RU2454239C2 - Probiotic gram-positive bacteria for human allergy prevention, inhibition or elimination - Google Patents

Abstract

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

Description

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

Allergic reactions to substances such as pollen (hay fever) or other plant components, cat hair and other animal hair, dust containing ticks from house dust mites, insect venom, foods such as milk or nuts, or perfumes and other cosmetics are well known and are a growing problem for an ever-increasing number of people.

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

In this autoimmune response, white blood particles, lymphocytes, play a significant role. One form of lymphocytes is T lymphocytes, or helper cells (TH cells), which secrete various mediators, cytokines, to control the immune response. Firstly, there are cytokines that reduce or prevent allergic reactions, and secondly, other cytokines that cause inflammatory, i.e. allergic, reactions with a stimulating effect as mediators on active cells, such as mast cells. According to this effect, all TH cells are divided into two groups, namely T _n 1 and T _n 2 cells. T _n 1 cells include anti-allergic cytokines, such as γ-interferon (IFN-γ) or interleukin 2 (IL-2). T _n 2 cells include mediators that cause allergic reactions, such as interleukins IL-3, IL-4, IL-5 and IL-10. Interleukin-4 stimulates mast cells to form a type E immunoglobulin antibody (IgE), which is present in very large quantities for allergies.

The ratio between the number of T _n 1 cells and T _n 2 cells is critical for the body's immune response to captured pathogens, it is significantly lower in patients with an allergic reaction than in healthy people. It is known that newborns or premature babies also have a very low value for this ratio so that the mother’s body does not mistakenly attack the baby’s cells.

Therefore, the balance of T _n 1-T _n 2 is an important characteristic of each person and is also 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 a decrease in infectious diseases create a predisposition for the difference in balance and therefore give rise to allergic hypersensitivity.

Therefore, even in infancy, an invasion of allergens can cause inflammatory reactions, such as a runny nose and swollen mucous membrane, and even elevated body temperature.

Numerous pharmaceutical preparations are known that fight or suppress symptoms that occur. Their disadvantage is that often they are very expensive, cause many unwanted side effects and must 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 therefore is readily available and recyclable, which provides low prices, which are already found in small quantities even among healthy people and therefore compatible with the human body certain limits and which does not attack allergic reactions at the symptom level, but at the launch stage by neurotransmitters.

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

The “probiotic” attribute 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, a second organism, namely a bacterium, is beneficial to the human body.

The bacterium itself is not damaged and therefore remains viable. This is because when taken orally, as many bacteria as possible pass unscathed through the stomach with its acids, enzymes and other digestive agents.

The term "probiotic" also implies that adverse effects on humans are minor or at least very small and that effects that are classified as beneficial predominate. The term "probiotics" means preparations of microorganisms that have a healing effect on the host when used in sufficient quantities. Probiotic lactic acid bacteria (Lactobacillus) have been used for a long time. Probiotics can be administered as specially prepared foods or in the form of pharmaceutical preparations.

The healing effects of probiotics are strain-specific in each case. It is known from medicine that probiotic bacterial strains undoubtedly increase the absorption of lactose, inhibit pathogens in the intestines and limit or suppress diarrhea.

Other bacterial strains increase protection against infection, lower cholesterol and reduce the risk of colon cancer. These effects have been successfully demonstrated in vitro before widespread use. Therefore, it is legitimate to confirm the healing effect of this invention by a number of laboratory experiments.

Through the experiments described below, it was estimated which cytokines from the human blood cell fraction (PBMC - peripheral mononuclear blood cells) are released under specific conditions. For one series of experiments, these blood 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 further series Dermatophagoids (Dpt) were added because those suffering from house dust allergies usually also have allergies to them. In this way, the invasion of an allergenic substance was simulated.

Interferon-γ was evaluated as a substituent for the T _n 1 reaction. A T _n 2 sample was recorded by isolated interleukins 4 and 5 (IL-4 and IL-5).

Factors that may be involved in the increased spread of allergic disease are thought to include modification of the intestinal flora or lack of bacterial stimulation in childhood. T _n 2 cytokines increase IgE production and stimulate mast cells. On the other hand, γ-interferon (IFN-γ), Tlcytokine contributes to the suppression of IgE synthesis. Thus, the imbalance of the expression "T _n 1 to T _n 2" can contribute to the launch and maintenance of allergic diseases. Lactobacillus bacteria, which are part of the natural intestinal microflora, are believed to reduce the frequency and severity of allergic manifestations and modulate the T _n 1 / T _n 2 response. The functional mechanism has not yet been explained.

