RU2536707C1 - Method for assessing local humoral anticholeraic immunity in vitro simulated in experimental animals - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves immunising with 5×10⁷ microbial cells Vibrio cholerae 5879; 10 days later, a common pool of anticholeraic Ig is recovered from enteric washing liquid. That is combined with producing the primary enterocyte culture from intact mice. Thereafter, the doubly-diluted anticholeraic Ig are applied in an amount of 3 ml on a monolayer of erythrocytes formed in wells of culture panels. The latter are added with alive choleraic vibrions of the strain Vibrio cholerae 5879, 40 m.c. per 1 erythrocyte and incubated for 3 hours. Then, the panels are washed three times, fixed with ethanol for 20 minutes and stained by Romanowsky-Giemsa. Local humoral anticholeraic immunity is assessed under the microscope by the number of vibrions stained in dark blue and attached to one erythrocyte.

EFFECT: method enables assessing specific local humoral immunity and intensifying the anti-adhesive activity of the common pool of anticholeraic immunoglobulin.

4 cl, 2 ex, 2 tbl

Description

The invention relates to medicine, in particular to the question of studying the release activity of cholera immunoglobulins and enhancing it to improve specific prophylaxis of cholera.

Cholera is an anthroponotic disease, so the development of animal models of infection has always met with serious difficulties. To date, experimenters have made numerous attempts to work out a model of infection in mice. There are models of generalized forms of cholera in mice, sealed mice. Given that cholera disease develops and proceeds in the small intestine, several experimental models have been developed that allow animals to obtain a pathogenetic picture of cholera disease that is similar to that in humans: a model of the tied loop of the small intestine of an adult rabbit, sucker rabbits.

However, currently there are no methods and models allowing to evaluate the effect of various drugs, for example immunomodulators, on the formation of local cholera immunity, in particular on the enhancement of antibody formation and anti-adhesive activity of cholera immunoglobulins (Ig). The development of such experimental methods and models is very relevant, since the first obstacle to many infections, including cholera, are protective factors acting on the surface of mucous membranes (epithelial cells, various bactericidal substances, secretory IgA, etc.) and preventing the adhesion of pathogens [12]. At the same time, the predominant value of the antibacterial component that prevents the attachment of cholera vibrios to the intestinal mucosa in the first stages of infection of the body is shown [3]. One of the main indicators of antibacterial immunity is the anti-adhesive activity of cholera Ig, the presence of which, along with antitoxic antibodies, indicates the adequacy of the formation of local humoral immunity [4, 5].

For the prototype, a method was chosen for evaluating the effectiveness of the use of the immunomodulator immunofan in experimental cholera (see patent RU No. 2481791, class A61B 10/00, A61K 38/08, A61P 43/00, G09B 23/28), which is that before infection animals in an isolated loop of the small intestine with a virulent strain they are injected with an immunomodulator immunofan in a dose of 0.2 μg, diluted in 0.3 ml of novocaine, in the amount of 10 injections. The effectiveness of the use of immunofan is assessed by the presence of an enteropathogenic effect in the experimental bandaged loops of the small intestine, the severity of which is evaluated in the crosses, and the presence of a cholerogenic effect, which is calculated by the formula K = Vzh / Np, where K is the loop extension coefficient, Vzh is the volume of fluid, Np - loop length, if there is no enteropathogenic effect in the experimental loops and K <1.0, consider using immunofan effective.

However, the objective of the known method was to evaluate the effect of immunomodulators on the development and severity of the pathogenetic picture of experimental cholera and does not allow studying in vitro the formation of cholera immunity, in particular the effects of various drugs, including immunomodulators, on the release activity of a common pool of cholera Ig from enteric wash liquids of white mice.

The purpose of the invention was to develop a method that allows to evaluate in vitro specific local humoral immunity in experimental cholera, as well as to enhance the anti-adhesive activity of the common pool of cholera Ig from the intestinal leaching fluids of white mice in the process of formation of cholera immunity.

This goal is achieved by the fact that in the known method for assessing local humoral anticholeron immunity in an experiment, white mice are immunized with 5 × 10 ⁷ Vibrio cholerae 5879 microbial cells, 10 days later a common pool of anti-cholera Ig is isolated from the small intestinal lavage fluids of mice, an additional primary culture of small intestine enterocytes is obtained from intact mice, then anti-cholera Ig in double dilution is applied in a volume of 3 ml onto a monolayer of enterocytes formed in the wells of the culture panels, then in the last nan FNF live Vibrio cholerae strain of Vibrio cholerae 5879, MK 40 1 enterocyte and incubated for 3 hours, washed three times after the panel, fixed with ethanol for 20 minutes and stained with Romanovsky-Giemsa, local humoral anticholera immunity is evaluated under a microscope according to the number of vibrioes stained in dark blue and attached to one enterocyte.

