Method of altering immunomodulating properties of lipopolysaccharides of plague bacteria in vitro

FIELD: chemistry.

SUBSTANCE: invention relates to a method of altering immunomodulating properties of lipopolysaccharides of plague bacteria in vitro, which involves obtaining preparations of lipopolysaccharides (LPS) and mouse toxin (MT) Yersinia pestis with subsequent formation of a LPS-MT complex thereof. A modified form of LPS-MT is used as an inducer of synthesis of cytotoxins TNF-α and IFN-γ. To this end, a test sample is prepared, to which LPS is added in amount of 5 mcg (50 mcl from working dilution of 100 mcl/ml) and MT is added in amount of 50 ng (5 mcl from working dilution of 10 mcg/ml); the sample is then incubated for 30 min at 37°C. The volume of the sample in eppendorfs is then brought to 100 mcl with sterile buffered physiological solution of NaCl and the obtained mixture is added a tray dimple containing a culture of human monocyte cell line U-937 (1×106 cells in a dimple); the latter is cultured in a medium of PRMI 1640 with simultaneous double control. Further, 1, 4, 20 hours after the beginning of combined incubation of the preparations of LPS with monocytes, quantitative accounting of the synthesised cytotoxins is carried out, wherein change in the immunomodulating properties of LPS of plague bacteria in vitro is determined from the amount of cytotoxins produced and the dynamics of their synthesis.

EFFECT: invention enables to alter immunomodulating properties of lipopolysaccharides of plague bacteria in vitro, which enables to realise toxic potential of the endotoxin of plague bacteria.

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The present invention relates to medical Microbiology, namely the pathogenic properties of the plague microbe, in particular for molecular mechanisms of toxic action, and can be used in clinical immunology, to improve treatment methods plague infection using toxic drugs and specific immunotherapy.

The results of studies of the chemical structure of lipopolysaccharides (LPS) Yersinia pestis, carried out in recent years, indicate that at low temperature cell cultivation plague microbe (28°C) lipid A LPS is represented mainly hexazinone form, LPS has pronounced immunostimulating properties. LPS isolated from bacteria Y.pestis grown at body temperature warm-blooded host (LPS), differs in chemical composition from LPS, its lipid A contains mainly three - and tetracyline molecules. LPS is a weak inducer of proinflammatory cytokines [1, 2]. At the same time, the pathogenesis of plague in any form is a stage of development of infectious-toxic shock, which is caused by the action of LPS of the pathogen.

It is known that the full manifestation of toxic effects of LPS depends not only on the specific chemical structure of the molecules, but also on their conformation [3]. Modulators Tox is ical properties of LPS on the level of conformation are specific proteins of microbial cells, associated with LPS [4], and in vivo proteins and lipoproteins of the microorganism, which is bonded with LPS, reinforce or neutralize their action [5, 6].

The basis of the present invention required the use of the developed by the applicant of a specific protein complex of the plague microbe "mouse" toxin with different forms of LPS Y.pestis (LPS-MT) (see Sokolov, H.E., tynianova V.I., Demidov, GV, Zyuzin VP, Plotnicki SUPERVISION, Dinkevich NICHOLAS, Bespalova I.A. Goncharov, E.K. "the Formation of the complex "mouse" toxin - lipopolysaccharide from Yersinia pestis". Journal of Biotechnology, 2000, No. 5, p.25-30), where it is confirmed that the formation of physico-chemical bonds between the MT and the FSC has a specific character and is accompanied by a change in the conformation of LPS molecules [7]. The correctness of this assumption is demonstrated in two ways: by Western blot turns and gel filtration. By the method of Western blot turns using specific antisera to MT and LPS are shown linking them together. Method gel filtration possible to prove the formation of complexes of LPS-MT and assess their quantitative ratio (100:1). It is established, that connection MT and LPS leads to conformational changes in the molecules of LPS and is reflected in the changing areas and the redistribution of elution profiles LPS. The dynamics of their changes under the influence of MT describes the logarithmic crooked is, a similar curve Michaelis.