Task: Our experimental task was to determine the effect of probiotic bacteria on the production of such cytokines T _n 1 and T _n 2 types of healthy people and patients with allergies to house dust mites and to explain the molecular basis of the effects of bacterial genomic DNA on T _n 1 / T _n 2 response to staphylococcus enterotoxin A (SEA) and Dermatophagoides pteronyssinus (Dpt) and compare them with the effects of live bacteria.

Methods: RVMS from patients with house dust mite allergies compared with the same from healthy donors were incubated for 24 and 48 hours with or without Dpt and SEA allergens. The effect of pre-incubation with live probiotic bacteria, as well as the effect of their genomic DNA, which was simultaneously added to the cultured and incubated for 24 hours, were evaluated by measuring the production of T _n 1 / T _n 2 cytokines.

results

Tested, viable, gram-positive, probiotic bacteria and their genomic DNA showed that they suppressed SEA and Dpt-stimulated secretion of T _n 2 cytokines (EL4 and EL5) and increased IFN-γ stimulation. This effect depended on the dosage and on the selected bacterial strain. There was no significant inhibition of control gram-negative Escherichia coli TG1. Probiotic bacteria reduced the production of IL-4 cytokines from allergic PBMCs, especially after repeated stimulation of SEA and Dpt, even more effectively than in healthy people. Inhibition of IL-5 in healthy subjects, on the other hand, is more clearly stated. Bacterial DNA also suppressed the release of IL-4 and IL-5, but only to a slightly lower degree. EL4 inhibition was more pronounced in the case of PBMC from allergic subjects than in healthy subjects, although this was the case for EL5.

Conclusion

T _n 1 / T _n 2 response to allergens modulated by tested probiotic bacteria, as well as their genomic DNA, showed anti T _n 2 activity. Therefore, various strains of probiotic bacteria and their genomic DNA may be useful in preventing allergic diseases.

1. Introduction

The theory of the causes of allergic diseases remains unclear, although experimental studies in humans show that genetic conditions contribute to the immune response of the intestinal mucosa, and intestinal bacteria contribute to the pathogenesis of allergic diseases. Some studies suggest that T _n 2 cytokines, especially IL-4, IL-5, 1L-9 and IL-13, play a significant role in pathogenesis by regulating IgE production and stimulating mast cells. The production of IL-4, IL-5, 1L-9 or IL-13 T _n 2 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 T _n 1 cytokine, has the ability to inhibit IgE synthesis. Defective IFN-γ expression is often associated with IgE-mediated allergies. The deregulation in the balance of T _n 1 / T _n 2 cytokines may be to a large extent responsible for triggering and maintaining allergic inflammatory processes in diseases such as bronchial asthma or atopic dermatitis.

It is proposed that, in particular, the profile of T _n 2 cytokine of newborn infants; aging, which usually reduces T _n 1 / T _n 2 balance; prescriptions of modern hygiene; intensive sterilization of food and / or changes in the intestinal flora of newborn infants caused by feeding with artificial drugs play the main role in changes in T _n 1 / T _n 2 balance. The acquired knowledge in this area prepared the basis for a number of new therapeutic approaches. One approach is to simply use probiotic bacteria to reproduce those cytokines that were not formed in sufficient quantities, or to reduce those cytokines that were formed on too large a scale because probiotic bacteria modulated T _n 1 / T _n 2 balance.

Probiotic bacteria are generally classified as safe and have been consumed by humans or animals. The bacteria Lactobacillus and Bifiobacterium are microorganisms most commonly found in the human gastrointestinal tract. Interest is in the growth of the immunostimulating effect of probiotic bacteria, especially their antiallergenic effect. The important role of bacteria in allergic diseases increases the possibility of preventing or treating these abnormalities by altering the intestinal microflora with probiotic treatment. Probiotic bacteria can act directly or indirectly due to changes in the endogenous flora or immune system. It was proposed to use the new expression "immunobiotics" for bacteria that improve health through the immune system of the mucous membrane, in contrast to the same, only with a local effect. Various immune responses to probiotics have been reported, such as secretion of inflammation-promoting cytokines, reproduction of lymphocytes, and the formation of nitrogen oxides. In addition, it was shown that all cells, some soluble components that were produced by LGG or DNA from LGG, cell wall components, such as Lactobacilli peptidoglycanic acid or lipoteichionic acid, cause inflammatory immune responses and immunobiotic activity. It has been demonstrated that not only live bacteria that are inserted into the intestinal tract, but also isolated probiotic DNA is active, even if it is injected subcutaneously.

