Probiotic gram-positive bacteria for human allergy prevention, inhibition or elimination

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly allergology and represents a pharmaceutical preparation for human allergy prevention, inhibition or elimination differing by the fact that an active ingredient is presented by genome DNA of probiotic, gram-positive bacteria and/or Lactobacillus gasseri PA 16/8 and/or Bifidobacterium bifidum MG 20/5 and/or Bifidobacterium longum SP 07/3 bacterial strains as viable and/or inactivated bacteria.

EFFECT: invention provides effective antiallergic action.

6 cl, 5 dwg

 

The invention relates to a pharmaceutical preparation for the prevention, suppression or elimination of allergic reactions in people.

Allergic reactions to substances such as pollen (hay fever) or other parts of plants, cat hair and wool other animals, dust, containing a selection of house dust mites, insect venom, foods, such as milk or nuts, or spirits, and other components of cosmetics, a well-known and are a growing concern for the increasing number of people.

The reason is that our own body's immune system reacts to these substances as if they were actually harmful invaders such as parasites. In addition, the degree of response is excessive.

In this autoimmune response, the white particles of blood, lymphocytes, play a significant role. One form of lymphocytes - T cells or helper cells (TH cells), which secrete a variety of mediators, cytokines, to control immune responses. There are, first, the cytokines that reduce or prevent allergic reactions, and, secondly, other cytokines, which cause inflammation, i.e. allergic reaction in the presence of a catalyst effect as mediators in existing cells, such as mast cells. According to this effect, all T cell units is alauda into two groups, namely, Tn1 and Tn2 cells. Tn1 cells include anti-allergic cytokines such as gamma interferon (IFN-γ) or interleukin 2 (IL-2). Tn2 cells include mediators that cause allergic reactions, such as interleukins IL-3, IL-4, IL-5 and IL-10. Interleukin-4 stimulates the fat cells to generate antibody immunoglobulin type E (IgE), which is present in very large quantities for allergies.

The ratio between the number of Tn1 cells and Tn2 cells is critical for the immune response of the body to the captured pathogens, it is significantly lower in patients with an allergic reaction than in healthy people. It is known that newborns or premature born babies also have a very low magnitude of this relationship to the mother's body by mistake did not attack the cells of the baby.

Therefore, the balance Tn1-Tn2 is an important characteristic of every person, and also is becoming more widely known.

In General, it is assumed that changes in the intestinal flora and/or lack of bacterial stimulation in very early childhood as a result of excessive hygiene and reduction of infectious diseases create a predisposition for dismissing the value of balance and therefore give rise to allergic hypersensitivity.

Therefore, even in ml is geneste the invasion of allergens can trigger inflammatory responses such as runny nose and swollen mucosa and even fever.

Numerous pharmaceutical drugs that cure or suppress the symptoms. Their disadvantage is that they are often very expensive, cause many unwanted side effects and should be taken by the patient continuously.

Against this background, the object of the invention is to identify an active substance that is already known can be obtained on an industrial scale and is therefore readily available and recyclable, which provides low rates, which already occurs in small quantities even in the midst of healthy people and therefore compatible with the human body within certain limits and which does not attack allergic reaction to the symptoms, but being run by the mediators.

To achieve this objective, the invention provides a pharmaceutical product, in which probiotic, gram-positive bacteria, such as Lactobacillus and Bifidobacterium are present as an essential active ingredient, namely as viable bacteria and/or inactivated bacteria and/or their genomic DNA.

The sign of "probiotic" is explained in Brockhaus as the coexistence of two organisms in favor of one partner without damaging the other. In the case of this invention,the second body, namely bacteria, useful for the human body.

Itself the bacterium is not damaged and therefore remains viable. It is that when oral intake as much as possible many bacteria pass unharmed through the stomach with his acids, enzymes and other digestive agents.

The term "probiotic" also implies that the adverse effects on the person are minor or at least very low and is dominated by the effects, which are classified as useful. The term "probiotic" means preparations of microorganisms that have a health effect on the host organism, when consumed in sufficient quantities. Probiotic lactic acid bacteria (Lactobacillus) has been used for a long time. Probiotics can be entered as specially prepared food or in the form of pharmaceutical preparations.

Health effects of probiotics are strain-specific in each case. Of medicine it is known that probiotic bacterial strains undoubtedly increase the absorption of lactose, inhibit pathogenic microorganisms in the gut and restrict or inhibit diarrhea.

Other bacterial strains increase protection against infection, lower cholesterol and reduce the risk of cancer of the colon the colon. These effects successfully demonstrated in vitro before their widespread use. Therefore lawful to confirm the health benefits of this invention, numerous laboratory experiments.

Through experiments, described below, were evaluated, what cytokines from fractions of human blood cells (RVMS peripheral mononuclear blood cells) are released under specific conditions. For one series of experiments, these cells were isolated from the blood of healthy people and for the second series of experiments - from the blood of allergic patients who were allergic to house dust.

For stimulation of type a staphylococcal enterotoxin (SEA) and in the further series Dermatophagoides (Dpt) was added, because suffer from allergies to house dust usually also are allergic to them. Thus simulated the invasion of allergenic substances.

γ-Interferon was measured as the Deputy for Tn1 reaction. Tn2 the sample was registered by selected interleukins 4 and 5 (IL-4 and IL-5).

Factors that may be involved in the increased prevalence of allergic disease believed to include the modification of the intestinal flora or lack of bacterial stimulation in childhood. Tn2 cytokines increase the production of IgE and stimulate fat cells. On the other hand, γ-interf the Ron (IFN-γ), Tzirakis contributes to suppression of the synthesis of IgE. Thus, the imbalance of the expression "Tn1 to Tn2 may contribute to the initiation and maintenance of allergic diseases. The bacteria Lactobacillus, which are part of the natural intestinal flora are believed to reduce the frequency and severity of allergic manifestations and modulate Tn1 / Tn2 response. The functional mechanism is not yet explained.