Intensive synthesis of proinflammatory cytokines at the stage of septic shock and at the same time weak cytokineinduced activity 37°-aqueous LPS in vitro experiments allowed us to carry out the modification of LPS Y.pestis in terms of the microorganism. In the literature there are no data on the immunomodulating action of LPS Y.pestis, modified with MT. This circumstance made it possible to formulate the task of the invention is the ability to change cytokineinduced properties of LPS using a specific protein Y.pestis - MT - and to evaluate this method by studying the dynamics of the synthesis of tumor necrosis factor (TNF-α) and interferon gamma (IFN-γ) by human monocytes under the influence of the original and modified forms LPS and LPS vaccine strain Y. pestis EV76 and virulent strain of Y.pestis 231 in vitro.

The aim of the invention is to develop ways of changing the immunomodulatory properties of lipopolysaccharides plague microbe in vitro.

This objective is achieved in that in the known method changes immunomodulatory properties of lipopolysaccharides plague microbe in vitro, including the production of preparations of lipopolysaccharide (LPS) and "mouse" toxin (MT) Yersinia pestis followed by the formation of their complex FSC-MT, a modified form which the CSO is used as the inducer of the synthesis of the cytokines TNF-α and IFN-γ, to do this, prepare a pilot test in which add LPS (5 μg (50 μl of the working dilution 100 mg/ml) and MT in the amount of 50 ng (5 μl from the working dilution of 10 µg/ml), thereafter, the sample is incubated for 30 min at 37°C, then the size of the sample in Eppendorf brought to 100 μl of sterile buffered physiological NaCl solution and the resulting mixture is introduced into the hole of the tablet containing the cell culture human monocytes line U-937 (1×106cells in the well), the latter is cultivated in an environment PRMI 1640 with simultaneous production of double control, then after 1, 4, 20 hours after the start of the joint incubation of LPS preparations from monocytes conduct quantitative account of the synthesized cytokines, while the number of the produced cytokines and the dynamics of their synthesis detect changes immunomodulatory properties FSC plague microbe in vitro. In addition, before making finished products FSC-MT in the wells of the last selected old growth environment and make 100 ál of fresh medium PRMI containing 4% FCS.

The method is as follows.

In work using drugs LPS isolated from bacteria Y. pestis grown at 28° and 37°C on LB agar (Difco, USA) for 48 h Bacterial mass inactivate thimerosal sodium. FSC produce by the method of Westphal. MT of Y.pestis is isolated and purified by the original method is I Marchenkova, LD50MT=5,6 (4,0-7,9) μg/mouse [8].

FSC and MT dissolved in distilled water and incubated for 24 hours at 4°C. Working dilution of the drug FSC - 100 µg/ml, MT - 10 µg/ml of the Sample prepared in sterile test tubes "Eppendorf". In the experimental sample type LPS (5 μg (50 μl), MT - 50 ng (5 μl). This dose of LPS preparations and MT eliminates the damage and loss of culture cells monocytes and induce the expression of cytokines TNF-α and IFN-γ. Samples contain or LPS with MT (pre-incubated for 30 min at 37°C to obtain a complex of LPS-MT), or only LPS. The size of the sample in Eppendorf brought to 100 μl of sterile buffered physiological NaCl solution. The resulting mixture is added to the wells containing cell culture.

Immunomodulating properties of LPS study on the model cells human monocytes line U-937. Cells human monocytes (1×106of cells per well) were cultured in 96-well tablet in the environment PRMI 1640 containing 8% fetal calf serum (FCS) and 100 μg/ml of gentamicin. Before making preparations LPS (100 µl of the prepared sample with a buffered saline solution) from the wells pipetted old growth environment and make 100 ál of fresh medium PRMI containing 4% FCS. Put two controls. In the first control hole making MT 50 ng, re is the query result dose not have a cytotoxic effect and stimulates the synthesis of cytokines by monocytes. The second control is buffered physiological solution, which also does not cause the synthesis of cytokines.