Undenatured CpG motifs in bacterial DNA have been reported to be mitogenetic for mouse B cells and cause the production of inflammatory cytokines by macrophages and tree cells (DC). Both CpG oligodeoxynucleotides (ODN) and bacterial DNA induce 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 causes the spread of B cells and the separation of unaffected and activated T cells in T helper cells 1 and 2. Specific CpG ODNs (D-type) are especially effective in activating NK cells and producing α-interferon ( IFN-α) by plasmacytoid tree cells, while other ODNs (type K) are particularly effective B cell activators. It has recently been found that bell-shaped receptor 9 (TLR9) plays a critical role in triggering cell activation with CpG DNA.

Experiments that were performed on mice that were sensitized to egg albumin showed that after the introduction of lactic acid bacteria into the stomach, specific IgE and T _n 2 profile-dependent inflammatory responses were suppressed. In addition, recent reports suggest that lactobacillus Rhamnosus GG can reduce the symptoms of allergic diseases in humans, just as innate immune responses can trigger them by activating transcription factors that are integrated into the cytokine signaling function. The introduction of this bacterial strain in infants and newborns has reduced the risk of atopic eczema in infants. In vitro experiments have shown that the excitation of PBMC or healthy donor monocytes by a variety of lactic acid bacteria causes the secretion of IL-12, which is a significant pro-T _n 1 cytokine and is involved in the management of allergic diseases. The functional mechanisms by which LAB affects the production of T _n 2 cytokines, which are responsible for initiating the development of allergic diseases, remain identifiable. Taking into account the presence of undenatured DNA sequences (CpG motifs) in the DNA chromosomes from Bifidobacterium and Lactobacillus, we decided in this work to study the effect of four bacterial strains of Lactobacillus and Bifidobacterium, as well as their chromosomal DNA on the production of IL-4, IL-5 and IFN-γ γ SEA- and Dpt-stimulated PBMCs of healthy and allergic test subjects. The studied Lactobacillus bacteria and Bifiobacterium showed _anti-TH 2 activity and pro TH ₁ cytokine, IFN-γ.

2. Methods and Material

2.1. Bacterial culture

The following bacterial strains were used in this study:

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

The strains of Bifidobacterium and Lactobacillus were used (0.02% inoculum material of the strains, which was anaerobically stored at minus 80 ° C in 30% glycerol) (anaerobic system, MACS-VA500 Workstation with an airlock and an airlock, Don Whitley Scientific Limited UK in MRS medium (MRS; Merck, Darmstadt, Germany) supplemented with 0.05% L-cysteine at 37 ° C for 16 hours Prior to collection and washing in PBS, successive solutions of freshly prepared cultures were applied to plates containing MRS - agar medium and cultured for counting. The counting colony forming units (CFU ml ^-1) probiotich cyclic groups of bacteria was obtained with repeated 10-fold dilutions on plates. The plates were incubated anaerobically at 37 ° C for 24-48 hours. Bacteria were then washed three times with PBS and brought to final concentrations of 10, ^10, 10, ⁷ and ¹⁰ January CFU 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 OD600 vs agar plates of freshly prepared, repeatedly dissolved cultures. In order to calculate the count of viable bacteria in freshly prepared cultures, the curves are equipped with logarithmic printouts, which received all values of 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 assembled as described above.

2.2. Obtaining genomic DNA from bacteria

Genomic DNA from pure cultures of probiotic bacteria was purified by extraction using phenol / chloroform / isoamyl alcohol (25: 24: 1). To obtain complete destruction of the cell, the method was slightly modified by prolonging the enzymatic lysis from 2 to 7 hours. Subsequently, the DNA was precipitated, sterilized with cold (-20 ° C) 95% ethanol and dissolved in twice distilled water (ddH ₂ 0). The concentration and purity of all DNA preparations were obtained by measuring OD ₂₆₀ absorption or OD _260/280 and OD _260/230 ratios. Only DNA with a ratio of OD _260/280 > 1.8 and OD _250/230 > 2 was used.