Objective: Our experimental goal was to determine the effect of probiotic bacteria on the production of these cytokines Tn1 and Tn2 types of healthy people and patients with allergies to house dust mites and explain the molecular basis of the effects of bacterial genomic DNA on Tn1/Tn2 response to the enterotoxin And Staphylococcus (SEA) and Dermatophagoides pteronyssinus (Dpt) and to compare them with the effects of live bacteria.

Methods: RVMS from patients with allergies to house dust mites in comparison with those from healthy donors were incubated for 24 and 48 hours with or without Dpt and SEA allergens. The effect of preincubation with live probiotic bacteria and the influence of their genomic DNA, which was simultaneously added to the cultured and incubated for 24 hours, were evaluated by measuring the production of Tn1/Tn2 cytokines.

Results/p>

Tested, viable, gram-positive, probiotic bacteria and their genomic DNA showed that they inhibited SEA and Dpt-stimulated secretion of Tn2 cytokines (EL4 and EL5) and increased the stimulation of IFN-γ. This effect depended on the dose and on the selected bacterial strain. Was not caused significant inhibition control gram-negative Escherichia coli TG1. Probiotic bacteria reduced the production of cytokines IL-4 from allergic RVMS especially after repeated stimulation of the SEA and Dpt even more effectively than in healthy people. On the other hand, inhibition of IL-5 in healthy subjects more clearly stated. Bacterial DNA is also suppressed the release of IL-4 and IL-5, but only to a slightly less degree. Inhibition of EL4 was more evident in the case RUMS from allergic subjects than in healthy subjects, although it was for EL5.

Conclusion

Tn1/ Tn2 response to allergens, modulated tested probiotic bacteria and their genomic DNA, showed anti Tn2 activity. Therefore, different strains of probiotic bacteria and their genomic DNA can be useful in the prevention of allergic diseases.

1. Introduction

Theory of the causes of allergic diseases remains unclear, although experimental studies on humans show is live, what genetic condition contribute to the immune response of the intestinal mucosa and intestinal bacteria contribute to the pathogenesis of allergic diseases. Some studies suggest that Tn2 cytokines, especially IL-4, IL-5, 1L-9 and IL-13, play a significant role in pathogenesis by regulating the production of IgE and stimulating the fat cells. The production of IL-4, IL-5, 1L-9 or IL-13 Tn2 lymphocytes at a high level can play a decisive role in the development and progress of allergic responses; in contrast, IFN-γ, the so-called Tn1 cytokine, has the ability to suppress IgE synthesis. Defective IFN-γ expression is often associated with IgE-mediated allergies. Rasagulla in the balance of Tn1/Tn2 cytokines may largely be responsible for initiation and maintenance of allergic inflammation in diseases such as bronchial asthma or atopic dermatitis.

Suggested that, in particular, the profile of Tn2 cytokine newborn babies; aging, which usually reduces Tn1/Tn2 balance; prescriptions of modern hygiene; intensive sterilization of food and/or changes in the intestinal flora of infants, caused by feeding artificial drugs play a major role in the changes of Tn1/Tn2 balance. Acquired knowledge in this area p is hotovely the basis for a number of new therapeutic approaches. One approach is simply to use probiotic bacteria to reproduce those cytokines that were not generated in sufficient quantities, or reduce those cytokines that have been formed in a too large scale due to the fact that the probiotic bacteria modulated Tn1/Tn2 the balance.

Probiotic bacteria are generally classified as safe and has been used by humans or animals. Bacteria Lactobacillus and Bifiobacterium - organisms, the most widespread in the gastrointestinal tract of man. Of interest is the growth of the immunostimulating effect of probiotic bacteria, especially their anti-allergic effect. The important role of bacteria in allergic diseases increases the ability to prevent or treat these deviations by changing the intestinal probiotic treatment. Probiotic bacteria can act directly or indirectly through changes in endogenous flora or the immune system. It was proposed to use a new expression "immunobiotic" for bacteria that improve health through immune system of the mucosa, in contrast to the same, with only local effect. It was reported on various immune responses to probiotics, such as secretion promoting inflammation cytokines, reproducing the government of lymphocytes and the formation of nitrogen oxides. In addition, it is shown that all cells, some soluble components, which were developed LGG or DNA from LGG, components of cell walls, such as peptidoglycan or liptakova acid Lactobacilli, causing inflammatory immune responses and immunobiotics activity. Demonstrated that not only are live bacteria that are introduced in the intestinal tract, but also isolated probiotic DNA is active, even if it is introduced subcutaneously.

It was reported that Undenatured CpG motifs in bacterial DNA are mitogeneticheskie for b-cells of mice and induce the production of inflammatory cytokines by macrophages and tree cells (DC). And the CpG oligodeoxynucleotide (one) and bacterial DNA trigger the production of interleukin 6 (IL-6), interleukin 12 (IL-12) and interferon-γ (IFN-γ) B-lymphocytes and natural killer cells of mice. In addition, CpG DNA induces proliferation of b-cells and the separation is not under the influence and activated T-cells into T-helper cells 1 and 2. Specific CpG one (D-type) is particularly effective in activating NK cells and the production of α-interferon (IFN-α) plasmacytoid tree cells, while the other one (type K) is particularly effective activators of b-cells. Recently installed that dear, bell-like receptor 9 (TLR9) plays a critical role in the launch of cleocin the th activation with CpG DNA.