To assess the dynamics of the synthesis of TNF-α and IFN-γ in the quantification of cytokines carried out after 1, 4 and 20 hours after the start of the joint incubation of LPS preparations from monocytes. The number of synthesized cytokines determine enzyme-linked immunosorbent assay using commercial test kits (Vector-best, Novosibirsk, Russia). The confidence interval calculated using the Student's t-Fisher (p=0.95).

Example 1 to illustrate modified forms of LPS vaccine strain Y.pestis EV76 to change the cytokine response of the cell culture human monocytes

In experiments comparing cytokineinduced ability source (LPS and LPS) and modified forms of LPS (LPS-MT and LPS-MT) vaccine strain Y.pestis EV76 (see Fig 1-4). In the control wells with MT and buffered saline solution is not observed to increase the content of cytokines TNF-α and IFN-γ during the experiment.

Figure 1 shows a graph of the dynamics of the synthesis of TNF-a cells of the monocytic line human U-937 under the influence of various forms LPS Ypestis EV76.

The abscissa shows the time of culturing monocytes with LPS preparations, on the y - axis the number of synthesized cytokines (PG/ml).

The cytokine activity of unstimulated monocytes is zero.

Figure 2 - the dynamics of the synthesis of IFN-γ by cells of the monocytic line human U-937 under the influence of various forms LPS Y.pestis EV76.

The abscissa shows the time of culturing monocytes with LPS preparations, on the y - axis the number of synthesized cytokines (PG/ml).

The cytokine activity of unstimulated monocytes is zero.

Figure 3 - Dynamics of the synthesis of TNF-α and macrophage cells lines human U-937 under the influence of various forms LPS Ypestis EV76.

The abscissa shows the time of culturing monocytes with LPS preparations, on the y - axis the number of synthesized cytokines (PG/ml).

The cytokine activity of unstimulated monocytes is zero.

Figure 4 - the Dynamics of the synthesis of IFN-y by cells of the monocytic line human U-937 under the influence of various forms LPS Ypestis EV76.

The abscissa shows the time of culturing monocytes with LPS preparations, on the y - axis the number of synthesized cytokines (PG/ml). The cytokine activity of unstimulated monocytes is zero.

Thus, from the graphs it is seen that quantification of cytokine synthesis, defined in dynamics, allows to detect changes cytokineinduced properties FSC plague microbe using MT and show the differences between the original and modified forms FSC Y.pestis EV76. The dynamics of the synthesis of TNF-α under the influence LPS characterized by the fact that the accumulation of this cytokine in the growth medium for 4 hours at 50±6,4 PG/ml with subsequent reduction of TNF-α on the 8 ħ 2.6 PG/ml to 20 hours of observation. Activation of monocytes conformationally altered form of LPS-MT reveals a different pattern: a significant amount of TNF-α recorded in the test samples during the first 60 min - 22±4,2 PG/ml after 4 hours its production is reduced to 10±2,8 PG/ml, and 20 hours the level of synthesis of TNF-α increased again up to 26±4.6 PG ml Drug LPS stimulates monocytes to the synthesis of IFN-γ, a high level of production of this cytokine is logged in 4 hours - 90±8,6 PG/ml with subsequent reduction in its quantity to 30±5,0 PG/ml to 20 hours. The synthesis of IFN-γ under the influence of a modified form LPC-MT characterized by sufficient "monotony", without sharp fluctuations in time, from 10±2,8 PG/ml to 30±5,0 PG/ml.

Cytokineinduced activity of drugs LPS Y.pestis EV76 by the number of produced cytokines TNF-α and IFN-γ and the dynamics of their synthesis is significantly different from the actions LPS-MT. LPS as its conformationally modified form LPC-MT, was weak inducers of synthesis of TNF-α. The synthesis of IFN-γ under the influence LPS increases in proportion to time the activation of monocytes and reaches its maximum value to 20 hours (62±7,0 PG/ml). Modified the form LPC-MT inhibits the synthesis of IFN-γ in monocytes.