DNA was purified from endotoxin using TritonX-114D and also tested for lipopolysaccharides (LPS) using the limulus amaebocytes test (QCL-1000, CAMBREX, Germany). The LPS content was less than 0.01 U endotoxins mkg ^-1 .

To determine the biological activity of LPS or other bacterial contaminants in DNA preparations, DNA was degraded by deoxyribonuclease I (Sigma). There was no apparent inhibition of cytokines by PBMC cells below the baseline when degraded DNA was added at a concentration of 75 μg ml ^-1 (measured before DANN destruction treatment).

2.3. PBMC isolation and culture

2.3.1. Preincubation of PBMC with probiotic bacteria and further stimulation with SEA and Dpt

Eight allergic patients and eight healthy donors were enrolled. Prior to registration, all test subjects submitted verbal and written consent for this study, as required by the Ethics Committee of the University of Kiel to use human subjects in the study. Venous blood was sent from donors to heparinized Vacutainers and dissolved in a 1: 1 ratio of 0.9% NaCl. Fresh peripheral blood mononuclear cells (PBMC) were then isolated from heparinized dissolved blood by centrifugation according to increasing density (1.077 g ml ^-1 ) (Lymphoprep, AXIS-SHIELD PoC AS, Oslo, Norway). The cells were converted into an emulsion in RPMI-1640 culture medium (Sigma, Munich, Germany), supplemented with 10% (v / v) activated high temperature (56 ° C, 1 h) fetal bovine serum, Gentamicin (50 μg ml ^{- 1} ) (Sigma), penicillin streptomycin (1%) and sodium pyruvate solution (0.23 mmol L ^-1 ) (Sigma) (ordered as complete medium). All components were purchased tested for endotoxins as required by LAL. Cells were cultured in complete medium at a concentration of 2 * 10 ⁶ cells ml ^-1 in a 24-well container. At the same time, four strains of live probiotic bacteria and gram-negative bacteria (E. coli TG1) were added to the cultures to stimulate. As required for bacterial counting, probiotic and other bacteria were viable during supplementation with cultures. Cells that were cultured only with medium served as an unstimulated control. Dosage-dependent tests 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 × 10 ⁶ mononuclear cells and 5 × 10 ⁴ , 5 × 10 ⁵ , 2 × 10 ⁶ , 5 × 10 ⁶ , 2 × 10 ⁷ and 5 × 10 ⁷ bacteria / ml, corresponding to 0.025, 0.25, 1, 2, 5, 10 and 25 bacteria per mononuclear the cage. They showed that maximum suppression was observed with 2 × 10 ⁷ CFU bacteria / ml, corresponding to a ratio of 10: 1 (bacteria to PBMC). This concentration was used in other experiments. The final ratio between RVMS and bacteria (ratio of bacteria to RVMS) was 10: 1 for healthy donors and for allergy sufferers. For control treatment, only culture medium was added to the PBMC solution. Then, and after incubation for three hours at 37 ° C, PBMCs were further stimulated with SEA (2 μg ml ^-1 ) or Dpt (2000SQ-E ml ^-1 equivalent to 2 μg dose ml ^-1 ) and in 5% wet CO ₂ incubator at 37 ° C for 48 hours. All experiments were performed in duplicate. After incubation for 48 hours, the culture medium was centrifuged at 4 ° C for 20 minutes at 1,000 × g. The cell-free supernatant was sterilized by passing through a 0.2 micron filter (Millipore, Germany) and stored at -80 ° C until use. Cell viability was determined before and after incubation with bacteria by exclusion with trypan blue.