Experiments carried out on mice that were sensitized to egg albumin, showed that after the introduction of lactic acid bacteria in the stomach-specific IgE and Tn2 profilowanymi inflammatory responses were suppressed. In addition, recent reports suggest that lactobacillus Rhamnosus GG may reduce the symptoms of allergic diseases in man, just as they can induce innate immune responses by activating transcription factors that are integrated into the signal function of cytokines. The introduction of this bacterial strain breastfeeding mothers and newborn infants has led to the suppression of the risk of atopic eczema in infants. In vitro experiments have shown that the excitation RVMS or monocytes of healthy donors a variety of lactic acid bacteria causes the secretion of IL-12, which is a significant Pro-Tn1 cytokine and is involved in managing the development of allergic diseases. The functional mechanisms by which LAB affects the production of the Tn2 cytokines, which are responsible for initiating the development of allergic diseases remain identifiable. Taking into account the presence of UN-denatured DNA sequences (CpG motifs) in the chromosomal DNA of Bifidobacterium and Lactobacillus, we decided in this work is to sledovat effect of four bacterial strains Lactobacillus and Bifidobacterium, as well as their chromosomal DNA on the production of IL-4, IL-5 and IFN-γ SEA - and Dpt-stimulated RVMS healthy and allergic test subjects. The studied bacteria Lactobacillus and Bifiobacterium showed anti-Tn2 activity and Pro-T1cytokine IFN-γ.

2. Methods and Material

2.1. Bacterial culture

In this study, we used the following bacterial strains:

Lactobacillus rhamnosus GG (92164), Lactobacillus gasseri PA 16/8), Bifidobacterium bifidum (20/5 MG), Bifidobacterium longum SP 07/3) and LgsB.bB.I (a mixture of Lactobacillus gasseri, Bifidobacterium bifidum and Bifidobacterium longum).

Strains of Bifidobacterium and Lactobacillus were used (0,02% grafting material strains, which anaerobic stored at minus 80°C in 30%glycerol) (anaerobic system, MACS-V500 Workstation with the airlock and the airlock, Don Whitley Scientific Limited, UK in MRS medium (MRS; Merck, Darmstadt, Germany), supplemented with 0.05% of L-cysteine at 37°C for 16 hours. Before collecting and washing in PBS consistent solutions of freshly prepared cultures were deposited on plates containing MRS - agar medium and cultivated for counting. Counting colony forming units (CFU ml-1) probiotic groups of bacteria was obtained with the use of repeated 10-fold digestions on a plate. Plates anaerobically incubated at 37°C for 24-48 hours. Bacteria are then washed three times with PBS and brought to a final concentration of 1010, 107and 10 1CFU ml-1. The bacterial suspension was stored at -80°C in MRS solution, containing 30%glycerol. For all bacterial strains normal growth curves were obtained by recording the count OD600 vs agar plates of freshly made repeatedly dissolved cultures. To calculate the count of viable bacteria in freshly prepared cultures, curves fitted logarithmic printouts, who received all values more than 98,5% (data not shown). Gram-negative E. coli TG1 (product number BU-00035) was purchased from Maxim Biothec Inc and grown in LB-medium for 18 hours at 37°C and harvested as above.

2.2. Obtaining genomic DNA from bacteria

Genomic DNA from pure cultures of probiotic bacteria purified by extraction using phenol/chloroform/isoamyl alcohol (25:24:1). To get the complete destruction of cells, the method was slightly modified by the extension of enzymatic lysis with 2 to 7 hours. Subsequently, the DNA was precipitated, sterilized cold (-20°C) 95%ethanol and dissolved in double-distilled water (ddH20). The concentration and purity of all DNA preparations were obtained by measuring OD260absorption or D260/280and OD260/230relations. Used only DNA with respect to OD260/280>1.8 and OD250/230>2.

DNA was purified from endotoxin with p the physical alteration of TritonX-114D and also studied at the content of lipopolysaccharide (LPS) using the limulus amaebocytes test (QCL-1000, CAMBREX, Germany). The LPS content was less than 0.01 U endotoxin IYP-1.

To determine the biological activity of LPS or other bacterial contamination in DNA preparations of DNA degraded by desoksiribonukleaza I (Sigma). There was no apparent suppression of cytokines by cells RPMS below the main level, when it was added degraded DNA with a concentration of 75 µg ml-1(measured before treatment DANN destruction).

2.3. Isolation RVMS and culture

2.3.1. Preincubate RVMS probiotic bacteria and further stimulation with SEA and Dpt

Eight of allergic patients and eight healthy donors were taken for the study. All test subjects before registering presented oral and written consent for this study, as required by the ethics Committee of the University of Kiel for use in the study of human subjects of subjects. Venous blood was sent from donors in heparinized Vacutainers and dissolved in a 1:1 ratio with 0.9% NaCl. Then mononuclear cells from fresh peripheral blood (RVMS) were isolated from heparinised dissolved blood by centrifugation according to increasing density of 1.077 g ml-1) (Lymphoprep, AXIS-SHIELD PoC AS, Oslo, Norway). Cells were converted to the emulsion in an environment of culture RPMI-1640 (Sigma, Munich, Germany), supplemented with 10% (vol./about.) the act is endorsed high temperature (56°C, 1 h) fetal bovine serum, Gentamicin (50 µg ml-1) (Sigma), penicillin streptomycin (1%) and pyruvate solution of sodium (0.23 mmol l-1) (Sigma) (ordered as complete medium). All components were purchased tested for endotoxins, as required LAL. Cells cultured in complete medium at a concentration of 2*106cells ml-1in the tank with 24 holes. At the same time to stimulate the cultures added four strains of live probiotic bacteria and gram-negative bacteria (E. coli TG1). As required for bacterial count, probiotic and other bacteria were viable at the time of addition to the cultures. Cells that are cultivated only with the environment, served as restimulating control. Tests on the dependence on dosage, performed for IL-4 and IL-5 as cultures with a final volume of 200 μl/well received in flat-bottomed 96-well microtiter plates (Nune, Roskilde, Denmark) with 2×106mononuclear cells and 5×104, 5×105, 2×106, 5×106, 2×107and 5×107bacteria/ml, corresponding 0,025, 0,25, 1, 2, 5, 10 and 25 of bacteria on mononuclear cell. They showed that the maximum suppression was observed with 2×107CFU bacteria/ml, corresponding to a ratio of 10:1 (bacteria to RVMS). This concentration was used in other experiments. Final Rel the decision between RVS and bacteria (the ratio of bacteria to RMS) was 10:1 for healthy donors and Allergy. For the control treatment only environment culture was added to a solution of RVMS. Then after incubation for three hours at 37°C, RVMS then stimulated with SEA (2 µg ml-1) or Dpt (2000SQ-E ml-1equivalent to 2 µg dose ml-1) and 5%uverennom CO2incubator at 37°C for 48 hours. All the experiments were performed in duplicate. After incubation for 48 hours, the medium culture was centrifuged at 4°C for 20 minutes at 1,000×g Not containing cell supernatant sterilized by passing through a filter with a pore size of 0.2 μm (Millipore, Germany) and stored at -80°C until use. Cell viability determined before and after incubation with bacteria by exception with Trifanova blue.