In this case, a common pool of cholera Ig is obtained from small intestinal leaching fluids, for this, two ligatures are applied at a distance of 5 cm from each other and 5 ml of physiological saline are injected into an isolated area, massaged and a syringe is taken which is precipitated with 50% ammonium sulfate, and the precipitate formed overnight at 4 ° C is centrifuged at 1000 rpm for 10 minutes.

In addition, the primary culture of small intestine enterocytes is obtained from intact white mice; for this, 5 ml of 0.2% ethylenediaminetetraacetic acid is injected into the lumen of the washed small intestine of animals, massaged for 30 minutes and the contents are pipetted, the cells obtained are washed three times with physiological saline, and suspended in culture medium with 10% fetal calf serum, 2 mm L-glutamine, 10 mm HEPES, 5 × 10 ⁵ M 2-mercaptoethanol, 100 IU / ml penicillin, 100 μg / ml streptomycin to a concentration of 150,000 cells / ml and seeded in wells 6 well culture s Costar panel after panel incubated in CO ₂ incubator for one day to form a sparse monolayer cells were then washed three times with fresh portions of medium without serum.

Moreover, for immunization of animals with strain Vibrio cholerae 5879, the latter was grown at 37 ° C for 24 hours on Marten agar (pH 7.6), followed by inactivation by boiling in a water bath for 30 minutes and checking for specific sterility.

The method is carried out in two stages:

Stage I - isolate a common pool of cholera Ig from small intestinal leaching fluids of white mice;

Stage II - the release activity of the cholera Ig obtained from stage I on the enterocyte monolayer is evaluated.

For stage I, two groups of experimental mice, 12 pieces each, were immunized orally with killed microbial cells (mk) Vibrio cholerae 5879 (5 × 10 ⁷ mk). Strain 5879 V. cholerae O1 of Serovar Inaba is obtained from the collection of living cultures of RostNIPCHI. The latter was pre-grown at 37 ° C for 24 h on Marten agar (pH 7.6). Inactivation of the bacterial culture is carried out by boiling in a water bath for 30 minutes. After checking for specific sterility, a suspension of bacterial cells is used to immunize animals.

Then, to the second experimental group of animals intramuscularly, daily, for 10 days, 0.2 ml of a 0.0005% solution of immunofan is administered. The first experimental group of immunized mice does not receive an immunomodulator.

After that, a common pool of cholera Ig is isolated from the washing liquids of the small intestine of the killed mice of each of the experimental groups. To do this, in white mice, 2 ligatures are placed on the small intestine at a distance of 5 cm from each other, 5 ml of physiological saline are injected into an isolated area of the small intestine, massaged for several minutes and the contents are taken with a syringe. Wash liquids precipitated with 50% ammonium sulfate. The precipitate formed overnight at 4 ° C is separated by centrifugation at 1000 rpm for 10 minutes. The precipitated proteins are dialyzed against distilled water (with a three-fold change of diluent), diluted to the original volume and sterilized by filtration through membrane filters with a pore diameter of 0.22 μm Schleicher & Schuell.

Stage II consists of: a) the isolation of the primary culture of enterocytes; b) in the assessment of the release activity of antichole Ig on a monolayer of enterocytes

a) the primary culture of enterocytes of the small intestine to evaluate the adhesive activity of live cholera vibrios is obtained from 10 intact white mice. For this, 5 ml of 0.2% ethylenediaminetetraacetic acid (EDTA) (Germany) is injected into the lumen of the washed small intestine of animals (Germany), carefully massaged for 30 minutes and the contents are aspirated with a pipette. Cells are washed three times with physiological saline, suspended in a DMEM growth medium (Germany) with 10% fetal calf serum, 2 mM L-glutamine (Russia), 10 mM HEPES (Switzerland), 5 × 10 ⁵ M 2-mercaptoethanol (USA) 100 UNITS / ml penicillin, 100 μg / ml streptomycin to a concentration of 150,000 cells / ml and plated in the wells of 6-well Costar culture panels (Netherlands). The panels are incubated in a CO ₂ incubator for 24 hours to form a rarefied monolayer, after which the cells are washed three times with fresh portions of serum-free medium.