2.3.2. Excitation with SEA, LPS and Genomic DNA

Fresh PBMCs from heparinized peripheral blood from four healthy donors were isolated by centrifugation according to increasing density (1,077 g ml ^-1 ) (Lymphoprep, AXIS-SHIELD PoC AS, Oslo, Norway). Cells were emulsified in RPMI 1640 culture medium (Sigma, Munich, Germany), supplemented with 10% (v / v) activated high temperature (56 ° C, 1 h) fetal bovine serum, Gentamicin (50 μg ml ^-1 ) (Sigma), streptomycin with penicillin (1%) and sodium pyruvate solution (0.23 mmol L ^-1 ) (Sigma) (complete medium). All components were purchased tested for endotoxin. All dosage dependence tests 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 × 10 ⁶ mononuclear cells and 5, 10, 25, 50, 75 and 100 μg of genomic DNA per ml. They showed that maximum suppression could be observed with 75 μg ml ^-1 DNA. This concentration was used in a further experiment. Cells were cultured in complete medium at a concentration of 2 * 10 ⁶ cells ml ^-1 in a 24-well plate (Nune, Roskilde, Denmark). At the same time, genomic DNA (75 μg ml ^-1 ) of probiotic bacteria, genomic DNA from calf (75 μg ml ^-1 Sigma, Munich, Germany) SEA (2 μg ml ^-1) and LPS from E. coli (20 μg ml ^-1 , Sigma, Munich, Germany) were added to the culture and incubated at 37 ° C, 5% CO ₂ -moistening for 24 hours. All experiments were performed in triplicate. After a 24-hour incubation, cell-free supernatants were collected and centrifuged at 1000g for 20 minutes at 4 ° C, sterilized by passing through a 0.2 μm pore filter (Millipore, Germany) and stored at -80 ° C in certain quantities up to analysis. Cell viability was determined before and after incubation with genomic DNA by elimination with trypan blue. In all experiments, 95-98% of PBMCs were viable. Dosage-dependent experiments performed on all cytokines showed that maximum suppression was observed at a concentration of 75 μg ml ^-1 . This concentration was used in further experiments.

2.4. Cytokine Assay by Enzyme-linked Immunosorbent Assay (ELISA)

The concentrations of IL-4, IL-5 and IFN-γ were quantified in cell-free supernatants using a special ELISA (BD OptEiA ™ kit of IL-4, 5 and human IFN-γ, Heidelberg, Germany). The sensitivity of the study was 0.5 μg ml ^-1 for IL-4, 0.5 μg ml ^-1 for IL-5 and 1 μg ml ^-1 for IFN-γ. The optical density values of the samples were read at 450 and 570 nm using an ELISA anode electric reactor. The experiments were repeated at least twice and performed in triplicate.

2.5. Statistical analysis

The experimental test data were produced by ± S.E.M and a non-parameterized statistical analysis was performed with a t-test. P values of less than 0.05 were considered statistically significant.

results

Lactobacillus and Bifidobacterium suppress IL-4 and IL-5 and initiate IFN-γ production via SEA or Dpt-stimulated PBMCs from healthy donors. It was shown that streptococcal superantigens cause a high concentration of IL-4 and IL-5 from PBMC from healthy donors, and lactic acid bacteria killed by high temperature were able to suppress the production of a type-2 cytokine profile. In addition, the study showed that allergic patients had elevated levels of IL-4 and IL-5. Dermatophagoides pteronyssinus, Dpt (in 83.8%) and Dermatophagoides farinae, Df (at 78.4%) are the most common causative allergens in patients with allergic rhinitis. Now we have confirmed these observations with another superantigen - staphylococcal enterotoxin A (SEA) and Dpt.

When live bacterial strains of Lactobacillus and Bifidobacterium were preincubated with PBMC, the resulting production of IL-4a by SEA and Dpt was greatly reduced compared to that induced by the positive control (without preincubation with Lactobacillus and Bifidobacterium). Interestingly, no significant suppression was observed when PBMCs were pre-incubated with a Gram-negative control strain TG1 (Figure 1). Although neither four strains of Lactobacillus and Bifidobacterium nor TG1 caused the main production of IL-4, all four strains caused the production of IFN-γ. When PBMCs with SEA and Dpt were stimulated, the concentration of IFN-γ output, in addition, greatly increased. A synergistic effect was observed not only with Lactobacillus and Bifidobacterium bacteria, but also with TG1. Therefore, Lactobacillus and Bifidobacterium are apparently able to influence the secretion of both T _n 1 and T _n 2 cytokines in opposite directions.

The production of cytokines depends on time and dosage.

