-reactions between antigens of BCG mycobacteria, pertussis bacteria and human brain tissue. J. Immunology. 1986, N 6, S. 55-58), which in turn includes a Thy-1 antigen marker of T-lymphocytes (G. Belokrylov et al. "Thy-1 antigen; as a stimulator of immunogenesis". J. Immunology, 1982, N 6, p.63). Based on the generality of antigenic determinants, we can assume the effect of anti-pertussis antibodies on Thy-positive cells. In our laboratory, experiments were carried out to study the mechanism of action of anti-pertussis antibodies on the immune system of adult immunoreactive organisms. For this, individual portions of antitussis antibodies were depleted by brain homogenates, thymocytes and washed suspension of pertussis microbes, and then the effect of depleted sera on the immune response was studied. The results of the experiments showed that native pertussis serum, as well as serum depleted by liver homogenates and erythrocytes, had a pronounced suppressive effect on the formation of humoral reactions to the thymus-dependent antigen. In the case when the depletion of pertussis serum was carried out by brain homogenates, thymocytes, and also a suspension of pertussis bacteria, suppression of immunological reactions was canceled. This suggests that the observed effect of immunosuppression is associated with a cross of antigenic determinants between brain tissue, thymocytes and microbial antigens of pertussis bacteria.

 The withdrawal of the effect after depletion of serum by thymocytes or a suspension of the brain indicates that the target for pertussis antibodies are T cells. This is likely to result in a deletion of antigen-reactive T cell precursors due to a mechanism called apoptosis associated with DNA degradation (Smider R. Lees R. ea // J. epx. Med, 1989, vol. 169, No. 6, p. 2149 ; Smith Ch. Williams GT Kingston R. ea // Nature. 1989, vol. 337, N 6203, p. 181-184).

 The following data presents the results on the development of optimal doses of antitussis serum, inducing a secondary immunodeficiency state according to the T-cell type.

 To study the effect of different doses of pertussis agglutinating serum on the formation of an immunodeficiency state in a macroorganism, white outbred mice were inoculated with pertussis agglutinating serum in doses of 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 0.14 0.15 0.16 ml. Control mice were inoculated with normal rabbit serum at the same doses. After a day, one part of the experimental animals was immunized with a thymus-dependent (EB) antigen, and the second part was thymus-dependent (V-antigen). After 5 days, the level of antibodies to EB and Vi antigen was determined in experimental and control animals. The results of the study, presented in Table 2, showed that the optimal dose for the induction of a secondary immunodeficiency state according to the T-cell type is the dose of pertussis agglutinating serum, which is 0.1 ml, which leads to a 64-fold decrease in the titer of antibodies to sheep erythrocytes, recorded in the hemagglutination reaction and does not affect the level of antibodies to the thymus-independent antigen. The administration of pertussis agglutinating serum in doses of 0.09-0.08 ml suppresses the level of humoral immunity to the thymus-dependent antigen by 8-16 times, and the use of pertussis serum in doses of 0.06-0.05 ml practically does not affect the humoral response to sheep erythrocytes . Increasing the dose of pertussis agglutinating serum from 0.11 to 0.16 gives the same effect as the dose of 0.10 ml, i.e. suppression of the humoral response to the thymus-dependent antigen corresponded to a 64-fold decrease in the titer of antibodies to sheep erythrocytes, without touching the level of antibody response to the thymus-dependent antigen.

 Thus, the optimal dose of pertussis agglutinating serum for the induction of secondary immunodeficiency in the T-cell type is a dose of 0.10 ml, since reducing the dose of pertussis serum from 0.09 ml and below was insufficient to reproduce the effect of immunosuppression, and increasing the dose Serums with 0.11 ml and above are considered inappropriate due to the unreasonably high consumption of the material used.

 The specificity of the immunosuppression of the humoral response to a thymus-dependent antigen, namely, pertussis agglutinating serum, was verified by experiments involving the use of antibacterial serum of a different specificity antitoxic antistaphylococcal serum. The research data are presented in table.3.

 As can be seen from the presented results, treatment of mice with antitoxic antistaphylococcal serum does not lead to the induction of an immunodeficiency state and the level of antibodies to the thymus-dependent antigen (EB) detected in the RGA is not inferior to the level of antibodies in mice treated with normal serum.

 PRI me R 1. Two outbred white mice were injected intraperitoneally once pertussis agglutinating serum at a dose of 0.09 ml. Two control mice were injected with normal rabbit serum at the same dose. After 24 hours, control and experimental animals were immunized with sheep erythrocytes and Vi antigen. 5 days later, the level of antibodies to EB and Vi antigen was determined.

 As can be seen from table 4, treatment of mice with pertussis agglutinating serum at a dose of 0.09 ml led to a 16-fold decrease in the level of antibodies to EB, which was insufficient to fully reproduce the effect of immunosuppression according to the T-cell type.

 PRI me R 2. Two outbred white mice were intraperitoneally injected once pertussis agglutinating serum in a dose of 0.10 ml. Two control mice were injected with normal rabbit serum at the same dose. After 24 hours, control and experimental animals were immunized with sheep erythrocytes and typhoid Vi antigen. 5 days later, the level of antibodies to EB and Vi antigen was determined in the blood serum of animals.

 As can be seen from table 5, treatment of mice with pertussis agglutinating serum at a dose of 0.10 ml led to a sharp inhibition of the humoral reaction to sheep erythrocytes. Antibodies in a mouse treated with normal serum were detected in titers of 1: 1280, whereas in a mouse treated with pertussis serum, the level of antibodies to EB decreased by 64 times and was detected in a titer of 1:20. The level of antibodies to the thymus-dependent antigen in both experimental and control mice did not differ. The inhibition of the humoral reaction to the thymus-dependent antigen testified to the development of a secondary immunodeficiency state according to the T-cell type.

 PRI me R 3. Two outbred white mice intraperitoneally, once administered pertussis agglutinating serum in a dose of 0.11 ml. Two control mice were injected with normal rabbit serum at the same dose. After 24 hours, control and experimental animals were immunized with EB and Vi antigen. 5 days later, the level of antibodies to EB to the Vi antigen was determined in the blood serum of animals.

 The results of the experiment, presented in table 6, indicate that the preliminary introduction of pertussis agglutinating serum at a dose of 0.11 ml into the body of the animal also leads to the development of a secondary immunodeficiency state. Antibody titers to a thymus-dependent antigen (EB) in a mouse treated with pertussis agglutinating serum were 64 times lower than in a control animal.

 Thus, the use of the proposed method in comparison with the prototype greatly simplifies the method and increases the reproducibility of modeling a secondary immunodeficiency state through the use of a standard preparation.

Claims (1)

 METHOD FOR SIMULATING A SECOND IMMUNO DEFICIENCY STATE BY T-CELL TYPE by immunizing an animal, characterized in that for the immunization they use cell pertussis agglutinating serum, which is administered at a dose of 0.5 g / kg of body weight once per day.

Images