Method for assessing local humoral anticholeraic immunity in vitro simulated in experimental animals

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

 

The invention relates to medicine, in particular for the study of anti-adhesive activity of cholera immunoglobulins and enhance it to improve specific prophylaxis of cholera.

Cholera is anthroponosis disease, so the development of models of infection in animals is always met serious difficulties. To date, the experimenters take numerous attempts to develop a model of infection in mice. Models are generalized forms of cholera in mice, sealed mouse. Given that the disease cholera develops and runs in the small intestine, were developed several experimental models that can be used in animals pathogenetic picture of cholera, similar to that in humans: a model tied loops of the small intestine of an adult rabbit, rabbit-suckers.

However, currently available methods and models to assess the effect of various drugs, such as immunomodulators, for forming a local cholera immunity, in particular the increase in the antibody productions and anti-adhesive activity of cholera immunoglobulins (Ig). The development of such experimental methods and models is very important as the first obstacle to many infections, including Ho is a career, are protective factors acting on the surface of mucous epithelial cells, various antibacterial agents, secretory IgA and other) and preventing the adhesion of pathogens [1, 2]. It is shown that the prevailing value of the antibacterial component, preventing the attachment of V. cholerae to the mucous membrane of the intestine in the early stages of infection of the organism [3]. One of the main indicators of antibacterial immunity is anti-adhesive activity of cholera Ig, which, along with antitoxic antibodies, indicating the adequacy of the formation of local humoral immunity [4, 5].

A prototype of the chosen method of evaluation of the effectiveness of immunomodulator imunofana in experimental cholera (see patent RU No. 2481791, CL AV 10/00, A61K 38/08, A61P 43/00, G09B 23/28), namely, that prior infection with animals in an isolated loop of the small intestine virulent strain they impose immunomodulator Imunofan dose of 0.2 µg dissolved in 0.3 ml of novocaine in the amount of 10 injections. Evaluating the effectiveness of imunofana carried out according to the presence of experienced tied the loops of the small intestine enteropathogenic effect, the severity of which is estimated at crosses, and the presence of choleragenic effect, which is calculated by the formula K=V/NP, where K is the coefficient of expansion loops, V - is the volume of the liquid, NP is the length of the loop, if the experimental loops enteropathogenic effect is absent and K<1,0, consider the use of imunofana effective.

However, the problem of the known method consisted in the evaluation of the action of immunomodulators on the development and severity of pathogenetic picture of the experimental cholera and does not give the possibility of studying in vitro the formation of cholera immunity, in particular the actions of various drugs, including immunomodulators, anti-adhesive activity total pool cholera Ig of the small bowel wash fluids of mice.

The purpose of the present invention was to develop ways to assess in vitro site-specific humoral immunity in experimental cholera, as well as to strengthen the anti-adhesive activity total pool cholera Ig of the small bowel washing liquids of white mice in the formation of cholera immunity.

This objective is achieved in that in the known method to assess local humoral cholera immunity in the experiment of white mice subjected to immunization with 5×107microbial cells of Vibrio cholerae 5879, after 10 days, allocate the total pool of cholera Ig of the small bowel wash fluid of mice additionally receive a primary culture of small intestine enterocytes from intechnica, then cholera Ig in a twofold dilution is applied in a volume of 3 ml on a monolayer of enterocytes, formed in the wells of the culture of the panels, and then at last put a live cholera Vibrio cholerae strain of Vibrio cholerae 5879, 40 M. K. 1 enterocyte and incubated for 3 hours, after panel washed three times, fixed with ethanol for 20 min and stained by Romanovsky - Institute, evaluation of local humoral cholera immunity are under the microscope by the number of vibrios, painted in dark blue color and adherent to one enterocyte.

The total pool of cholera Ig obtained from the wash fluid of the small intestine, this impose on the last two ligatures at a distance of 5 cm from each other and injected into the isolated section 5 ml of physiological solution, massaged and syringe take the liquid, which is precipitated by 50% ammonium sulfate, and formed overnight at 4°C the precipitate centrifuged at 1000 rpm for 10 minutes

In addition, primary cultures of enterocytes of the small intestine receives from intact white mice in the lumen flushed the small intestine of animals injected with a syringe and 5 ml of 0.2% ethylenediaminetetraacetic acid, massage 30 min and suck the contents of the pipette, the received cells are washed three times with saline, suspended in culture medium with 10% AMB the optional calf serum, 2 mm L-glutamine, 10 mm HEPES, 5×105M 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin to a concentration of 150,000 cells/ml and seeded in wells of a 6-hole culture panels Costar, after the panel incubated in CO2the incubator during the day for the formation of a sparse monolayer, the cells are then washed three times with fresh portions of medium without serum.

