RU2108096C1 - Compound stimulating during experiment synthesis of immunoglobulins - Google Patents

Abstract

FIELD: medicine; may be used as stimulator of B-system immunity. SUBSTANCE: invention involves application, as stimulator of immunity B-system, compound 1-hydroxyhermatran (monohydrate). Proposed compound promotes synthesis of IgG, IgA, IgM immunoglobulins and intensifies synthesis of IgE. EFFECT: higher efficiency. 11 tbl

Description

 The invention relates to medicine, namely immunology, and can be used in experimental studies to study the mechanism of influence of biologically active substances on the body's immune system, in particular on the B-system, as well as in clinical practice as a medicine.

 Currently, the effect of various immunomodulators of a chemical and biological nature (taktivin, myelopid, diutsifon, etc.) on the indicators of the immune status of humans and laboratory animals has been well studied, and many drugs are already used in clinical practice for the treatment of various diseases associated with impaired immune systems .

 Particular attention, as promising for practical use, is given to the group of germanium compounds, which are increasingly used as agents with a different spectrum of biological activity and low toxicity.

 Germatranes stand out from this group of biologically active substances, the representatives of which exhibit anticarcinogenic, antiviral, neurotropic, etc. activity [1].

One of the representatives of the named group of organogermanium compounds is 1-hydroxyhermatran (monohydrate) (C ₆ H ₁₅ NO ₅ Ge), which is known and for which antihypoxic activity has been established [2].

1-hydroxyhermatran (monohydrate) (or germatranol) is a white crystalline powder, readily soluble in water, DMSO, DMFS. The molecular weight is 254.01. It is characterized by extremely low toxicity. According to various definitions, it has an LD ₅₀ of 6,000 to 10,000 mg / kg body weight.

 The objective of the invention was to find among known germanium-organic compounds compounds that have a stimulating effect on the B-system of immunity and its use with this activity.

 The problem was solved using the well-known compound - 1-hydroxyhermatran (monohydrate) (germatranol), which, as a result of a comprehensive experimental study, revealed a stimulating effect on the synthesis of immunoglobulin, in particular IgA, IgM, IgG.

 A cumulative test to evaluate the B-system of immunity is to study the ability of B-lymphocytes to perform their main function: to synthesize immunoglobulins IgA, IgM, IgG. To do this, in vitro synthesis of immunoglobulins is induced using T-B-mitogen-mitogen-pacifier (PWM).

 The authors of this application studied the effect of the drug germatranol (Ge) on the main parameters of the immune system: the functional activity of phagocytes, T- and B-lymphocytes.

In accordance with the task, the effect of germatranol on the following immunity indicators was studied:
1. proliferative response of lymphocytes to T-mitogen: phytohemagglutinin (PHA);
2. synthesis of immunoglobulins in vitro;
3. phagocytic activity of peripheral blood cells;
4. luminol-dependent chemoluminescent response of phagocytic cells;
1. The proliferative response of lymphocytes to PHA.

 Blood sampling was carried out in the morning on an empty stomach. Used heparinized (20 units / ml) blood taken from the ulnar vein. Mononuclear cells (MK) were isolated using a one-stage density gradient Ficoll-Paque ("Pharmacia"). The cell viability after isolation was 98-99% (according to the staining method with a 0.1% solution of trypan blue Serva). MK in complete culture medium (PCS) :( RPMI-1640 with the addition of 101)% inactivated fetal calf serum (ETS), 10 mM HEPES buffer (all "Flow"), 2 mM L-glutamine (Institute of Poliomyelitis and Viral Encephalitis AMN (RF) and 40 μg / ml gentamicin (Pharmachim, Bulgaria) at a concentration of 2 million / ml.

Mononuclear cells were spread into a 96-well round-bottom plate ("Linbro") at 100 thousand per well in triplets. Only PKC was added to the 1st triplet, and PHA (60 μg / ml Serva) was added to the 2nd triplet. These triplets were used as control. To the 3rd, 4th, 5th and 6th triplets, respectively, germatranol was added at a final concentration of 50, 5, 0.5 and 0.05 μg for the entire incubation period, i.e. 72 h. Cells were incubated in a CO ₂ incubator. The final volume of each well is 150 μl. Proliferative response levels were evaluated radiometrically by incorporation of 3H-thymidine (1 μK / well) delivered 6 hours before the end of incubation. Data on triplets was averaged. The number of emitted pulses per minute (imp / min), stimulation indices (IS) were estimated as the ratio of induced proliferation to spontaneous.

 2. Synthesis of immunoglobulins in vitro.

 The functional state of immunocytes was evaluated by the ability of MNCs to synthesize Ig during prolonged cultivation. This method makes it possible to assess the state of B-cells transforming into plasma Ig-synthesizing cells, T-helpers that transmit an activating signal to B-cells and, to a lesser extent, T-suppressors that control activation mechanisms.

