Means for stimulating the differentiation of t-lymphocytes
(57) Abstract:The invention relates to medicine, in particular to the medical industry, and can be used for treating immunodeficiencies involving violation of the process of differentiation of T-lymphocytes. The essence of the invention is the use of a peptide of the formula Lys-Glu-Glu-Leu-Asn-Glu as a means of stimulating the differentiation of T-lymphocytes. The technical result is the high specific activity of the proposed peptide and reducing the risk of side effects. 3 table. The invention relates to medicine and medical industries and is the use of a peptide of the formula Lys-Glu-Glu-Leu-Asn-Glu as a means of stimulating the differentiation of T-lymphocytes.Differentiation of T-lymphocytes is a process of formation of functionally complete T-cells from precursor cells, which occurs mainly in the Central body of the cellular immune system - thymus. Differentiation ends with the formation of Mature T-lymphocytes that have specific membrane markers (antigens CD3, CD4, CD8), missing at early stages of development . One of the mechanisms of formation is such cases, stimulants differentiation may lead to normalization of immune status.Known substances peptide that stimulates the differentiation of T-lymphocytes. These include drugs, obtained from the thymus of animals, such as thymosin , T-activin , the adjuvant . These drugs consist of complex substances peptide, which is able to regulate different stages of proliferation and differentiation of T-lymphocytes. Use them as drugs is difficult due to the complexity of the methods of preparation, low output active substances, considerable variability in their physical and chemical properties, as well as due to the presence of unwanted side effects due to the presence of natural preparations of thymus ballast of high molecular weight components. It is preferable to use low molecular weight synthetic peptides. They are effective in very small doses and do not have harmful effects on the body.Known synthetic peptides having the ability to stimulate the differentiation of T-lymphocytes. These include peptide thymopentin Arg-Lys-Asp-Val-Thr (Goldstein G. , Audhya T. K. Thymopoietin to Thymopentin: Experimental Studies. Survey of immunologic Research. (1985), 4: suppl. 1, pp. 1-10); analogues thymopentin (patent EP 1450798, FROM 07 TO 7/64, 1985; U.S. patent 5013723 And 61 To 37/02, 1991); the peptide Tyr-Gly (U.S. patent 5100663 And 617/00, 1995); the Tripeptide Arg-Lys-Glu possessing immunostimulatory activity and acts primarily on T-cell immunity (U.S. patent 4874844, 07 K 5/08, 1989); similar serum thymic factor of General formula A-Gly-Gly-Ser-Asn-B-C-NH-R, where a is Glu, Gln, Ala-Lys-Ser or Glu-Ala-Lys-Ser; and - Gly, Phe, Leu, Ala (patent EP 0201646, FROM 07 TO 07/06, 1986).Known means timogen, possessing the ability to stimulate the differentiation of T-lymphocytes [5,6]. Timogen synthesized based on a selection of thymalin by high performance liquid chromatography immunoactive faction, representing the dipeptide Glu-Trp. This substance is the technical essence and the achieved effect is closest to the proposed solution and its prototype. According to the prototype synthetic peptide Glu-Trp dose-dependently and selectively stimulates the differentiation of precursor T cells into Mature T-cells, but does not affect b-cell differentiation. However, this peptide has low biological activity that requires the use of large doses of the drug and cause the appearance of undesirable side effects.The present invention is directed to expanding Arsenal and increase biological activity funds, is the quality tools, having the ability to stimulate the differentiation of T-lymphocytes, it is proposed to use synthetic peptide of structural formula Lys-Glu-Glu-Leu-Asn-Glu. The peptide was synthesized by standard methods of solid-phase synthesis by increasing peptide chain from N-Terminus . Lys-Glu-Glu-Leu-Asn-Glu - acidic, hydrophilic peptide with MM 761 D.In experimental study of the specific activity of the claimed funds held in comparison with timagenes identified by its ability to stimulate