2.3.2. The excitation SEA, LPS and genomic DNA

Fresh RUMS from heparinized peripheral blood from four healthy donors allocated by centrifugation in accordance with the increasing density of 1.077 g ml-1) (Lymphoprep, AXIS-SHIELD PoC AS, Oslo, Norway). Cells were transformed into the emulsion in the medium RPMI 1640 culture (Sigma, Munich, Germany), supplemented with 10% (vol./about.) activated high temperature (56°C, 1 h) fetal bovine serum, Gentamicin (50 µg ml-1) (Sigma), penicillin streptomycin (1%) and pyruvate solution of sodium (0.23 mmol l-1) (Sigma) (complete medium). All components of b is whether purchased tested for endotoxin. All tests on the dependence on dosage, performed for IL-4 and IL-5 as cultures with a final volume of 200 μl/well, were obtained in flat-bottomed 96 - well microtiter plates (Nune, Roskilde, Denmark) with 2×106mononuclear cells and 5, 10, 25, 50, 75 and 100 µg of genomic DNA per ml They showed that the maximum suppression could be observed with 75 μg DNA ml-1. This concentration was used in further experiments. Cells cultured in complete medium at a concentration of 2*106cells ml-1in a 24-hole plate (Nune, Roskilde, Denmark). At the same time, genomic DNA (75 µg ml-1) probiotic bacteria, genomic DNA from calf (75 µg ml-1Sigma, Munich, Germany) SEA (2 µg ml-1)LPS from E. coli (20 µg ml-1, Sigma, Munich, Germany) was added to the culture and incubated at 37°C, 5% CO2-hydration for 24 hours. All the experiments were performed in triplicate. After 24-hour incubation, the supernatant without cells were collected and centrifuged at 1000g for 20 minutes at 4°C, sterilized by passing through a filter with 0.2 μm pores (Millipore, Germany) and stored at -80°C in certain amounts to the analysis. Cell viability determined before and after incubation with genomic DNA by removing Trifanova blue. In all experiments, 95-98% RVMS were viable. A pilot phase of the coefficients in dependence on dosage, which is true for all cytokines, showed that the maximum suppression was observed at a concentration of 75 µg ml-1. This concentration was used in further experiments.

2.4. The study of cytokines by antispyzone immunosorbent assay (ELISA)

The concentration of IL-4, IL-5 and IFN-γ quantified in the supernatant fluids without cells through specific ELISA (BD OptEiA™ set IL-4, 5, and IFN-γ person, Heidelberg, Germany). The limits of sensitivity studies were 0.5 μg ml-1for IL-4, 0.5 μg ml-1for IL-5 and 1 µg ml-1for IFN-γ. The values of optical density of the samples were read at 450 and 570 nm on the anode electric reactor ELISA. Experiments repeated at least twice and performed in triplicate.

2.5. Statistical analysis

Experimental test data were produced by ±S.E.M and was made parameterless statistical analysis with t-test. By P-values less than 0.05 were considered as statistically significant.

Results

Lactobacillus and Bifidobacterium inhibit IL-4 and IL-5 and start the production of IFN-γ by SEA or Dpt-stimulated RUMS from healthy donors. It is shown that streptococcal superantigen cause a high concentration of IL-4 and IL-5 from RUMS from healthy donors and killed by the high temperature of the lactic acid is acteria were able to suppress production of the cytokine profile of type-2. In addition, research has shown that allergic patients had increased levels of IL-4 and IL-5. Dermatophagoides pteronyssinus, Dpt (83.8 percent) and Dermatophagoides farinae, Df (78,4%) are the most common causative allergens in patients with allergic rhinitis. Now we have confirmed these observations with other superantigen - staphylococcal enterotoxin A (SEA) and Dpt.

When live bacteria strains Lactobacillus and Bifidobacterium preincubation with RVMS received production of IL-4a with SEA and Dpt is very reduced in comparison with that which was caused by the positive control (without preincubation with Lactobacillus and Bifidobacterium). Interestingly, there were no significant suppression when RVMS preincubation with gram-negative control strain TG1 (figure 1). Although neither four strains of Lactobacillus and Bifidobacterium, nor TG1 has not caused the main production of IL-4, all four strains caused the production of IFN-γ. When RVMS with SEA and Dpt was stimulated, the concentration of IFN-γ release, in addition, it has increased. A synergistic effect was observed not only with Lactobacillus and Bifidobacterium bacteria, but also with TG1. Therefore Lactobacillus and Bifidobacterium, apparently, able to influence the secretion of Tn1, and Tn2 cytokines in opposite directions.

The production of cytokines depends on the time and dosages.

Other experiments performed with four strains of Lactobacilus and Bifidobacterium showed that the suppression of Tn2 cytokines depends on the time and dosages. When RVMS stimulated with probiotic bacteria before addition SEA, growth inhibitory effect on the production of the Tn2 cytokine was increased with the ratio of bacteria to RVMS. Maximum growth inhibition was observed at 2×107CFU bacteria ml-1, corresponding to a ratio of 10:1 (bacteria to IUD) (figure 2, a and b).