b) in order to evaluate the release activity of anticcholic Ig, the latter in double dilution in a volume of 3 ml are applied on a panel with a monolayer of enterocytes. At the same time, cholera Ig obtained from animals of the experimental group, where only immunization with strain V. cholerae 5879 was carried out, was injected into some wells, and the general pool of Ig isolated from the group of animals immunized and treated with immunofan was administered. In control wells, saline is added to the enterocytes. Then, live cholera vibrios of strain Vibrio cholerae 5879 (for 1 enterocyte - 40 m.k.) are introduced into all wells and incubated for 3 hours in a CO ₂ incubator. The panels are washed three times with fresh portions of saline, fixed with ethanol for 20 minutes and stained according to Romanovsky-Giemsa. To do this, 3.8 g of dry Romanovsky paint is dissolved in 250 ml of pure ethyl alcohol. Shake the solution for 4 days, then add 250 ml of glycerol and shake again for 4 days. The panels are stained with a working paint solution (1 ml of paint: 10 ml of distilled water) for 20 minutes. After exposure, the wells are washed three times with distilled water, dried, counted under a microscope (eyepiece 10, lens 90), the number of dark blue colored attached to 200 enterocytes of vibrios and determine their average value.

The presence of severity of anti-adhesive activity is judged by a decrease in the number of vibrioes attached to one enterocyte by at least 2 times in comparison with the control.

During immunization, the number of attached vibrios decreases by 2 times in comparison with the control on the 14th day of the post-immunization period, and the addition of immunotherapy reduces this period to 7 days, which indicates an increase in the anti-adhesive activity of anticholeria antibodies under the influence of the immunofun immunomodulator.

Thus, this method allows to determine the anti-adhesive activity of a common pool of cholera Ig obtained from small intestinal leaching fluids of immunized mice at different times after immunization, as well as to evaluate the effect of immunomodulators, in particular immunofan, on the increase in the ability of these antibodies to inhibit adhesion of cholera vibrios to enterocytes in vitro .

Example 1. Evaluation of the release activity of a common pool of anti-cholera Ig obtained from enteric wash liquids of immunized mice.

Studied the release activity according to the technology of the proposed method (see "The method is carried out ...") cholera Ig isolated from the small intestine immunized with MK strain V. cholerae 5879 mice. At different times after immunization (see table 1), a decrease in the average number of vibrios attached to one enterocyte on day 5 was revealed, which indicated the beginning of the synthesis of specific antibodies. On day 14, the release activity of these Ig significantly increased compared to the start of immunization and continued to increase until the end of the observed period.

Table 1 Day after immunization The number of vibrios attached to one enterocyte The control 25.6 ± 0.4 3 24.6 ± 0.4 5 18.9 ± 0.3 7 15.4 ± 0.3 fourteen 12.6 ± 0.3 21 11.4 ± 0.2 28 10.3 ± 0.2

Thus, the example shows that already on the 14th day after immunization, the average number of vibrioes attached to enterocytes decreased by 2 times compared with the control, which indicates the presence of a sufficient number of specific cholera antibodies. The proposed method makes it possible to evaluate their anti-adhesive activity and allows us to judge the adequacy of the formation of local humoral anticholeria immunity.

Example 2. The study of the effect of the immunomodulator immunofan on the release activity of the common pool of cholera Ig obtained from the enteric leaching fluids of immunized mice during the formation of local cholera immunity.

The study of the effect of the immunomodulator immunofan on the ability of cholera Ig to inhibit the adhesion of vibrios at different times of the post-immunization period showed (see table 2) that the anti-adhesive activity of antibodies isolated from the intestinal mucosa of immunized and immunomodulating mice was manifested to a greater extent and earlier than in immune animals. On day 7, the average number of vibrioes attached to one enterocyte in samples containing washing liquids obtained from animals after immunization and immunocorrection was significantly lower than in wells with Ig isolated from animals immunized and not receiving immunofan. This trend continued throughout the observation period (up to 28 days). A 2-fold decrease in the average number of vibrioes attached to enterocytes in comparison with the control already on the 7th day of the post-immunization period indicated an increase in the anti-cholera Ig anti-adhesive activity under the influence of the immunomodulator.

table 2 Day after immunization The number of vibrios attached to one enterocyte The control 25.6 ± 0.4 3 23.8 ± 0.4 5 13.3 ± 0.3 7 10.5 ± 0.2 fourteen 8.6 ± 0.2 21 7.8 ± 0.2 28 6.3 ± 0.2

Thus, using the proposed method in an in vitro model, the stimulating effect of the immunofan immunodulator on the production of anti-cholera Ig and on the increase in their anti-adhesive activity was revealed. The anti-adhesive activity of immunoglobulins prevents the adhesion of cholera vibrios to enterocytes, providing the most significant level of protection of the mucous membrane of the small intestine.