And for immunization of animals strain of Vibrio cholerae 5879 last grown at 37°C for 24 h on agar Martin (pH 7,6) with subsequent inactivation by boiling in a water bath for 30 min and checking for specific sterility.

The method is carried out in two stages:

Phase I: produce a common pool cholera Ig of the small bowel wash liquids white mice;

Phase II - evaluate the anti-adhesive activity obtained under the first stage of cholera Ig on the monolayer of enterocytes.

For conducting the first stage of the two groups of experimental mice of 12 pieces in each oral subjected to immunization killed microbial cells (M. K.) Vibrio cholerae 5879 (5×107M. K.). Strain 5879 V. cholerae O1 of serovar Inaba receive from the collection of live cultures of Rostopchin. The last pre-grown at 37°C for 24 h on agar Martin (pH 7,6). Inactivation of bacterial culture performed by boiling in a water bath for 30 minutes After checking on specific sterilized is of a suspension of bacterial cells used for immunization of animals.

Then the second experimental group of animals intramuscularly, daily for 10 days, injected with 0.2 ml of 0.0005% solution imunofana. The first experimental group immunized mice, immunomodulator, it will not.

Then allocate the total pool of cholera Ig from the wash fluid of the small intestine of dead mice of each experimental group. To do this, white mice in the small intestine impose 2 ligatures at a distance of 5 cm from each other, is introduced into the isolated section of the small intestine 5 ml of physiological solution, gently massage a few minutes and take the contents with a syringe. The washing liquid is precipitated by 50% ammonium sulfate. Formed overnight at 4°C the precipitate was separated by centrifugation at 1000 rpm for 10 min. the Precipitated proteins cialiswhat against distilled water (three times the change of diluent used), was diluted to the original volume and sterilized by filtration through membrane filters with a pore diameter of 0.22 μm Schleicher&Schuell.

Stage II is: a) in the allocation of primary cultures of enterocytes; b) in the evaluation of anti-adhesive activity of cholera Ig on the monolayer of enterocytes

a) primary culture of enterocytes of the small intestine to evaluate the adhesive activity of living V. cholerae get 10 intact white mice. To do this in the lumen flushed the small intestine of animals injected with syringe 5 ml,2% ethylenediaminetetraacetic acid (EDTA) (Germany), gently massaged for 30 min and suck the contents of the pipette. Cells are washed three times with saline, suspended in a nutrient medium, DMEM (Germany) with 10% fetal calf serum, 2 mm L-glutamine (Russia), 10 mm HEPES (Switzerland), 5×105M 2-mercaptoethanol (USA), 100 U/ml penicillin, 100 μg/ml streptomycin to a concentration of 150,000 cells/ml and seeded in wells of a 6-hole culture panels Costar (the Netherlands). Panel incubated in CO2the incubator during the day for the formation of a sparse monolayer, after which the cells washed three times with fresh portions of medium without serum.

b) to assess anti-adhesive activity of cholera Ig last two-fold dilution in a volume of 3 ml is applied to the panel by a monolayer of enterocytes. In some wells contribute cholera Ig obtained from animals of the experimental group, where he was held only immunization strain of V. cholerae 5879, and other common pool Ig selected from the group of animals immunized and treated Imunofan. In the control wells to enterocytes add saline solution. Then all wells make a living cholera Vibrio cholerae strain of Vibrio cholerae 5879 (1 enterocyte - 40 M. K.) and incubated for 3 hours in CO2-incubator. Panel washed three times with fresh portions of saline, fixed with ethanol for 20 min and paint what about the Romanovsky - From the Institute. For this purpose, 3.8 g of anhydrous Romanovsky dissolved in 250 ml of pure ethyl alcohol. Within 4 days the solution is shaken out, then add 250 ml of glycerol and again shaken for 4 days. Panel dye working solution of the dye (1 ml of ink : 10 ml distilled water) for 20 minutes After exposure, the wells washed three times with distilled water, dried, and counted under a microscope (ocular 10, the lens 90) number painted in dark blue color, adherent to 200 enterocytes vibrios and determine their average value.

About the presence of expression of anti-adhesive activity is judged by the decrease in the number of adherent to one enterocyte vibrios not less than 2 times in comparison with the control.

After immunization, the number of adherent cholerae is reduced in comparison with control 2 times at 14 day postimmunization period, and the addition of immunotherapy reduces this period to 7 days, indicating an increasing anti-adhesive activity of cholera antibodies under the influence of immunomodulator imunofana.