This effect was also observed for the production of IL-4 and IL-5. For example, strains of L. gasseri the percentage of growth inhibition of production of IL-4 rose from 40,19%±3% (ratio 1:1) to 54 22% ± (ratio 10:1) in response to SEA. The percentage of suppression of the production of IL-5 increased from 43,77%±8% (ratio 1:1) to 73,17%±5% (ratio 10:1). In addition, the percentage of inhibition of production of IL-4 in response to Dpt in RVMS from healthy donors increased from 34,61%±4% (ratio 1:1) to 47,33%±5% (ratio) of 10:1. The percentage of growth inhibition of production of IL-5 increased from 45,72%±4% to 77,59%±6%. In contrast to Tn2 cytokine, our tested strains caused the main production of IFN-γ, which, apparently, depends on the ratio of bacteria/cell. It is remarkable that it was possible to show that probiotic bacteria have a significant impact on the production of IFN-γ in response to stimulation of the SEA (figures 1 and 2).

Time dependent inhibitory growth effect of live probiotic bacteria on the production is tion of IL-4 and IL-5

RUMS from healthy donors (n=4 2×106ml-1) stimulated with SEA (2 μg/ml). Mononuclear cells or preincubation for 1, 3 and 5 hours strains L.GG (ratio 10:1) before incubation to stimulate SEA, or they are simultaneously stimulated strains L.GG, and superantigen or L.GG was added after 1, 3 and 5 hours after SEA stimulation, and cell culture preincubation for 24, 48 and 72 hours. When the above time values supernatant without cells collected after centrifugation and sterilization stored at -20°C. the Measured concentration of IL-4 and IL-5 showed that the maximum growth inhibition was observed for the 3 hours preincubation RVMS live bacteria before initiation of the SEA. Similarly, the maximum growth inhibition was observed for 48-hour incubation (data not shown). This period was used in further experiments.

Lactobacillus and Bifidobacterium inhibit the production of Tn2 cytokines from allergen-stimulated RUMS from allergic patients.

The ability of Lactobacillus and Bifidobacterium to correct the imbalance between Tn1 and Tn2 cytokines evaluated in a more relevant model of excitation allergens. When RVMS from patients allergic to D. pteronyssinus) preincubation with a variety of viable strains of Lactobacillus and Bifidobacterium, the production of IL-4 and IL-5, Apuseni after specific stimulation D. pteronyssinus allergens, significantly reduced, especially depending on the relationship of bacteria/cell. This effect on IL-5 does not depend on the type of strain studied probiotic bacteria. Suppression of IL-5 was consistent with, for example, in the case .b. - 57.31%±to 4.52% in the case of B.I. - 63,77%±5% (figure 1).

In addition, similar effects were observed when RVMS from allergic patients were stimulated by the SEA. In this case, although the production of IL-4 and IL-5 is very high compared with the production, which was observed with D. pteronyssinus (figure 1), tested probiotic bacteria reduced the secretion of IL-5 to 71,29%±4.10% for L.GG and to 77,3%±3,52% for L.gasseri (figure 1). Co-incubation with Lactobacillus and Bifidobacterium very increased production of IFN-γ (figure 1). Thus, the reduction in the production of Tn2 cytokines and increased production of IFN-γ due to RVMS from allergic patients Lactobacillus and Bifidobacterium, apparently, are anti-Tn2 activity. In this context it is interesting to note that the effect of inhibiting the growth of Lactobacillus and Bifidobacterium on the production of IL-4 by RVMS from allergic test subjects more than in healthy test subjects. In contrast, the inhibitory growth effect of these bacteria on the production of IL-5 by RVMS from healthy donors more than for allergic test subjects (figure 1). In contrast, inhibitory costeffect B.I and LgsBbBI on the production of IL-4 by Dpt-stimulated RUMS from allergic test subjects more than he from healthy test subjects (p<0.05), the inhibitory effect L.GG, .b. and B.I on the production of IL-4 SEA-stimulated RUMS from allergic test subjects was greater than that of healthy test subjects (p<0,01). In contrast, the inhibitory growth effect of L. gasseri, .b and B.1 on the production of IL-5 Dpt-stimulated RUMS from healthy test subjects was greater than that for allergic test subjects (p<0.05), the effect of suppressing LgsBbBI on the production of IL-5 from SEA-stimulated RUMS from healthy test subjects was greater than that for allergic patients (p<0.01, figure 1).

Genomic DNA from probiotic bacteria inhibits the production of IL-4 and IL-5 SEA-stimulated RVMS healthy test subjects, depending on the time and dosages.

To evaluate the effect of inhibiting the growth of bacterial DNA in comparison with animal DNA and LPS, RVMS from four healthy test subjects incubated with genomic DNA from four strains of probiotic bacteria, L. GG, L. gasseri, .b., B.1 and LgsBbBI (a mixture of L-gasseri, .b. and B.I), LPS and animal DNA (DNA calf thymus). Suppression of Tn2 cytokines was observed with the use of test sets BDoptEIA to demonstrate and quantify the production of cytokines. As shown in figure 4, DNA from probiotic bacteria normally inhibits RusPromAvto IL-4 and IL-5 in response to stimulation through SEA or Dpt. In contrast, LPS did not reduce the production of IL-4 and IL-5; similar to DNA, available from LPS calf thymus, did not reduce the production of IL-4 and IL-5 (data not shown).

