RU2052192C1 - Method of selection of preparation for immunocorrection - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method for selection of preparation for immunocorrection includes preliminary determination of modifying effect of preparations on expression of complex of differentiating antigens CK1,4,8,38, HLAl on the basis of the sum of units calculated for each marker by formula (O-K x 100)/K, with K being equal to or less than lower limit of normal value and (K-O x 100)/O with K being equal to or larger than the normal value upper limit, where K and O are percent of cells carrying given antigen before and after incubation with preparation. In this event, if preparation optimally correcting relation CD4⁺/CD8⁺ of cells has not gained the maximum number of units, selection between it and preparation which most intensively increases expression of complex of receptors and changing to the increase of above indicated relation, is carried out with the help of formula given in invention description, and in case, it changes towards decrease of the above indicated relation, with the help of formula given in invention description. In case of result being less than 1.5 selected is the preparation which has gained the largest number of units. In initial change of relation CD4/CD8 cells being equal to or larger than 2 or equal to or less than 0.7, selection coefficient of the formula equals 1.2. EFFECT: higher accuracy of selection and increased efficiency of use of immunicorrecting preparation. 2 cl, 6 tbl

Description

 The invention relates to medicine, in particular immunology, in pediatrics and can be used in practical health care and research institutions in order to improve the accuracy of choice and increase the effectiveness of the use of immunocorrectors.

 Known methods for determining the need for the introduction of immunocorrective agents based on the determination in vitro of their individual modifying reaction of the surface of the blood mononuclear cells of the subject. For the most part, the degree of increase in the reaction of T-rosette-forming cells (reflects changes in the expression of CD2-T cell antigen on lymphocytes) is used as a criterion for determining the need for the introduction of an immunostimulant under the influence of this immunocorrector.

 Closest to the claimed is a method that is selected as a prototype. After isolation of the mononuclear cells, they are incubated with immunocorrecting drugs. The degree of increase in T-ROCK in each sample judges the sensitivity of this patient to the corresponding drug.

 The disadvantages of the prototype is, on the one hand, the fact that this method of selecting and prescribing an immunocorrector does not take into account the initial state, modifying the effect of drugs on expression, the ratio of other receptors that take part in the processes of cellular recognition, cooperation in the process of the immune response, from the presence of which, the ratio depends on the intensity, other characteristics of the immune response. It is known that the presence of various differentiation antigens is associated with certain functions of immunocompetent cells. Another disadvantage of the method used is that the methodological approach used to determine the reaction of mononuclear cells in the reaction of rosette-forming cells is a method that establishes subpopulations of cells indirectly, thereby less accurate and reproducible than the determination of differentiation markers using monoclonal antibodies. In the prototype there are no specific indicators to assess the degree of increase of T-ROCK. In connection with these shortcomings, the choice of an immunocorrector using the used prototype method may not be possible or this choice will not be optimal.

 The technical result is to increase the accuracy of choice and increase the effectiveness of the drug.

×100 при К (нижней границы нормы и

×100 при К) верхней границы нормы, где К и O клеток, несущих данный антиген соответственно до и после инкубации с препаратом, причем, если препарат, оптимально корректирующий соотношение СД4-/СД8- клеток, не набрал максимальное число баллов, то выбор между ним и препаратом, наиболее интенсивно увеличивающим экспрессию комплекса рецепторов и изменяющим в сторону повышения вышеуказанное соотношение, осуществляют по формуле

в сторону понижения по формуле

где СД4^-/СД8^- оптим оптимальное соотношение;
Б_макс максимальное количество баллов, и при результате (1,5 назначают препарат, набравший большее число баллов. При исходном изменении соотношения СД4^-/СД8^-) 2 или (0,7 коэффициент изменения разности соотношения СД4^-/СД8^- к изменению разности баллов равен <1,2).The technical result is achieved by preliminarily determining the effect of drugs on the expression of a complex of differentiating antigens, for example, SD 3, 4, 8, 38, HLA1, based on the total points calculated for each of the markers according to the formula

× 100 at K (the lower limit of the norm and

× 100 at K) the upper limit of the norm, where K and O cells carrying this antigen, respectively, before and after incubation with the drug, and if the drug, which optimally corrects the ratio of CD4- / CD8-cells, did not score the maximum number of points, then the choice between him and the drug, the most intensively increasing the expression of the complex of receptors and changing upward the above ratio, is carried out according to the formula

downwards according to the formula

where SD4 ^- / SD8 ^{- the} optimal ratio;
B _max the maximum score, and if the result is (1.5 prescribers receiving the higher the number of points at the reference change ratio of CD4 ^-. / CD8 ^-) or 2 (0,7 rate of change ratio difference CD4 ^- / CD8 ^- to points difference change equals <1,2).