Other experiments performed with four strains of Lactobacillus and Bifidobacterium showed that the suppression of T _n 2 cytokines depends on time and dosage. When PBMCs were stimulated with probiotic bacteria prior to SEA supplementation, the growth inhibitory effect on the production of T _n 2 cytokine increased with the ratio of bacteria to PBMC. The maximum growth inhibition was observed at 2 × 10 ⁷ CFU bacteria ml ^-1 , corresponding to a ratio of 10: 1 (bacteria to IUD) (figure 2, a and b).

This effect has also been observed for the production of IL-4 and IL-5. For example, for L. gasseri strain, the percentage of inhibition of growth in IL-4 production rose from 40.19% ± 3% (1: 1 ratio) to 54, 22% ± (10: 1 ratio) in response to SEA. The percentage of inhibition of IL-5 production increased from 43.77% ± 8% (1: 1 ratio) to 73.17% ± 5% (10: 1 ratio). In addition, the percentage of inhibition of IL-4 production in response to Dpt in PBMC from healthy donors increased from 34.61% ± 4% (ratio 1: 1) to 47.33% ± 5% (ratio) 10: 1. The percentage of inhibition of growth in IL-5 production increased from 45.72% ± 4% to 77.59% ± 6%. Unlike the T _n 2 cytokine, our tested strains caused the main production of IFN-γ, which, apparently, depends on the ratio of bacteria / cells. Remarkably, it was possible to show that probiotic bacteria significantly affect the production of IFN-γ as a response to SEA stimulation (figures 1 and 2, C).

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

PBMCs from healthy donors (n = 4 in 2 × 10 ⁶ ml ^-1 ) are stimulated with SEA (2 μg / ml). Mononuclear cells were either preincubated for 1, 3, and 5 hours with L.GG strains (10: 1 ratio) before incubation before stimulation with SEA, or they were simultaneously stimulated with L.GG strains, and superantigen or L.GG was added after 1, 3, and 5 hours after SEA stimulation, and cell cultures were preincubated for 24, 48 and 72 hours. At the aforementioned time values, cell-free supernatants were collected and, after centrifugation and sterilization, were stored at -20 ° C. Measured concentrations of IL-4 and IL-5 showed that maximum growth inhibition was observed for 3-hour pre-incubation of PBMCs with live bacteria before SEA stimulation. Similarly, maximum growth inhibition was observed for 48-hour incubation (data not shown). This gap was used in further experiments.

Lactobacillus and Bifidobacterium inhibit the production of T _n 2 cytokines from allergen-stimulated PBMCs from allergic patients.

The ability of Lactobacillus and Bifidobacterium to correct the imbalance between T _n 1 and T _n 2 cytokines is then evaluated in an even more relevant model of allergen excitation. When PBMCs from patients (allergic to D. pteronyssinus) are preincubated with various viable strains of Lactobacillus and Bifidobacterium, the production of IL-4 and IL-5, started after specific stimulation of D. pteronyssinus by allergens, is significantly reduced, especially depending on the bacterial / cell ratio. This effect on IL-5 is independent of the type of strain of the probiotic bacteria studied. The suppression of IL-5 was consistent, for example, in the case of B.b. - 57.31% ± 4.52% and in the case of BI - 63.77% ± 5% (figure 1).

In addition, similar effects were observed when PBMCs from allergic patients were stimulated by SEA. In this case, although the production of IL-4 and IL-5 is very high compared with the production that was observed with D. pteronyssinus (Figure 1), the tested probiotic bacteria reduced the secretion of IL-5 to 71.29% ± 4.10% for L.GG and up to 77.3% ± 3.52% for L..gasseri (Figure 1). Co-incubation with Lactobacillus and Bifidobacterium greatly increased the production of IFN-γ (Figure 1). Thus, by reducing the production of T _n 2 cytokines and intensifying the production of IFN-γ due to PBMCs from allergic patients, Lactobacillus and Bifidobacterium appear to exhibit anti-T _n 2 activity. In this context, it is interesting to note that the effect of the growth inhibitory Lactobacillus and Bifidobacterium on the production of IL-4 due to PBMC from allergic test subjects is greater than for healthy test subjects. In contrast, the growth inhibitory effect of these bacteria on IL-5 production due to PBMC from healthy donors is greater than for allergic test subjects (Figure 1). In contrast, the growth inhibitory effect of BI and LgsBbBI on IL-4 production due to Dpt-stimulated PBMCs from allergic test subjects is greater than that from healthy test subjects (p <0.05), and the inhibitory effect of L.GG, B .b. and BI for the production of IL-4 by SEA-stimulated PBMCs from allergic test subjects was greater than for healthy test subjects (p <0.01). In contrast, the growth inhibitory effect of L. gasseri, B.b, and B.1 on IL-5 production by Dpt-stimulated PBMCs from healthy test subjects was greater than for allergic test subjects (p <0.05), and the suppression effect LgsBbBI for the production of IL-5 from SEA-stimulated PBMCs from healthy test subjects was greater than for allergic patients (p <0.01, figure 1).