Using the present invention allows due to the possibility of studying the release activity of a common pool of cholera Ig from small intestinal leaching fluids of immunized mice to evaluate in vitro specific local humoral immunity in experimental cholera.

In addition, this method allows you to study the increase in the synthesis of cholera antibodies and the strengthening of their anti-adhesive activity under the influence of immunomodulators directly in the intestine, where the macroorganism interacts with the cholera pathogen.

Thus, the proposed method makes it possible to assess the adequacy of the formation of local cholera immunity by the anti-adhesive activity of specific antibodies, as well as the effect of various drugs on the immune response to the cholera pathogen, which opens up new approaches to improving the vaccine prophylaxis of this disease.

Information sources

1. Kostyukova N.N. The initial stage of the infectious process is colonization and ways to prevent it // Zh. microbiol., epidemiol., immunobiol. - 1989. - No. 9. - S.103-110.

2. Williamson E., Westrich G.M., Viney I.L. Modulating dendritic cells to optimize mucosal immunization protocols // J. Immunol. - 1999. - Vol. 163, No. 7. - P.3668-3675.

3. Yamamoto, T., Fibert M.J., Bradley S.R. et al. Adherence to human small intestines of capsulated Vibrio cholerae 0139 // FEMS Microbiol. Lett. - 1994. - Vol. 119, No. 1-2. - P.229-235.

4. Chelyadinova A.V. The study of the biological properties of cholera vibrios 0139 serogroup: Author. dis ... cand. honey. sciences. - 2000. - 18 p.

5. Jerborn M., Nordstrom I., Kilander A. et al. Local and systemic immune responses to rectal administration of recombinant cholera toxin B subunit in humans // Infect. Immun. - 2001. - Vol.69, No. 6. - P.4125-4128.

Claims (4)

1. A method for evaluating local humoral anticholeron immunity in an experiment, characterized in that white mice are immunized with 5 × 10 ⁷ Vibrio cholerae 5879 microbial cells, 10 days later a common pool of cholera Ig is isolated from the small intestine wash liquids of mice, and an additional culture of small intestine enterocytes is obtained from intact mice, then anti-cholera Ig in double dilution is applied in a volume of 3 ml onto a monolayer of enterocytes formed in the wells of the culture panels, then live cholera vibrios are applied to the latter strain Vibrio cholerae 5879, 40 m.k. 1 enterocyte and incubated for 3 hours, washed three times after the panel, fixed with ethanol for 20 minutes and stained with Romanovsky-Giemsa, local humoral anticholera immunity is evaluated under a microscope according to the number of vibrioes stained in dark blue and attached to one enterocyte. 2. The method according to claim 1, characterized in that the total pool of anti-cholera Ig is obtained from leaching fluids of the small intestine; for this, two ligatures are applied to the latter at a distance of 5 cm from each other and 5 ml of physiological saline are injected into an isolated area, massaged with a syringe a liquid is taken which is precipitated with 50% ammonium sulfate, and the precipitate formed overnight at 4 ° C is centrifuged at 1000 rpm for 10 minutes. 3. The method according to claim 1, characterized in that the primary culture of enterocytes of the small intestine is obtained from intact white mice, for this purpose, 5 ml of 0.2% ethylenediaminetetraacetic acid is injected into the lumen of the washed small intestine of animals, massaged for 30 minutes and the contents are aspirated with a pipette, the obtained cells are washed three times with physiological saline, suspended in a nutrient medium with 10% fetal calf serum, 2 mM L-glutamine, 10 mM HEPES, 5 × 10 ⁵ M 2-mercaptoethanol, 100 IU / ml penicillin, 100 μg / ml streptomycin to a concentration 150,000 cells / ml and seeded in Lu ki 6-well tissue culture Costar panels after the panels are incubated in CO ₂ incubator for one day to form a sparse monolayer cells were then washed three times with fresh portions of medium without serum. 4. The method according to claim 1, characterized in that for the immunization of animals with strain Vibrio cholerae 5879 the latter is grown at 37 ° C for 24 hours on Marten agar (pH 7.6), followed by inactivation by boiling in a water bath for 30 min and checks for specific sterility.