 The essence of the method is as follows.

при К< нижней границы нормы и

при К> верхней границы нормы, оценка первоначальной экспрессии дифференцировочных маркеров может осуществляться как исходя из так и абсолютного количества клеток в мкл крови. После подсчета баллов и анализа соотношения СД4^-/СД8^- клеток, изменившегося под влиянием апробируемых препаратов, для иммунокоррекции выбирают препарат, набравший наибольшее число баллов и наилучшим образом изменивший в заданную сторону соотношение СД4^-/СД8^- клеток. Если препарат оптимально корректирующий соотношение СД4^-/СД8^- клеток, не набрал максимальное число баллов, выбор между ним и препаратом наиболее интенсивно увеличивающим экспрессию комплекса рецепторов и изменяющим в сторону повышения, понижения указанное соотношение производят соответственно исходя из формул

где СД4^-/СД8^- оптим оптимальное соотношение;
Б_макс максимальное количество набранных баллов, и при результате <1,5 назначают препарат, набравший большее число баллов. При исходном, более резком изменении соотношения СД4^-/СД8^- клеток >2 или <0,7 коэффициент выбора указанных формул равен <1,2. Если инкубация с отдельными препаратами не дает полностью желаемого эффекта изменения экспрессии рецепторов, инкубацию мононуклеаров, при необходимоси совместного использования иммунокорректоров, проводят с соответствующими смесями их (каждый препарат используется в дозе близкой к терапевтической). Выбор при необходимости совместного назначения иммунокорректоров осуществляется таким же самым способом.Mononuclear cells are isolated from a blood sample using a density gradient. A suspension of mononuclear cells is standardized (the concentration is adjusted to 1-2x10 cells / ml) and individual portions of them (0.02 ml each) are incubated for 30 minutes with 0.02 ml of the corresponding immunocorrector in concentrations close to therapeutic (take 3-5 drugs, if necessary and more, for example: thymalin 0.5 μg / ml; T-activin 0.1 μg / ml; thymoptin 0.1 μg / ml) and medium 199. The presence of receptors is detected by incubation with the corresponding antisera in an indirect or direct immunofluorescence reaction. The modifying effect of drugs on the expression of a complex of differentiating antigens (for example, CD3, 4, 8, 38, HLA1) is determined based on the sum of points calculated in the ratio of each of the markers according to the formula

when K <the lower limit of the norm and

when K> the upper limit of the norm, the initial expression of the differentiation markers can be evaluated both on the basis of the absolute number of cells in μl of blood. After scoring and analysis of the ratio of CD4 ^- / CD8 ^- cells, which changed under the influence of tested drugs, for immunocorrection, choose the drug that scored the most points and best changed the ratio of CD4 ^- / CD8 ^- cells in the given direction. If the drug optimally corrects the ratio of CD4 ^- / CD8 ^- cells, has not scored the maximum number of points, the choice between it and the drug that most intensively increases the expression of the receptor complex and changes upward, lowering this ratio is carried out accordingly based on the formulas

where SD4 ^- / SD8 ^{- the} optimal ratio;
B _{max is the} maximum number of points scored, and with a result of <1.5, the drug with the most points is prescribed. In the initial, sharper change in the ratio of CD4 ^- / CD8 ^- cells> 2 or <0.7, the selection coefficient of these formulas is <1.2. If incubation with individual drugs does not give the completely desired effect of changes in the expression of receptors, the incubation of mononuclear cells, if necessary, the use of immunocorrectors, is carried out with their respective mixtures (each drug is used in a dose close to the therapeutic). The selection, if necessary, of the joint appointment of immunocorrectors is carried out in the same way.