Time-dependent effect of inhibiting the growth of bacterial DNA on the production of IL-4 and IL-5 by SEA-stimulated RUMS from healthy test subjects

Several experiments were performed to determine the temporal profile of the response on the genomic DNA. RUMS from healthy donors (n=4, 2×106ml-1) stimulated SEA (2 µg ml-1and either preincubation within one, three and six hours with genomic DNA from L.GG or .b. (30 µg ml-1before SEA stimulus, or stimulated simultaneously with genomic DNA. Superantigen or genomic DNA added after 1, 3 and 6 hours after SEA stimulus, and cell cultures incubated for periods of 24, 48 and 72 hours. Genomic DNA from L.GG and .b. selected as a representative for two strains of Lactobacillus and Bifidobacterium. When the above time periods supernatant without cells collected after centrifugation and sterilization stored at -80°C. the Measured concentrations of IL-4 and IL-5 showed that the maximum growth inhibition was observed at tohours incubation RVMS with genomic DNA and the SEA (data not shown), similar to the maximum suppression was achieved after incubation for 24 hours. This time was used to draft the experiments. Genomic DNA from .b. has been confirmed as a more effective inhibitor of IL-4 than genomic DNA from L.GG. On the contrary, it is confirmed that the genomic DNA from L.GG is a more effective inhibitor of IL-5 than genomic DNA from .b.

Dependent on the dosage effect of inhibiting the growth of bacterial DNA on the production of IL-4 and IL-5 from SEA-stimulated RUMS from healthy test subjects

To investigate the effect of inhibiting the growth of bacterial DNA on the inhibition of IL-4 and IL-5 SEA-stimulated RUMS from healthy test subjects, genomic DNA from each strain introduced in RVMS culture in various concentrations, namely in the range from 5 to 105 µg ml-1to determine which dosage has the largest effect of inhibition on the production of IL-4 and IL-5 (figure 4). This dosage was chosen based on the results of the tests described above, which showed that the production of cytokines by immune cells can be demonstrated after the addition of at least 3 μg ml-lE.coli. DNA and optimal stimulation required for bacterial DNA at a concentration of>50 μg ml-1. As shown in figure 4, the maximum inhibition of IL-4 and IL-5 was observed when genomic DNA was used at a concentration of 75 µg ml-1. The other three sample answers cytokine could be clearly differentiated. Genomic DNA: All strains suppressed p is izvodstvo IL-4 and IL-5 at a concentration of 75 µg ml -1except .b.-DNA and L.GG-DANN, which suppressed the production of IL-4 and IL-5 at a concentration of 65 μg ml-1.

All genomic DNA suppressed the production of IL-4 and IL-5 to a lower steady-state levels when added in a concentration range of from 5 to 45 μg ml-1. In contrast, all genomic DNA showed dependent on dosage intensification of the production of IL-4 and IL-5 when it was used in a concentration range of from 85 to 105 µg ml-1. Compared with other genomic DNA, .b and L.GG DNA is likely to be more effective inhibitors of the production of IL-4 and IL-5 in response to SEA-stimulation. In addition, genomic DNA from .b. showed the lowest inhibitory effect on the production of IL-5 at a concentration of 105 µg ml-1.

The inhibitory growth effect of genomic DNA from probiotic bacteria on the production of IL-4 and IL-5 SEA-stimulated RVMS of allergic test subjects

To investigate the inhibitory growth effect of genomic DNA from probiotic bacteria, RVMS (2×106ml-1) at 75 µg ml-1from L.GG DNA, Bi DNA and LgsBbBI, DNA incubated for 24 hours. Three other sample of inhibiting the production of IL-4 in response to SEA-stimulation could be clearly distinguished: genomic DNA from L.GG and LgsBbBI showed the strongest effect of inhibition, namely 37,4%±4% and 39,56%±5.1%, respectively. L.gasseri DNA showed the lowest is to function effectively inhibiting (19.14 per cent±2%) and B.b.B.I. showed a similar pattern (24,47%±3.28 per cent). On the contrary L. gasseri DNA shows the largest inhibitory growth effect on the production of IL-5 in response to SEA-stimulation (61,41%±3,74%), DNA LgsBbBI showed the lowest inhibition (46,31%±4%) and genomic DNA of L. GG, .b. and V.I. showed an identical pattern of suppression (data not shown). In addition, we compared the inhibitory effect of genomic DNA with live bacteria, as well as the effect of suppression of genomic DNA for production of IL-4 and IL-5 in response to SEA stimulation between healthy and allergic test subjects. The inhibitory growth effect of live bacteria on the production of IL-4 in healthy and allergic test subjects is higher than the influence of their genomic DNA. In contrast, the inhibitory growth effect of genomic DNA in allergic test subjects more than in healthy test subjects (with the exception of L. gasseri). Therefore, genomic DNA is more effective in reducing the production of IL-4 in response to SEA stimulation in allergic test subjects.

To put it briefly, the inhibitory growth effect of live bacteria on the production of IL-5 in response to SEA stimulation in RVMS from healthy and allergic patients is the same as the effect on production of IL-4 (e.g., inhibitory effect of live bacteria was higher than their genomic DNA in healthy and allergic test subjects). what about the inhibitory growth effect of genomic DNA for production of IL-5 in healthy donors was more than for allergic test subjects. Therefore, the genomic DNA of more effective inhibitors for IL-5 in healthy donors. Relative excitation Dpt, the inhibiting effect of the genomic DNA of L. GG and LgsBbBI on the production of IL-4 Dpt-stimulated RVMS of allergic test subjects was greater than in the case of healthy test subjects (p>0,05). A similar pattern was obtained for genomic DNA from L. gasseri B.b. and B.I. in Addition, the effect of inhibition of genomic DNA from L. gasseri, .b. B.1 and LgsBbBI on the production of IL-5 from Dpt-stimulated RUMS from healthy test subjects was greater than in the case of allergic test subjects. But inhibitory growth effect L.GG DNA on the production of IL-5 in healthy test subjects was the same as for allergic test subjects. Therefore, genomic DNA from L.GG can modulate the production of IL-5 Dpt-stimulated RUMS from healthy and allergic test subjects in the same way.