Conclusion: comparing the dynamics of the synthesis of cytokines TNF-α and IFN-γ by human monocytes under the influence of the original forms of LPS (LPS and LPS), confirmatio is but modified forms (LPS-MT and LPS-MT), we clearly recorded differences in their cytokineinduced properties, respectively, and immunomodulatory. Therefore, the proposed method changes cytokineinduced properties FSC plague microbe using MT was confirmed in this example.

Example 2, illustrating the ability of conformationally altered LPS virulent strain F.pestis 231 to change the cytokine response of the cell culture human monocytes

Compare, as in the previous example, cytokineinduced ability source (LPS and LPS) and modified forms of LPS (LPS-MT and LPS-MT) virulent strain of Y.pestis 231. It should be noted that LPS strain Y.pestis EV76 used in the first example, differs according to the chemical structure of lipid A and sugars cow area from FSC natural highly virulent strain of Y.pestis 231.

The results of this example are presented in figure 5-8. Figure 5 shows a graph of the dynamics of the synthesis of TNF-a cells of the monocytic line human U-937 under the influence of various forms LPS Ypestis 231.

The abscissa shows the time of culturing monocytes with LPS preparations, on the y - axis the number of synthesized cytokines (PG/ml).

The cytokine activity of unstimulated monocytes is zero.

6 - the Dynamics of the synthesis of IFN-γ by cells of the monocytic line human U-937 under the influence of various forms LPS Ypestis 231.

The abscissa shows the time of culturing monocytes with LPS preparations, on the y - axis the number of synthesized cytokines (PG/ml).

The cytokine activity of unstimulated monocytes is zero.

7 - Dynamics of the synthesis of TNF-α and macrophage cells lines human U-937 under the influence of various forms LPS Ypestis 231.

The abscissa shows the time of culturing monocytes with LPS preparations, on the y - axis the number of synthesized cytokines (PG/ml).

The cytokine activity of unstimulated monocytes is zero.

Fig - Dynamics of the synthesis of IFN-γ by cells of the monocytic line human U-937 under the influence of various forms LPS Y.pestis 231.

The abscissa shows the time of culturing monocytes with LPS preparations, on the y - axis the number of synthesized cytokines (PG/ml).

The cytokine activity of unstimulated monocytes is zero.

From the graphs it is seen that LPS-MT and LPS-MT different from their original forms on the dynamics of the synthesis of the cytokine TNF-α. Conformationally modified form LPC-MT causes the greatest synthesis of TNF-α after 4 hour (20±4,3 PG/ml), and LPS after 1 hour (12±3,4 PG/ml). A modified form LPC-MT, as it turned out, is the most potent inducer of the synthesis of proinflammatory cytokine TNF-α. The maximum number produced TNF-α through the 4 hour equals 68±8,2 PG/ml, which is almost 3 times higher compared with the level of activity of the parent drug LPS (28±52 PG/ml).

The dynamics of the synthesis of IFN-γ under the influence of modified forms of LPS are also different. If LPS induces rapid response cells and leads to the synthesis of the greatest amount of this cytokine (170±13,0 PG/ml) after one hour, it is a modified form completely suppresses its production by monocytes. This feature has conformationally modified form LPC-MT. The greatest synthesis of IFN-γ observed only after one hour of incubation (40±6,2 PG/ml), and after 4 hours, IFN-γ is completely absent in the growth medium, which was maintained until the end of observation (20 hours). LPS unlike a modified form LPC-MT induces the synthesis of IFN-γ by monocytes throughout the experiment and the maximum number recorded in 20 h (55±7,2 PG/ml).

Thus, creating with a specific protein Y.pestis MT complex FSC-MT, you can change cytokineinduced properties FSC plague microbe in vitro.