Coefficients 1.5 and 1.2 are selected on the basis of experimental studies, by calculations showing that the prevalence of expression changes in the total amount of the studied receptors over changes in the ratio of CD4 ^- / CD8 ^- cells is 1.5 times more significant for the effect than changes in expression and the ratio of these 2 receptors. With a sharper change in the ratio of CD4 ^- / CD8 ^- cells (> 2 and <0.7), the importance of changing the expression of these receptors increases, therefore, the coefficient determining the difference in the modifying effect of the drug on the expression of the sum of receptors to the change in the ratio of 2 effector subpopulations of mononuclear cells is reduced to 1 , 2.

 Immunocorrection is carried out by the selected drug according to commonly used schemes: for example, thymalin is administered IM deeply at a dose of 0.1-0.2 mg / kg of body weight 3-5 injections daily for 1 injection; T-activin and thymoptin are administered once a day IM, at a dose of 1 μg / kg of body weight 3-5 injections on the 1st daily or with an interval of 7-10 days.

 Examples of specific performance of the method.

 PRI me R 1. G.E. 03/26/88, M. Lenin, 153-44.

DS: Thymus hyperplasia. Acquired immunodeficiency, BDF (see table 1)
Based on the fact that the expression of receptors under the influence of thymalin in vitro increases most (140.21 points) and this drug most optimally (up to 1.06) corrects the ratio of CD4 ^- / CD8 ^- cells, initially reduced to 0.7, this patient the administration of thymalin at a dose of 0.1 mg / kg weight i.m. 1 time per day N 5 was prescribed. During (3 courses) immunocorrective therapy and after it for 10 months, I did not suffer from intercurrent infections. Before treatment, frequent respiratory infections 1-2 times a month. The intensity of in vivo receptor expression in the last study was: CD4 29% CD3 78% CD8 24% CD38 22% HLAI 84% Ratio CD4- / SD8-1.2.

 PRI me R 2. A.O. 09/05/1975 Mr. M. st. Troyanovsky, 70/13.

 DS: Atopic dermatitis. Allergic rhinus sinusopathy, FDI (see table. 2).

A change in the expression of the studied markers was found, a slightly increased ratio of CD4 ^- / CD8 ^- cells due to a sharper decrease in the detection of CD8 antigen. This ratio was normalized by Timoptin and T-activin. However, the intensity of changes in the expression of the studied antigens was more pronounced in T-activin. Differences in the change in the ratio of CD4 ^- / CD8 ^- when using 2 of these drugs were insignificant in relation to variations in the expression of the studied markers. The coefficient of the formula was 1.4. In connection with the foregoing, the administration of T-activin N 3 is prescribed.

During immunocorrective therapy, the child's condition improved. Allergic manifestations decreased, rhinorrhea disappeared, I did not suffer from intercurrent diseases. The intensity of marker expression was CD4 29% CD3 69% CD8 21% CD38 28% HLA1 81% The ratio of CD4 ^- / CD8 ^- 1.43. These changes in T-ROCK of mononuclear cell populations under the influence of in vitro immunomodulators gave in all cases an increase in the expression of receptors by 18-20% and the choice of the drug according to these data is impossible.

 PRI me R 3. H.O. 04/25/90, D. st. Embankment 1776-83.

 DS: Timomegaly 2 tbsp. Secondary immunodeficiency state, FDB (see table 3).

The child has a secondary immunodeficiency state with a sharp decrease in CD4 and CD8 receptors. The ratio of diabetes ^- / diabetes8 ^- cells is also sharply reduced to 0.6. Incubation of blood mononuclear cells revealed that thymoptin and thymalin correct the expression of receptors, increasing the indicated ratio. The coefficient of choice between these drugs is 1.1, which is <1.2, therefore, thymalin N 3 is prescribed to the above patient. Before treatment, frequent acute respiratory viral infections, prolonged, constant subfibrillation. After the first course of therapy, subfibrillity disappeared. ARVI was not sick. Expression of CD3 receptors 72% CD4 17% SD8 16% SD38 20% HLA1 90% Ratio of CD4 ^- / SD8 ^- mononuclear cells 1.06. Immunocorrective therapy continued.

PRI me R 4. R.E. 11/21/89; M. Mounting 54/4
DS: FDB. Timomegaly 1 tbsp. (see table 4).

