RU2481791C2 - Method of assessing efficiency of immunomodulator imunofan application in experimental cholera - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to experimental medicine, in particular to study of immunomodulator efficiency for prevention and treatment of cholera. Before infection rabbits weighing 1.5-2.0 kg are given 10 injections of imunofan in dose 0.2 mcg, diluted in 0.3 ml of novocaine, every second day. After that animals are kept without food for 24 hours. After that, infection is performed by introduction of virulent strain of cholera vibrio into isolated loop of small intestine. Loops of small intestine are taken out, ligatures are applied, equal 12 cm long parts of intestine are tied up with 4-5 cm intervals between them under ether anesthesia. 1 ml of 0.9% sodium chloride solution is introduced into one ligated loop for control, with 10⁹ cells of 24-hour culture of Vibrio cholerae 5879 being introduced into another experimental loop. After that, abdominal wall is sutured and 18 hours later animals are killed for the further study and estimation of efficiency of imunofan application. Estimation is carried out by presence in experimental tied up loops of small intestine of enteropathogenic effect, whose expression is estimated in crosses, and presence of cholerogenic effect.The latter is calculated by formula: K=V_liq/L_loop, K is coefficient of loop stretching, V_liq is liquid volume, L_loop is loop length. If enteropathogenic effect is absent in experimental loops and K<1.0, application of imunofan is considered to be efficient.

EFFECT: method ensures objective assessment of imunofan efficiency in case of cholera.

2 ex, 2 tbl

Description

The present invention relates to medicine, in particular to the question of studying the effectiveness of the use of immunomodulators for the prevention and treatment of especially dangerous infections, namely cholera.

Currently, there is a problem in laboratory studies of especially dangerous infections, such as cholera, to accurately mimic the disease in experimental animals to determine their pathogenesis and to assess the possibility of using immunotherapy in this disease.

There is a method of evaluating the effectiveness of the use of the immunomodulator thymalin in acute purulent surgical diseases (see patent RU No. 2147436, class A61K 35/26, AK 35/00, 04/20/2000), which consists in determining the level of leukocytes in the patient’s blood, and in the absence of its increase relative to the norm, the patient’s condition is regarded as secondary immunodeficiency and thymoline is prescribed, after which the number of leukocytes in 1 μl of blood is calculated according to a certain formula, and if the thymalin (CE) treatment efficiency coefficient is> 1.2, then I consider the treatment effective.

However, this known method cannot be used for cholera due to the fact that this disease refers to especially dangerous intestinal infections, therefore, another way is needed to evaluate the effectiveness of immunomodulators in this infection.

The purpose of the invention is to develop a method to evaluate the effectiveness of the immunomodulator immunofan in cholera-infected experimental animals by its effect on the presence of enteropathogenic and cholerogenic effects in an isolated loop of the small intestine of adult rabbits.

This goal is achieved by the fact that before the infection of animals in an isolated loop of the small intestine of adult rabbits weighing 1.5-2.0 kg every other day, they inject immunomodulator immunofan in a dose of 0.2 μg, diluted in 0.3 ml of novocaine in the amount of 10 injections, then the animals are kept without food for 24 hours, after which, under ether anesthesia, loops of the small intestine are removed, ligatures are applied, identical sections of the intestine are 12 cm long with 4-5 cm intervals between them, while 1 ml of 0.9% is introduced into one ligation loop solution sodium lorida to control, in another pilot loop - 10 ⁹ cells of 24-hour culture of Vibrio cholerae 5879, after the abdominal wall was sutured, and after 18 hours the animals are sacrificed for further study and evaluation of the effectiveness of imunofana, which is carried out by the presence in the test tied loops of the small intestine enteropathogenic effect, the severity of which is evaluated in crosses, and the presence of a cholerogenic effect, the latter is calculated by the formula

Where

K is the coefficient of stretching the loop;

V _W - the volume of liquid;

L _p - the length of the loop,

if the enteropathogenic effect is absent in the experimental loops and K <1.0, the use of immunofan is considered effective.