To assess the ability of B cells to synthesize Ig, 50, 5, 0.5, and 0.05 μg, respectively, of germatranol were added to 150 thousand responding cells. To assess the ability to synthesize Ig cells at the same concentration, they were incubated with PWM at a dose of 125 μg / ml ("Sigma"). Cells were cultured for 10 days in a CO ₂ incubator ("Flow") at 37 ^° C in round bottom plates ("Linbro"). At the end of the cultivation, the plates were centrifuged (10 min, 400 g) and the contents of IgA, IgG and IgM were determined in supernatants by the enzyme immunoassay (in ng / ml). For this, anti-isotypic goat antibodies against IgA, IgG, and IgM (10 μg / ml, Sigma) in phosphate buffer (pH 7.4) were added to wells of 96-well flat-bottom plates (Greiner) and left for 18 h at 4 ^o C. After washing four times with phosphate buffer, the test samples and standard serum (with 2 μg / ml in steps of "2" to 1.9 ng / ml) were introduced into triplicate wells and incubated for 60 min at 37 ^° C in phosphate buffer with tween-20 (0.05%). After washing four times, a goat anti-human Ig conjugate labeled with horseradish peroxidase (Sigma) labeled at a dilution of 1: 1000 was added to the wells and left for 60 min at 37 ^° C. Then a substrate mixture (0.15 M phosphate-citrate buffer, pH) was added to the wells. = 5.0 with 0.04% orthophenyleneamine and 0.012% hydrogen peroxide) at 100 μl / well and left at room temperature for 30 minutes. The substrate-enzyme reaction was stopped by adding 2 N sulfuric acid at 50 μl / well. The concentration of Ig in standard solutions and test samples was determined in accordance with the optical density of the reaction mixture on a Multiscan-MCC440 spectrophotometer, wavelength 492 nm. Using computer programs, the results of studies of standard solutions were used to construct a calibration curve for the regression analysis, and the Ig content in the test samples in ng / ml was calculated using the formula describing this curve.

 3. Phagocytic activity of peripheral blood cells.

 The principle of the technique is that the number of fluorescent polymorphonuclear leukocytes, clearly identified in a separate window by direct and side scattering of the laser beam, absorbing FITC-labeled yeast is recorded on a flow laser cytometer.

For this purpose, a leukocyte suspension is prepared with a concentration of polymorphonuclear leukocytes of 10-6 / ml on medium 199. A leukocyte suspension is obtained by the standard method of spontaneous sedimentation of red blood cells in the presence of 1% gelatin. Simultaneously prepare a suspension of baker's yeast on PBS. To standardize the size of baker's yeast, they are grown in Saburo's nutrient medium overnight. A well-washed suspension of baker's yeast on PBS at a concentration of 20 million / ml is incubated with an equal volume of FITC-PBS at a final concentration of the latter of 2 mg / ml for 1 hour at room temperature with constant shaking. At the end of the incubation, the yeast is washed thoroughly in PBS at least 10 times. A final concentration of 4-10 ml per PBS is prepared, which can be stored at -20 ^o C for at least a month.

 In the experiment, 0.1 ml of the cell suspension is mixed with 0.1 ml of autologous plasma, 0.1 ml of baker's yeast suspension (concentrations indicated above) and 0.2 ml of medium 199. When testing the immunomodulating effect, instead of medium 199, germatranol was administered in the appropriate dose in 0.1 ml volume and 1 ml of 199 medium was added.

The mixture is incubated for 60 minutes at 37 ^° C. with constant shaking. At the end of the incubation, 0.5 ml of 2% paraformaldehyde is added until the result is measured on a cytometer. In the process of cytometry, when compiling a program, a neutrophilic window and a yeast window are released. Windows should be clearly separated from each other. Control samples should not give the number of events in the neutrophilic window more than 100. The introduction of experimental samples shows the number of events in the neutrophilic window and the percentage of fluorescent cells that absorb FITC-labeled yeast.

 Taking into account the number of events in the neutrophilic window in control samples, as well as the number of events and the percentage of fluorescent cells in the same window, the phagocytic index (PI) is calculated in the experiment.

 4. Luminol-dependent chemoluminescent response of phagocytic cells.

 Blood sampling was carried out in the morning on an empty stomach. Used heparinized (20 units / ml) blood taken from the ulnar vein. A leukocyte suspension was obtained by the standard method of spontaneous sedimentation of red blood cells in the presence of 1% gelatin. Neutrophils were isolated using a double density gradient Ficoll-Pague ("Pharmacia"). The viability of the cells after isolation was 98-99% (by staining with 0.1% trypan blue Serva). The isolated neutrophils were washed three times with Hanks solution with phenol red (Institute of Poliomyelitis and Viral Encephalitis of the Academy of Medical Sciences of the Russian Federation) and then resuspended in a sterile cooled medium 199 (Institute of Poliomyelitis and Viral Encephalitis of the Academy of Medical Sciences of the Russian Federation). The resulting neutrophil suspension was adjusted to a concentration of 1.8 million / ml.