The child has a secondary immunodeficiency state with an increase in the thymus gland. The ratio of CD4 ^- / CD8 ^- cells is reduced to 0.7. Incubation of blood mononuclear cells found that thymalin, thymoptin and T-activin correct the expression of receptors, increasing the above ratio. The coefficients between the preparation of the formula, optimally correcting CD4 ^- / CD8 ^- markers timalina and two other preparations Timoptin, T-activin are respectively 1.5 and 9.7. Based on the fact that the initial ratio of T-helpers and T-suppressors of 0.7 with a coefficient of 1.5 or more, thymalin was prescribed, which more optimally corrects the ratio of these effector subpopulations.

After the 1st course of therapy, ARVI was not sick. Expression of receptor LED3 LED4 70% 14% 12% CD8 SD38 14% HLA1 81% Ratio CD4 ^- / CD8 ^- mononuclear is 1.17.

PRI me R 5. Sh.I. 4.06.85 M. Comintern 34,
DS: Atopic dermatitis. BHA (see table 5).

The child has a secondary immunodeficiency state. The ratio of CD4 ^- / CD8 ^- cells is increased to 2. By incubation of mononuclear cells, it was found that all drugs used, especially T-activin and thymoptin, correct the above ratio, reducing it. The selection coefficient calculated by the formula is 1.2, so the child was prescribed 5 injections of T-activin. After the course of therapy, the manifestations of allergic dermatitis decreased, acute respiratory viral infection was sick. Duration, severity of the course of the disease is much easier than usual (t, respectively, 2 days and 5-6 days 38.5 and up to 39.5-40 ^about C). Expression of receptor CD3 5 CD4% CD8% 20 14% 36% HLA1 SD38 74% Ratio CD4 ^- / CD8 ^- 1.43 mononuclear cells.

 The given examples show that the use of the T-ROCK reaction immunocorrector or the change in the expression of the DM2 marker to assess the potential effect of the marker does not always provide a sufficient and objective assessment of the effect of immunostimulants on the mononuclear receptor apparatus as a result of possible variability of the methods (mismatch of absolute numbers of cells having a receptor according to the results of T ROCK and identified using monoclonal antibodies example 1), and due to the objective impossibility of establishing the optimal drug on the results of changes in the expression of T-cell receptors of T2DM (almost equal stimulation by various immunocorrective drugs of T2DM receptors - example 3), the possibility of obtaining incorrect orientations based on the results of evaluating the immunocorrecting drug by one marker (example 1: according to the results of the T-ROCK reaction, thymalin administration is not shown , the seemingly best effect belongs to T-activin), etc.

 Conducted immunocorrection according to the proposed method in comparison with the prototype method allowed to improve the state of immunological parameters (see table. 1), to provide a more intensive (p <0.05) reduction in the incidence of acute respiratory viral infections in children suffering from frequent diseases.

 Clinical and immunological parameters of children with altered reactivity, immunocorrection of which was carried out using various schemes, are given in table.6.

Thus, the advantages of the proposed method is that it allows you to make the optimal choice of immunocorrector. Application of the method reduces the number of cases in the group of children suffering from frequent compared to the prototype method is more than 3 times, more optimum correction causes immune status (see Table 6 CD4 ^-., CD8 ^- receptors, tends to increase the constant portion HAL1 receptors) .

Claims (2)

1. METHOD FOR SELECTING A DRUG FOR IMMUNO CORRECTION, including determining in vitro the modifying effect of immunocorrections, characterized in that the effect of the drugs on the expression of the complex of differentiating antigens CD3, 4, 8, 38, HlAl is preliminarily determined, the total score for each marker is calculated by the formula

when K ≅ the lower limit of the norm and according to the formula

at K ≥ the upper limit of the norm, where K and 0 are the percentage of cells carrying this antigen, respectively, before and after incubation with the drug, and if the drug that optimally corrects the ratio of CD4 ⁺ / CD8 ⁺ cells does not score the maximum number of points, choose between it and the drug, the most intensively increasing the expression of the complex of receptors and changing upward the specified ratio, is carried out according to the formula

and downward - according to the formula

where CD4 ⁺ / CD8 $\genfrac{}{}{0ex}{}{\text{+}}{\text{optim}}$ - optimal ratio;
B _{m and to a} - the maximum number of points,
and with a result of less than 1.5, a drug with a large number of points is prescribed. 2. The method according to claim 1, characterized in that when the initial change in the ratio of CD4 ⁺ / CD8 ⁺ cells> 2 or <0.7, the coefficient of change in the difference in the ratio of CD4 ⁺ / CD8 ⁺ to the change in the difference in points is equal to or less than 1.2.

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