The method is as follows:

Adult rabbits weighing 1.5-2 kg are injected with an immunomodulator immunofan in a dose of 0.2 μg, diluted in 0.3 ml of novocaine, every other day in an amount of 10 injections. Control animals did not receive an immunomodulator. Animals after immunotherapy and control rabbits are kept without food for 24 hours. Under ether anesthesia, a midline abdominal incision is made and loops of the small intestine are removed. Ligatures are applied 60 cm proximal to the appendix, dressing identical sections of the intestine 12 cm long with 4-5 cm intervals between them. 1 ml of 0.9% sodium chloride solution is introduced into one ligated loop (control loop), 10 ⁹ cells 24 -hour culture Vibriocholerae 5879 grown in peptone water and suspended in 1 ml of peptone solution (experimental loop). After this, the abdominal wall is sutured. After 16-18 hours, the animals are killed with an air embolism and opened.

The effectiveness of the use of immunofan is judged by the presence and severity of enteropathogenic and cholerogenic effects.

The enteropathogenic effect is evaluated by the pathoanatomical picture in the experimental bandaged loop of the small intestine, namely: the presence of edema of the mucosa and submucous membranes, hemorrhages and necrosis of the integument villi. The severity of this effect is evaluated in crosses. The absence of such violations indicates the effectiveness of the use of the immunomodulator immunofan.

The cholerogenic effect is estimated by the coefficient of stretching of the experimental loop, calculated by the formula

Where

K is the coefficient of stretching the loop;

V _W - the volume of liquid;

L _p - the length of the loop,

if K <1.0, then the use of an immunomodulator is effective.

Thus, the effectiveness of the use of immunofan is judged by the absence of enteropathogenic (there is no edema of the mucous and submucous membranes, hemorrhages and necrosis of the integumentary epithelium of the villi) and cholerogenic effects (K <1.0) in the experimental isolated loops of experimental animals.

In animals treated with immunofan, enteropathogenic and cholerogenic effects were absent.

Example 1. The effect of immunofan on the enteropathogenic effect in the small intestine of adult rabbits infected with cholera.

Pre-five adult rabbits weighing 1.5-2 kg are injected with an immunomodulator immunofan in a dose of 0.2 μg, diluted in 0.3 ml of novocaine, every other day in the amount of 10 injections. Five adult rabbits (control animals) did not receive immunofan. Animals after immunotherapy and control rabbits are kept without food for 24 hours. Then, using the model of an isolated loop of the small intestine of an adult rabbit, the animal is infected with a 24-hour culture of Vibrio cholerae 5879 grown in peptone water and suspended in 1 ml of peptone solution according to the above scheme. After this, the abdominal wall is sutured. After 18 hours, the animals are sacrificed by an air embolism and opened.

Then, the pathological picture is evaluated in the experimental bandaged loops of the small intestine of animals from the experimental and control groups. In animals that did not receive immunofan, pronounced (+++ and ++++) edema of the mucous and submucous membranes, hemorrhages and necrosis of the integumentary villi epithelium were observed, which indicated the presence of an enteropathogenic effect. In rabbits that were injected with an immunomodulator immunofan, no pathomorphological changes were detected, that is, there was no enteropathogenic effect.

The results of the effect of the immunomodulator on the severity of the enteropathogenic effect in case of cholera in an isolated loop of the small intestine of an adult rabbit are presented in table 1.

Thus, the absence of an enteropathogenic effect in experimental animals infected with cholera treated with immunofan immunotherapy indicates the effectiveness of this immunomodulator for cholera.

Example 2. The effect of immunofan on the severity of the cholerogenic effect in adult rabbits infected with cholera.