Discussion

Allergies - hypersensitivity reactions of the immune system to specific substances called allergens (such as pollen, insect venoms, pharmaceuticals or food). Some experimental studies show that allergic diseases may be associated with the imbalance of Tn1/Tn2 cytokines with a relative clause is eloholma T n2 cytokines and the lack of Tn1 cytokines. In fact, IL-4, IL-5, IL-3 and IL-9 are involved in the initiation and maintenance of allergic reactions. It is assumed that Tn2 cytokines, preferably involved in allergic diseases, because the shift of the immune response of Tn2 to Tn1 largely evident in most types of allergic diseases. The promising results obtained in the treatment of allergic patients with probiotic bacteria, prompted us to explain the interaction between Tn1/Tn2 cytokines and allergies. Therefore, one strategy in the treatment of allergies is to change Tn1/Tn2 the balance of the administration of probiotic bacteria to restore Tn1/Tn2 balance. Particularly interesting results regarding the ability of living or dead probiotic bacteria significantly reduce the number of IL-4 and increase the number SFN-γ cytokines.

In addition, this study has shown that the protective effect of probiotic bacteria could be associated with release of soluble factors, which alter the permeability of the epithelium and protects against pathogenic bacterial invasions. These data raise the question, could this soluble factor or other bacterial cytoplasmic components, such as DNA, would the ü involved in the launch and modulation of cytokines. From this point of view, we investigated the effect of live bacteria and pure genomic DNA from strains L.GG L. gasseri, .b., V.I. and LgasBbBI (a mixture of L-gasseri. b. and B.1) on the release of cytokines IL-4 and IL-5 using models of human culture. The results show that different strains probiotics bacteria, as well as their genomic DNA, cause significant changes in the profile of cytokines that are active are highlighted in vitro from SEA or Dpt-stimulated RVMS. In addition, they can modulate Tn1/Tn2 balance, reducing the production of the Tn2 cytokines and increasing the production of Tn1 cytokines. Therefore, these probiotic bacteria can be useful effect in allergic diseases as a result of their effect of inhibition on the production of the Tn2 cytokines.

To explore this effect, we first stimulated RUMS from healthy and allergic test subjects with SEA or Dpt (as producing Tn2 cytokine cell model). Many researchers reported that RVMS allocate Tn2 cytokines after excitation by superantigens. When in this study, SEA or Dpt-stimulated RVMS were preincubation with live probiotic bacteria and their genomic DNA, production of the Tn2 cytokines was reduced, but not in the presence of E. coli. In addition, this effect Engibarov what I was dose dependent so that at a concentration of 2×107CFU ml-1(according to the ratio of 10:1 to bacterial RVMS), the effect of inhibition of live bacteria reached a maximum relative production of IL-4 and IL-5. Such dependent dosage effect is also reported in vitro to produce IL-10 and IL-12 by monocytes from healthy test subjects after incubation with certain strains of gram-positive bacteria. This study also showed that not only live probiotic bacteria and their genomic DNA can inhibit the production of IL-4 and IL-5 SEA - or Dpt-stimulated RVMS. The most important point of this study that, first, it is an advantageous effect of genomic DNA or probiotic bacteria on the cells of allergic patients who are sensitive to the clamp household dust; that is, microscopic organisms that are found in homes. They are the main cause of allergies from dust. All other messages are only associated with the beneficial effects of living or dead probiotic bacteria on allergic diseases caused by food allergies or aeroallergens. Our data suggest that probiotic bacteria and their genomic DNA are direct or indirect regulation of the signaling pathway, which is required to suppress Tn2 cytokines, because the effect of inhibition was observed, even to the GDS probiotic bacteria and their genomic DNA was added before or simultaneously (for genomic DNA) with SEA - or Dpt stimulation. Some experimental studies suggest that the modulation of the production of IL-4 and IL-5 is a multifactorial process, and some cytokines, such as IFN-γ described as potential regulators of the production of IL-4. In addition, it is reported that IL-12 is a prominent cytokine in the call to the production of IFN-γ human RVMS. In our study, probiotic bacteria and their genomic DNA is able to increase the production of IL-12 VRMS (data not shown).

In this respect, bacterial DNA was added to SEA-stimulated culture RUMS and, interestingly, this experiment showed diverse production of IL-4 and IL-5 cytokines after initiation of bacterial DNA and SEA processing. Bacterial DNA from probiotic bacteria reduced the secretion of IL-5 is much greater than that of IL-4. Our data indicate that genomic DNA from probiotic bacteria may act as an inhibitor of the production of IL-4 and IL-5 SEA - and Dpt-stimulated RUMS from healthy and allergic test subjects. In addition, this study reports that killed a high temperature probiotic bacteria reduce the production of IL-4 and IL-5 SEA-stimulated RVMS. Taking into account that the process of obtaining, gastrointestinal tract and high temperature (70°C) affect the viability of bacteria, the results of these studies shows which indicate the possible effect of inhibiting genomic DANN, which is killed by high temperature bacteria and added to RVMS. Various articles describe the response of various cytokines on CPG motifs present in bacterial DNA, such as genomic DNA from L.GG. It would be interesting to know whether certain to strain the release of cytokines, which is demonstrated in this study, attitudes toward genomic sequences and CPG motifs each strain.

As far as we know, this message is the first that shows the effects of immunomodulation genomic DNA of probiotic bacteria from SEA - or Dpt-stimulated RUMS from healthy and allergic test subjects.

The observed differences in the rate and magnitude of inhibition of IL-4 and IL-5 in response to bacterial DNA and SEA - or Dpt excitation - interesting information about the impact of living or dead probiotic bacteria and/or bacterial components on the immune response. Growing knowledge about probiotics are exciting, but in the near future should be installed, what probiotic DNA (individual strains or a combination) has the strongest effect on a specific disease. Next, you want well-organized selective clinical trials have identified the role of genomic DNA from probiotic bacteria as prophylactic and therapeutic agents in the Bud the future.