Thus, this method allows you to determine the anti-adhesive activity total pool cholera Ig obtained from the small bowel wash liquids immunized mice at various times after immunization, and to assess the impact immunomodulator is s, in particular imunofana, to increase the ability of these antibodies to inhibit adhesion of V. cholerae to the enterocytes in vitro.

Example 1. Evaluation of anti-adhesive activity total pool cholera Ig obtained from the small bowel wash liquids immunized mice.

Studied anti-adhesive activity on the technology of the proposed method (see "How is...") cholera Ig isolated from the small intestine immunized M. K. strain V. cholerae 5879 mice. At various times after immunization (see table 1) revealed a decrease in the average number of adherent to one enterocyte vibrios on the 5th day, testified to the beginning of the synthesis of specific antibodies. 14 day anti-adhesive activity of these Ig significantly increased in comparison with the beginning of the immunization and continued to increase until the end of the observed period.

Table 1
Day after immunizationThe number of Vibrio cholerae attached to one enterocyte
Control25,6±0,4
324,6±0,4
518,9±0,3
715,4±0,3
1412,6±0,3
2111,4±0,2
2810,3±0,2

Thus, the example shows that at 14 days after immunization, the average number attached to enterocytes vibrios decreased compared with control 2 times, indicating the presence of a sufficient amount of a specific cholera antibodies. The proposed method gives the opportunity to evaluate their anti-adhesive activity and allows you to judge the adequacy of the formation of local humoral cholera immunity.

Example 2. Study of the effect of the immunomodulator imunofana on the anti-adhesive activity total pool cholera Ig obtained from the small bowel wash liquids immunized mice in the formation of local cholera immunity.

Study of the effect of the immunomodulator imunofana on the ability of cholera Ig to inhibit adhesion of Vibrio cholerae in different terms postimmunization period showed (see table 2) that the anti-adhesive activity of the antibodies isolated from swabs of the mucosa of the small intestine immunized and treated with immunomodulator mice, manifested in a greater extent and and earlier, than in immune animals. On day 7, the mean number of adherent to one enterocyte vibrios in the samples containing the washing liquid derived from animals after immunization and immune disorders was significantly lower than in the wells with Ig isolated from animals immunized and not receiving Imunofan. This trend was maintained during all periods of observation (up to 28 days). Decrease 2 times the average number attached to enterocytes vibrios in comparison with the control already on day 7 postimmunization period testified about the increase in anti-adhesive activity of cholera Ig under the influence of immunomodulator.

Table 2
Day after immunizationThe number of Vibrio cholerae attached to one enterocyte
Control25,6±0,4
323,8±0,4
513,3±0,3
710,5±0,2
148,6±0,2
217,8±0,2
28 6,3±0,2

Thus, using the proposed method to model in vitro revealed a stimulating effect of immunodulatory imunofana products cholera Ig and to increase their anti-adhesive activity. Anti-adhesive activity of immunoglobulin prevents adhesion of V. cholerae to the enterocytes, providing the most significant level of protection of the mucous membrane of the small intestine.

The use of the present invention allows for the possibility of studying the anti-adhesive activity total pool cholera Ig of the small bowel wash liquids immunized mice to evaluate the in vitro site-specific humoral immunity in experimental cholera.

In addition, this method allows to study the increased synthesis cholera antibodies and strengthening their anti-adhesive activity under the influence of immunomodulators directly into the intestine, where the interaction of the microorganism with the causative agent of cholera.

Thus, the proposed method allows to assess the adequacy of the formation of local cholera immunity for anti-adhesive activity of specific antibodies, as well as the influence of various drugs on the immune response to the causative agent of cholera, which opens up new approaches to improving immunization e is th disease.

Sources of information

1. Kostyukova N. N. The initial stage of infection is colonization and ways of its prevention, Zh. microbiol., Epidemiol., immunobiol. - 1989. No. 9. - S. 103-110.

2. Williamson E., G. M. Westrich, and I. L. Viney 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 Environ. Lett. - 1994. - Vol.119, No. 1-2. - P. 229-235.

4. Chelyadinova A. Century, the Study of the biological properties of Vibrio cholerae 0139 serogroup: author. dis... Kida. the honey. Sciences. - 2000. - 18 S.