As in example 1, immunotherapy with immunofan is performed in five adult rabbits. Five rabbits (control animals) did not receive immunofan. Then, in experimental and control animals, after 24-hour fasting, loops of the small intestine were bandaged as described above. 1 ml of 0.9% sodium chloride solution (control loop) was introduced into one ligated loop, 10 ⁹ cells of a 24-hour Vibriocholerae 5879 culture grown in peptone water and suspended in 1 ml of peptone solution were injected into another (test loop). After 18 hours, the animals are sacrificed by an air embolism and opened.

The effect of immunofan on the cholerogenic effect is judged by the coefficient of extension of the experimental loop (table 2). In the group of control animals in the infected test loop, a pronounced cholerogenic effect was observed (K> 1.0), in experimental rabbits receiving immunotherapy, this effect was absent (K <1).

The data presented in table 2 indicate that the use of immunomodulator immunofan with cholera is effective.

Using the present invention allows to evaluate the effectiveness of the use of immunomodulators, in particular immunofan, in experimental animals infected with cholera, using for this an isolated model of an isolated loop of the small intestine of adult rabbits. The results obtained by studying the effectiveness of the immunomodulator immunofan from the proposed invention in experimental animals allow us to recommend this immunomodulator for the prevention and treatment of cholera in humans.

Table 1 Animals Pathomorphological changes (++++) receiving immunofan: 1 rabbit (weight 1.5 kg) not identified 2 rabbit (weight 1.7 kg) not identified 3 rabbit (weight 2.0 kg) not identified 4 rabbit (weight 1.95 kg) not identified 5 rabbit (weight 1.65 kg) not identified Control (not receiving Munofan): 1 rabbit (weight 1.58 kg) ++++ 2 rabbits (weight 1.70 kg) ++++ 3 rabbit (weight 2.0 kg) +++ 4 rabbits (weight 1.85 kg) ++++ 5 rabbit (weight 1,60 kg) +++

table 2 Animals Fluid volume Loop length Loop Stretch Ratio (K) receiving immunofan: 1 rabbit (weight 1.5 kg) 1,2 12.0 0.10 2 rabbit (weight 1.7 kg) 1,0 11.9 0.08 3 rabbit (weight 2.0 kg) 1.4 11.8 0.12 4 rabbit (weight 1.95 kg) 1,2 12.0 0.10 5 rabbit (weight 1.65 kg) 1.15 11.5 0.10 Control (not receiving immunofan): 1 rabbit (weight 1.58 kg) 10.9 10.0 1.10 2 rabbits (weight 1.70 kg) 11.6 10.1 1.15 3 rabbit (weight 2.0 kg) 12.0 9.8 1.22 4 rabbits (weight 1.85 kg) 10.6 10.5 1.00 5 rabbit (weight 1,60 kg) 11.2 11.3 1.00

Claims (1)

A method for evaluating the effectiveness of the use of an immunoformant immunofan in experimental cholera, which involves infection of animals in an isolated loop of the small intestine of adult rabbits with a virulent strain of cholera vibrio, while before the infection with cholera, an animal weighing 1.5-2.0 kg is injected every day with an immunomodulator immunofan in a dose of 0.2 mcg, diluted in 0.3 ml of novocaine, in an amount of 10 injections, then the animals are kept without food for 24 hours, after which, under ether anesthesia, loops of the small intestine are removed, ligatures are applied, ligated other sections of the intestine 12 cm long with intervals between them 4-5 cm, while 1 ml of 0.9% sodium chloride solution is introduced into one ligated loop for control, and 10 ⁹ cells of a 24-hour Vibrio cholerae 5879 culture are introduced into another experimental loop, after the abdominal wall is sutured and after 18 hours the animals are slaughtered for subsequent study and evaluation of the effectiveness of the use of immunofan, which is carried out by the presence of 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, according to Lednov calculated by the formula

where K is the coefficient of stretching the loop;
V _W - the volume of liquid;
L _p - the length of the loop,
if the enteropathogenic effect is absent in the experimental loops and K <1.0, the use of immunofan is considered effective.