Genomic DNA from probiotic bacteria inhibits the production of IL-4 and IL-5 by SEA-stimulated PBMCs of healthy test subjects depending on time and dosage.

To evaluate the growth inhibitory effect of bacterial DNA compared with animal DNA and LPS, PBMCs from four healthy test subjects were incubated with genomic DNA from four strains of probiotic bacteria, L. GG, L. gasseri, B.b., B.1 and LgsBbBI ( a mixture of L-gasseri, B.b. and BI), LPS and animal DNA (calf thymus DNA). The suppression of T _n 2 cytokines was observed using BDoptEIA test kits to demonstrate and quantify cytokine production. As shown in FIG. 4, DNA from probiotic bacteria typically inhibits PBMCs to produce IL-4 and IL-5 as a response to stimulation by SEA or Dpt. In contrast, LPS did not reduce the production of IL-4 and IL-5; similarly, calf thymus LPS-free DNA did not reduce the production of IL-4 and IL-5 (data not shown).

The time-dependent effect of growth inhibitory bacterial DNA on the production of IL-4 and IL-5 due to SEA-stimulated PBMCs from healthy test subjects

Several experiments were performed to determine the time profile of the response to genomic DNA. PBMCs from healthy donors (n = 4, 2 × 10 ⁶ ml ^-1 ) were stimulated by SEA (2 μg ml ^-1 ) and either preincubated for one, three and six hours with genomic DNA from L.GG or B.b. (30 μg ml ^-1 ) before the SEA stimulus, or stimulated simultaneously with genomic DNA. Superantigen or genomic DNA was added 1, 3, and 6 hours after the SEA stimulus, and cell cultures were incubated for periods of 24, 48, and 72 hours. Genomic DNA from L.GG and B.b. selected as representative for two strains of Lactobacillus and Bifidobacterium. At the above time periods, cell-free supernatants were collected and stored after centrifugation and sterilization at -80 ° C. The measured concentrations of IL-4 and IL-5 showed that the maximum growth inhibition was observed at t _o hours of incubation of PBMCs with genomic DNA and SEA (data not shown), similarly, the maximum inhibition was achieved after incubation for 24 hours. This time was used for further experiments. Genomic DNA from B.b. has been confirmed as a more effective inhibitor of IL-4 than genomic DNA from L.GG. On the contrary, it was confirmed that genomic DNA from L.GG is a more effective inhibitor of IL-5 than genomic DNA from B.b.

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

In order to study the effect of growth-inhibiting bacterial DNA on the inhibition of IL-4 and IL-5 by SEA-stimulated PBMCs from healthy test subjects, genomic DNA from each strain was introduced into PBMC culture in various concentrations, namely in the range from 5 to 105 μg ml ^-1 to determine which dosage has the greatest inhibition effect on the production of IL-4 and IL-5 (Figure 4). This dosage range was selected based on the results of the tests described above, which showed that the production of cytokines by immune cells can be demonstrated after supplementing with at least 3 μg ml- ^{l of} E. coli. DNA and the optimal stimulation required by the 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 . Three other patterns of cytokine responses could be clearly differentiated. Genomic DNA: All strains suppressed the production of IL-4 and IL-5 at a concentration of 75 μg ml ^-1 except B. 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 DNAs suppressed the production of IL-4 and IL-5 to lower steady state levels when added in a concentration range of 5 to 45 μg ml ^-1 . In contrast, all genomic DNAs showed dosage-dependent intensification of IL-4 and IL-5 production when they were used in a concentration range from 85 to 105 μg ml ^-1 . Compared to other genomic DNAs, B.b and L.GG DNA are obviously more effective inhibitors of IL-4 and IL-5 production in response to SEA stimulation. In addition, genomic DNA from B.b. showed the lowest inhibitory effect on IL-5 production at a concentration of 105 μg ml ^-1 .