The use of the invention allows to study the dynamics of the synthesis of proinflammatory cytokines by human monocytes original and modified forms FSC Y.pestis in vitro.

The results obtained confirm that the conformational change in the LPS molecules of the plague microbe affect their immune-stimulating properties and change the toxicity of these drugs. The specificity of the IMM is nonodorous steps FSC plague microbe is shown at different stages of development of infectious process in man, what makes for the strongest violation of innate immunity.

Model conformational modifications FSC will continue to develop techniques specific antitoxic treatment of plague, which can be applied in the clinic simultaneously with antibacterial drugs.

Sources of information

1. Kawahara, K., Tsukano N., Watanabe, H. et al. Modification of the structure and activity of lipid A in Yersinia pestis lipopolysaccharide by growth temperature. Infect. Immun. 2002, 70 (8): 4092-4098.

2. Montminy, S., N. Khan, S. McGrath et al. Virulence factors of Yersinia pestis are overcome by a strong lipopolysaccharide response. Nature Immun. 2006, 7(10): 1066-1073.

3. Schromm, A., Brandenburg K., Loppnow H. et al. J. Immunol. The charge of endotoxin molecules influences their conformation and IL-6-inducing capacity. 1998; 161: 5464-5471.

4. Mayer K.R., Truchot A.T., Ward J et al. Cellular and molecular aspects of endotoxin reactions. Amsterdam: Elsevier. 1990; 145-156.

5. Bauer, M.E. et R.A. Welch Pleiotropic effects of a mutation in rfacon Escherichia coli hemolisin. Infect. Immun. 1997; 65(6): 2218-2224.

6. Li J., Clinkenbeard K.L. Lipopolysaccharide complex with Pasteurella haemolytica leukotoxin. Infect. Immun. 1999; 67 (6):2920 - 2927.

7. Sokolova, H.E., tynianova V.I., Demidov, GV, Zyuzin VP, Plotnicki SUPERVISION, Dinkevich NICHOLAS, Bespalova I.A. Goncharov, E.K. Education complex "mouse" toxin - lipopolysaccharide from Yersinia pestis. Biotechnology. 2000;(5):25-30.

8. Sokolova, H.E. Mechanisms of activation of toxic substances plague: author. dis..., kand. Biol. of Sciences, Rostov-on-don, 2002. - 18 S.

9. Zyuzin VP, tynianova V.I., Demidov, G.V. Sokolova, H.E., Scripture W., Bespalov IGOR, Borodin T.N., EN the Sims B. I., Mechaniken BN. The toxicity of lipopolysaccharides isolated from 28 and 37°C were cultures of Yersinia pestis. Biotechnology. 2003; (6):20-24.

1. How to change the immunomodulatory properties of lipopolysaccharides plague microbe in vitro, including the production of preparations of lipopolysaccharide (LPS) and "mouse" toxin (MT) Yersinia pestis followed by the formation of their complex FSC-MT, characterized in that a modified form of the LPS-MT is used as the inducer of the synthesis of the cytokines TNF-α and IFN-γ, prepare for this test sample, which adds LPS (5 μg (50 μl of the working dilution 100 mg/ml) and MT - 50 ng (5 μl from the working dilution of 10 µg/ml), thereafter, the sample is incubated for 30 min at 37°C, then the size of the sample in Eppendorf brought to 100 μl of sterile buffered physiological NaCl solution and the resulting mixture is introduced into the hole of the tablet containing the cell culture human monocytes line U-937 (1×106cells in the well), the latter is cultivated in an environment PRMI 1640 with simultaneous production of double control, then after 1, 4, 20 hours after the start of the joint incubation of LPS preparations from monocytes conduct quantitative account of the synthesized cytokines, while the number of the produced cytokines and the dynamics of their synthesis detect changes immunomodulatory properties FSC plague ICRI is BA in vitro.

2. The method according to claim 1, characterized in that before making finished products FSC-MT in the wells of the last selected old growth environment and make 100 ál of fresh medium PRMI containing 4% FCS.



 

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