the differentiation of T-lymphocytes in much lower concentrations. This is confirmed by the following examples.Example 1. The influence of the peptide Lys-Glu-Glu-Leu-Asn-Glu on the expression of E-receptors "trypsinisation" thymocytes.With the purpose of obtaining a suspension of lymphoid cells in the thymus of intact Guinea pigs male homogenized in medium 199. Then, the resulting suspension of thymocytes were treated with a proteolytic enzyme trypsin (Sigma), destroying a large part of E-receptors on the surface of cells to rabbit erythrocytes, resulting in a reduced percentage of cells detected in the reaction of rosethorne (T-lymphocytes). In suspensions of intact thymocytes and thymocyte-treated trypsin, aralica [8, 9]. The peptide Lys-Glu-Glu-Leu-Asn-Glu and timogen were made in culture trypsinisation" of thymocytes in the concentration range from 0.0004 to 0.05 µg/ml.Found that treatment with trypsin leads to a decrease of more than 2 times the number of thymocytes that have on the surface of E-receptors. The addition of a peptide Lys-Glu-Glu-Leu-Asn-Glu in a concentration of from 0.05 to 0.002 μg/ml and thymogen at a concentration of 0,05 - 0,01 µg/ml resulted in restoration of the number of E-receptors in lymphoid cells of the thymus (table 1).Thus, the minimum concentration at which the inventive tool enhances the expression of E-receptors of thymocytes in 5 times less than that of thymogen, indicating a higher specific activity of the peptide Lys-Glu-Glu-Leu-Asn-Glu.Example 2. The influence of the peptide Lys-Glu-Glu-Leu-Asn-Glu on the expression differencirovannyh antigens to T-lymphocytes of patients with burns in vitro.The specific activity of the proposed drug was evaluated by the ability to stimulate the expression differencirovannyh antigens are markers of Mature T-lymphocyte. The level of expression of differencirovannyh antigens was assessed by indirect membrane immunofluorescence with monoclonal antibodies LT3, LT4, LT8 to differe is already in the final stages of differentiation of T cells. The study was performed on lymphocytes of patients with burns, because you know that if you burn the sickness disturbed differentiation of T-lymphocytes in peripheral blood decreases the number of Mature forms of T cells [11,12]. Lymphocytes of patients with burns IIIB-IV degree 15-30% of the body surface was allocated on the density gradient of ficoll-urografin. The obtained cell suspension was incubated with the peptide Lys-Glu-Glu-Leu-Asn-Glu and timagenes in a concentration of from 0.0004 to 0.05 µg/ml for 2 h at 37oWith full nutrient medium RPMI-1640 (Flow) supplemented with 5% fetal calf serum ("Flow"), 2mm L-glutamine (Flow), 40 µg/ml gentamicin ("Pharmachem") and 5mm HEPES-buffer. As control was used saline solution in an appropriate volume. The obtained data are presented in table 2.Found that patients with burns sharply reduced the number of all the studied subpopulations of lymphocytes, excluding OWLS+cells. Timogen restored content D3+and CD4+lymphocytes at concentrations of 0.05 and 0.01 µg/ml, and the inventive tool was active at doses from 0.05 to 0.002 μg/ml, Both tools significant change in the number of CD8+lymphocytes did not cause (PL. 2).Thus, the peptide Lys-Glu-Glu-Leu-Asn-Glu is of its high specific activity.Example 3. The influence of the peptide Lys-Glu-Glu-Leu-Asn-Glu on the expression differencirovannyh antigens to T-lymphocytes in thymectomized mice in vivo. The biological activity of the proposed drug was studied on the model of thymectomy  . It is known that the thymus is the differentiation of T-lymphocytes and its removal is accompanied by disruption of this process. At three months of age produced a surgical removal of the thymus in mice. The control group of animals was subjected to false operation playback of all stages of surgical intervention except for removal of the thymus. At 1 month after surgery thymectomized animals were injected with the peptide Lys-Glu-Glu-Leu-Asn-Glu and timogen in doses of 0,0008 to 0.1 µg/kg body weight for 7 days. The control were thymectomized animals treated with saline in the same volume. The number of Mature