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

1. The method of estimating local humoral cholera immunity in the experiment, characterized in that the white mice subjected to immunization with 5×107microbial cells of Vibrio cholerae 5879, after 10 days, allocate the total pool of cholera Ig of the small bowel wash fluid of mice additionally receive a primary culture of small intestine enterocytes from intact mice, then cholera Ig in a twofold dilution is applied in a volume of 3 ml on a monolayer of enterocytes, formed in the wells of the culture of the panels, and then at last put a live cholera Vibrio cholerae strain of Vibrio cholerae 5879, 40 M. K. 1 enterocyte and incubated for 3 hours, after panel washed three times, fixed with ethanol in ECENA 20 min and stained by Romanovsky - The Institute, evaluation of local humoral cholera immunity are under the microscope by the number of vibrios, painted in dark blue color and adherent to one enterocyte.

2. The method according to p. 1, characterized in that the total pool of cholera Ig obtained from the wash fluid of the small intestine, this impose on the last two ligatures at a distance of 5 cm from each other and injected into the isolated section 5 ml of physiological solution, massaged and syringe take the liquid, which is precipitated by 50% ammonium sulfate, and formed overnight at 4°C the precipitate centrifuged at 1000 rpm for 10 minutes

3. The method according to p. 1, characterized in that the primary culture of small intestine enterocytes get from intact white mice in the lumen flushed the small intestine of animals injected with a syringe and 5 ml of 0.2% ethylenediaminetetraacetic acid, massage 30 min and suck the contents of the pipette, the received cells are washed three times with saline, suspended in culture medium with 10% fetal calf serum, 2 mm L-glutamine, 10 mm HEPES, 5×105M 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin to a concentration of 150,000 cells/ml and seeded in wells of a 6-hole culture panels Costar, after the panel incubated in CO2the incubator during the day for the formation of represenromania, the cells are then washed three times with fresh portions of medium without serum.

4. The method according to p. 1, characterized in that for the immunization of animals strain of Vibrio cholerae 5879 last grown at 37°C for 24 h on agar Martin (pH 7,6) with subsequent inactivation by boiling in a water bath for 30 min and checking for specific sterility.



 

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2 cl, 1 dwg

FIELD: medicine.

SUBSTANCE: invention represents a method for preparing weakly positive reference serum containing AD and AY subtypes of HBsAg involving donor serum sampling with ELISA, titration of the positive serum containing AD & AY subtypes of HBsAg in a dissolving stabilised solution, genotyping, thermal degradation test for establishing panel shelf life, differing by the fact that the positive reference serums containing AY and AD subtypes of HBsAg are dissolved to the range of the concentrations of 0.05 to 0.025 IU/ml and viscosity equal to that of normal human serum; and the dissolving stabilised solution is presented by negative defibrinated and lipid-free serum containing no markers of HBV infection which is to be detected by the test system of interest.

EFFECT: invention provides detection of HBV infection at the earlier stage of the disease and enables creating and controlling the test system quality.

5 cl, 2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method for making a panel of serums for the purpose of quality control of diagnosing hepatitis B with the certified low concentration of HBsAg subtypes AD and AY which involves the analysis of donor serums for the presence of anti-HIV, anti-HCV and anti-HBs antibodies and the nonspecific HBsAg binding; the selection of donor serums with said parameters not found; the use of the selected serums to prepare diluting solutions with a stabilising additive introduced; the certification of the monopreparations of HBsAg subtypes AD and AY by titration dilutions of international standards and the preparation of a reference panel from the certified monopreparations of HBsAg subtypes AD and AY within the range of 0.01 to 0.5 IU/ml; the lyophilisation of the serums followed by thermal degradation panel testing to determine a shelf life. The use of a new formulation of the stabilising additive containing proline 200-250 mM and benzoic acid 0.05-0.08 wt %, and the selection of an optimum temperature storage conditions promote the prolonged activity preservation at annual loss (at +4°C) making less than 0.5%.

EFFECT: higher shelf life.

3 cl, 5 tbl, 1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: what is offered is an expression construct for expression of single- or multipass transmembrane polypeptides in a bacterial host cell. Said construct contains a protein-coding polynucleotide, a strictly sensitive promoter of lower basal activity in the host-cell, and a leader sequence comprising a translation initiation enhancer. The strictly sensitive promoter comprises at least one positive control element and at least one negative control element. One or more positive and negative control elements represent a heterologous control element. Besides, what is offered is a method for producing the expressed transmembrane polypeptide and a method of recovering it form the host cell.

EFFECT: methods provide producing the high-yield transmembrane polypeptides either of native conformation, or in a soluble form.

53 cl, 28 dwg, 5 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new bacitracin compounds with antibiotic activity

,

wherein at least one of R1, R2 and R3 represents -CH=CH2 and wherein R1, R2 and R3 independently represent -H, -CH3 or -CH=CH2.

EFFECT: preparing the new bacitracin compounds.

13 cl, 8 dwg, 7 tbl, 5 ex

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