Further details and features of the invention are explained in detail below in relation to the examples with the results of several experiments. However, they are not intended to limit the invention, but only for his explanation. In schematic form

Figure 1

Modulation of cytokine RVMS without (Wednesday) or the stimulation of the SEA and Dpt

Figure 2

Dependent on the dosage effect of inhibition of living probiotic bacteria on

the production of IL-4 (a), IL-5 (b) and (IFN-γ) SEA stimulation RVMS healthy test subjects.

Figure 3

Cancelled

Figure 4

The inhibitory growth effect of genomic DNA from probiotic bacteria on the production of IL-4, IL-5 and IFN-γ SEA - or Dpt-stimulated RUMS from healthy (n=5) and allergic (n=5) tested subjects.

Figure 5

Dependent on the dosage of the inhibitory growth effect of genomic DNA for production of IL-4 (a), IL-5 (b) and IFN-γ (C) RVMS cells from healthy test subjects.

Detailed figures show:

Figure 1

Modulation of cytokine RVMS without (environment) or to the stimulation of the SEA and Dpt.

RUMS from healthy (n=8) and allergic (n=8) test subject were preincubation within 3 hours (2×105cells/ml) with medium without (RVMS) or with four strains of probiotic bacteria, LgB.b. B.1 (a mixture of probiotic bacteria and gram-negative bacteria control (E-coli TG1) if Rel is against bacteria cells 10:1 before stimulation with SEA-superantigen (2 µg ml -1), Dpt (2 µg ml-1or not.

IL-4, IL-5 and IFN-γ were quantified after hours incubation in the supernatant fluids particular study ELISA. Asterisks indicate specific suppression or stimulation (*p<0,05, **p<0,01) production of Tn1 and Tn2 cytokines compared to control.

Figure 2

Dependent on the dosage effect of inhibition of living probiotic bacteria on the production of IL-4 (a), IL-5 (b) and (IFN-γ) SEA - stimulation RVMS healthy test subjects. RUMS from four healthy donors (2×106cells ml-1) were cultivated with four strains of probiotic bacteria, L. GG, L. gasseri, B.b., B.I. in concentrations of 5×104, 5×105, 2×106, 5×106, 2×107, 5×107corresponding 0,025, 0,25, 1, 2,5, 5, 10 and 25 the relationship of bacteria to the cells for 3 hours before stimulation with 2 μg ml-1SEA. After 48 h incubation, IL-4, IL-5 and IFN-γ were measured by specific ELISA. A string of error messages indicates standard error of mean values.

Figure 3 is cancelled.

Figure 4

The inhibitory growth effect of genomic DNA from probiotic bacteria on the production of IL-4, IL-5 and IFN-γ SEA - or Dpt-stimulated RUMS from healthy (n=5) and allergic (n=5) tested subjects. RUMS from five healthy and five allergic test subjects cultivated for 48 h, the owls (2×10 6cells ml-1) with genomic DNA from 4 strains of probiotic bacteria, L.GG, L. gasseri, B.b., B.I. and LgBbBI (a mixture of L. gasseri, B.b and B.I). At a concentration of 75 µg ml-1before stimulation with SEA (2 µg ml-1) or Dpt (2000 SQ-EML-1corresponding to the introduction of 2 μg per ml-1). Environment and LPS (100 ng ml-1) was used as control. IL-4, IL-5 and IFN-γ were quantified after 24 h incubation in the supernatant fluids specific ELISA.

Asterisks indicate a remarkable inhibition (*p<0,05, **p<0,01) cytokine production compared with control (medium). Data are expressed as the mean +/-SEM.

Figure 5

Dependent on the dosage of the inhibitory growth effect of genomic DNA for production of IL-4 (A), IL-5 (b) and IFN-γ (C) PBMC from healthy test subjects.

RUMS from 4 healthy donors cultured with various concentrations (5-105 mg ml-1bacterial genomic DNA. The production of IL-4 and IL-5 (B) measured after 24 h incubation. Data are expressed as the mean +/-SEM.

1. Pharmaceutical preparation for the prevention, suppression or elimination of allergic reactions in humans, characterized in that the active component is present in the genomic DNA probiotic, gram-positive bacteria and/or bacterial strains Lactobacillus gasseri PA 16/8, and/or Bifidobacterium bifidum 20/5 MG, and/or Bifidobacterium longum SP 07/3 as a viable and/or inactivated bacteria.

2. Pharmaceutical drug to offset the balance Tn-Tn in the body in the direction of increasing T and/or reduce Tn, characterized in that the active component is present in the genomic DNA probiotic, gram-positive bacteria and/or bacterial strains Lactobacillus gasseri PA 16/8, and/or Bifidobacterium bifidum 20/5 MG, and/or Bifidobacterium longum SP 07/3 as a viable and/or inactivated bacteria.

3. A food product or an additive to the food product, characterized in that the active component is present in the genomic DNA probiotic, gram-positive bacteria and/or bacterial strains Lactobacillus gasseri PA 16/8, and/or Bifidobacterium bifidum 20/5 MG, and/or Bifidobacterium longum SP 07/3 as a viable and/or inactivated bacteria.

4. Using genomic DNA probiotic, gram-positive bacteria and/or bacterial strains Lactobacillus gasseri PA 16/8, and/or Bifidobacterium bifidum 20/5 MG, and/or Bifidobacterium longum SP 07/3 as a viable and/or inactivated bacteria for the treatment and/or prevention and/or reduction of risk of allergic reactions.

5. Using genomic DNA probiotic, gram-positive bacteria and/or bacterial strains Lactobacillus gasseri PA 16/8, and/or Bifidobacterium bifidum 20/5 MG, and/or Bifidobacterium longum SP 07/3 as a viable and/or inactivated bacteria for the treatment and/or prevention and/or reduction of risk of bias Tn-Tn balance the body in the direction of increasing T and/or reduce T.

6. The pharmaceutical preparation according to claim 1 or 2, characterized in that it is prepared for oral use, and/or for administration in the form of suppositories, or subcutaneous injection, or intravenous injection, or as a liquid for inhalation.



 

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