T-lymphocytes, with clusters of differentiation of CD4 (T-helper) and CD8 (cytotoxic T lymphocytes), was determined in the reaction of direct membrane immunofluorescence using monoclonal antibodies labeled with FITC, F7400 and F7525 to molecules CD4 and CD8 mouse respectively (Sigma, Sweden). It is known that CD4 and CD8 are differentsirovannym marker for T cells and determined Tvout in pre-T cells. The results are presented in table 3.It was determined that thymectomized mice dramatically reduced the number of T-lymphocytes bearing on its surface differencirovanie antigens CD4 and CD8. Introduction peptide Lys-Glu-Glu-Leu-Asn-Glu in the whole range of doses thymectomized animals leads to an increase in the number of Mature T-lymphocytes, with clusters of differentiation on its surface, and the maximum activity of the proposed tools registered in a concentration of 0.02 μg/kg body weight. A similar effect is achieved by the introduction of thymogen in doses of from 0.004 to 0.1 µg/kg with a maximum activity at a dose of 0.1 µg/kg body weight. Thus, the peptide Lys-Glu-Glu-Leu-Asn-Glu in vivo stimulates the differentiation of T-lymphocytes at concentrations 5 times smaller than the known peptide timogen.Thus, in experimental study of the specific activity of the claimed funds established that the peptide Lys-Glu-Glu-Leu-Asn-Glu has the property to stimulate the differentiation of T-lymphocytes. Compared with timagenes the claimed peptide Lys-Glu-Glu-Leu-Asn-Glu capable of stimulating the differentiation of T-cells 5 times smaller doses. Reducing the dosage of the inventive means to achieve a similar">The proposed tool can be used for the prevention and treatment of a wide spectrum of immunodeficiency involving violation of the process of differentiation of T-lymphocytes.Sources of information
1. Cetlinski S. A. , Kalinina N. M. Immunology for the doctor. - SPb. LLP, Izd-vo "Hippocrates". 1998. - 156 S.2. Goldstein, A. L., on A., Zatz M. M. et al. Purification and biological activity of thymosin, a hormone of the thymus gland // Proc. Nat. Acad. USA 1972. Vol. 69. # 7. P. 1800-1803.3. Arion C. J. T-activin and its immunobiological properties: author. dis. Doc. Biol. Sciences. - M - 1990. - 52 S.4. Mashkovsky M. D. Medicines. - M.: Medicine, 1988, 2, 9 main, C. 168-175.5. Yakovlev, M., Khavinson C. H., Morozov Century, and other Comparative study of the biological activity of thymalin and synthetic peptide thymus // proc. Dokl. scient. proc. Biochemistry medicine". - L., 1988, S. 217 - 218.6. Khavinson C. H., Sinkevich N. In., Grey S. C. Timogen.- SPb, 1991. - 48 S.7. Steward J. M. , Young, J. D. Solid phase peptide synthesis, San Francisco, 1969.8. Nekam, K., and Fudenberg, H. H., Strelkanskas A. J. Identification of "activ" T-lymphocytes among effector cells in guinea pigs // Immunopharmacol. - 1982. - Vol.5. - 1.- P. 85-94.9. Stadecker, M. J., Bishop, G., H. H. Wortis Rosette formation by guinea pig thymocytes and thymus derived lymphocytes with rabbit red blood cells // J. Immunol.- 1973. Vol. 111. 6. - P. 1834-1837.12. Belotsky S. M. , Borisova, So, Shastina T. I. and other Characteristics of phagocytes, T - and b-lymphocytes In annealed // Immunology. - 1983. - 6. - S. 51-54.13. Immunological methods / edited, Primes. TRANS. with it. A. P. Tarasova. - M.: Medicine, 1987, 472 S. The peptide of structural formula Lys-Glu-Glu-Leu-Asn-Glu, stimulating the differentiation of T-lymphocytes.
FIELD: medicine, cardiology.
SUBSTANCE: the suggested method should be performed at the background of medicinal therapy with preparations out of statins group, tevetene, polyoxidonium and conducting seances of plasmapheresis by removing 800 ml plasma twice weekly with N 5 due to additional intramuscular injection of immunophan 0.005%-1.0 with N 10 and fluimucyl 300 mg intravenously daily with N 5-10, total course of therapy lasts for 2 mo. The method provides modulation of leukocytic functional activity, moreover, due to altered cytokine profile and, thus, through disintegration of protein-lipid complexes participating in the development of atherosclerotic platelets.
EFFECT: higher efficiency of therapy.