The study of the chemiluminescent activity of neutrophils was carried out on a Luminometer 1251 (LKB) instrument at 37 ^° C in two stages: spontaneous luminescence and zymosan-induced luminescence.

 To record spontaneous luminescence, 100 μl of a solution of luminol, 300 μl of a suspension of neutrophils and 100 μl of a solution of germatranol with concentrations of 0.05, 0.5, 5, and 50 μg / ml in 199 medium were added to each measuring cuvette. As a negative control, instead of germatranol 100 μl of 199 medium was added. The background value of spontaneous luminescence was estimated from the luminescence of a neutrophil-free sample. Registration of spontaneous luminescence of neutrophils was carried out for 60 minutes.

 To evaluate the induced chemiluminescence, either 100 μl of a suspension of ethmosan particles (Biolar), opsonized with normal human pool serum at a concentration of 20 mg / ml in Hanks solution, was added to the cuvettes, or to estimate the background value equal in volume to the amount of Hanks solution. Registration of induced luminescence was performed for 60 minutes.

где ИХ - величина индуцирвованной хемилюминесценции;
СХ - величина спонтанной хемилюминесценции;
ФИ - фоновые значения индуцированной люминесценции;
ФС - фоновая величина спонтанной люминесценции.The chemiluminescence stimulation index (IP) was estimated by the formula

where THEM is the magnitude of induced chemiluminescence;
CX is the value of spontaneous chemiluminescence;
FI - background values of induced luminescence;
FS is the background value of spontaneous luminescence.

 1. The proliferative response of lymphocytes to PHA. The effect of germatranol in doses of 50, 5, 0.5, and 0.05 μg / ml on the proliferative response of donor peripheral blood lymphocytes to PHA was tested (Tables 1, 2, 3, 4). It turned out that at a dose of 50 μg / ml, germatranol does not significantly affect the proliferative response of lymphocytes (Table 1). However, in lower doses, germatranol always has a weak stimulating effect (Tables 2, 3, 4) on this indicator of the functional activity of T-lymphocytes.

 2. Synthesis of immunoglobulins in vitro. The drug germatranol had a multidirectional effect on the synthesis of various classes of Ig in vitro (Tables 5, 6, 7). At the highest doses (50 and 5 μg / ml), germatranol caused a suppression of the synthesis of the main classes of immunoglobulins: G, A, M. At a dose of 0.5 μg / ml, it practically did not affect the synthesis of immunoglobulins. When applying a dose of 0.05 μg / ml, germatranol had a pronounced stimulating effect on the synthesis of the main classes of immunoglobulins. A particularly strong effect was observed with the use of germatranol with respect to the synthesis of IgA and IgM, where germatranol has a stable stimulating effect. The stimulating effect of germatranol was also observed with respect to IgG, but here it is less pronounced. The effect of germatranol on IgG synthesis in donor 2 is interesting (Table 6): in this donor, in vitro IgG synthesis is reduced. Germatranol completely restores the ability to synthesize this immunoglobulin. Interesting is the effect of germatranol on the synthesis of minor immunoglobulins: IgE and IgD. Germatranol caused a pronounced increase in IgE synthesis (Table 8) in almost all donors. The synthesis of IgD under the influence of germatranol was increased in only one donor (Table 9).

 3. Phagocytic activity of peripheral blood cells. The use of two doses of germatranol: high and medium, showed the absence of the effect of this drug on the absorption capacity of phagocytic cells (Table 10).

 4. Luminol-dependent chemiluminescent response of phagocytic cells. The results of the study of the effect of germatranol on the formation of reactive oxygen species, recorded by luminol-dependent chemiluminescence, are presented in table. 11. It turned out that in high doses - 50 μg / ml, germatranol somewhat suppresses spontaneous chemiluminescence, i.e. functional activity of phagocytic cells. At lower doses - 5, 0.5 and 0.05 μg / ml, germatranol does not affect this indicator. Germatranol does not affect the induced chemiluminescence in any of the tested doses.

 Thus, the studies performed revealed a pronounced ability of germatranol to stimulate the synthesis of immunoglobulins in vitro. This is manifested in increased production of IgG, IgA, IgM. There is also an increase in the synthesis of IgE under the influence of this drug. The enhancing effect is detected when only low doses of the drug are used, which speaks in favor of its positive physiological effect.

 The results suggest that the drug is a stimulant of the B-system of immunity, while it does not have the ability to stimulate the T-system of immunity and phagocytosis.

Claims (1)

 The use of 1-hydroxyhermatran monohydrate as a means of stimulating the synthesis of immunoglobulins in an experiment.

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