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

To investigate the growth inhibitory effect of genomic DNA from probiotic bacteria, PBMC (2 × 10 ⁶ ml ^-1 ) at 75 μg ml ^-1 , from L.GG DNA, Bi DNA and LgsBbBI, the DNA was incubated for 24 hours. Three other IL-4 production inhibition patterns in response to SEA stimulation could be clearly distinguished: genomic DNA from L.GG and LgsBbBI showed the strongest inhibition effect, namely 37.4% ± 4% and 39.56% ± 5. 1%, respectively. L.gasseri DNA showed the lowest inhibition effect (19.14% ± 2%) and BbBI showed a similar sample (24.47% ± 3.28%). In contrast, L. gasseri DNA shows the largest growth inhibitory effect on IL-5 production as a response to SEA stimulation (61.41% ± 3.74%), LgsBbBI DNA showed the lowest suppression (46.31% ± 4%) and genomic DNA from L. GG, B.b. and B. i showed an identical suppression pattern (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 on the production of IL-4 and IL-5 as a response to SEA stimulation between healthy and allergic test subjects. The growth inhibitory effect of living bacteria on IL-4 production by healthy and allergic test subjects is higher than the effect of their genomic DNA. In contrast, the growth inhibitory effect of genomic DNA in allergic test subjects is greater than in healthy test subjects (with the exception of L. gasseri). Therefore, genomic DNAs are more effective in reducing the production of IL-4 as a response to SEA excitation in allergic test subjects.

In short, the growth inhibitory effect of living bacteria on IL-5 production as a response to SEA excitation in PBMCs from healthy and allergic patients is the same as the effect on IL-4 production (for example, the inhibitory effect of living bacteria was higher than their genomic DNA in both healthy and allergic test subjects). But the growth inhibitory effect of genomic DNA on IL-5 production in healthy donors was greater than for allergic test subjects. Therefore, genomic DNAs are more effective inhibitors for IL-5 in healthy donors. Regarding Dpt excitation, the inhibitory effect of genomic DNA from L. GG and LgsBbBI on the production of IL-4 by Dpt-stimulated PBMCs from allergic test subjects was greater than in the case of healthy test subjects (p> 0.05). A similar sample 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. B.1 and LgsBbBI for the production of IL-5 from Dpt-stimulated PBMCs from healthy test subjects was greater than in the case of allergic test subjects. But the growth inhibitory effect of L.GG DNA on IL-5 production 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 PBMCs from healthy and allergic test subjects in the same way.

Discussion

Allergies are hypersensitive reactions by the immune system to specific substances called allergens (such as pollen, insect poisons, pharmaceuticals or food). Some experimental studies show that allergic diseases can be associated with an imbalance in T _n 1 / T _n 2 cytokines with a relative superiority of T _n 2 cytokines and a lack of T _n 1 cytokines. In fact, IL-4, IL-5, IL-3, and IL-9 are involved in triggering and maintaining allergic reactions. It is believed that T _n 2 cytokines are preferably involved in allergic diseases because a shift in the immune response of T _n 2 to T _n 1 is largely evident in most types of allergic diseases. The encouraging results obtained in the treatment of allergic patients with probiotic bacteria prompted us to explain the interaction between T _n 1 / T _n 2 cytokines and allergies. Therefore, one strategy in allergy therapy is to alter the T _n 1 / T _n 2 balance by prescribing probiotic bacteria to restore the T _n 1 / T _n 2 balance. Of particular interest are the results regarding the ability of living or killed probiotic bacteria to significantly reduce the amount of IL-4 and increase the amount of SFN-γ cytokines.

In addition, the present study proved that the protective effect of probiotic bacteria could be associated with the release of soluble factors that alter the permeability of the epithelium and protect against pathogenic bacterial invasions. These data raise the question of whether this soluble factor or other cytoplasmic bacterial components, such as DNA, could be involved in triggering and modulating cytokines. From this point of view, we investigated the effect of living bacteria and pure genomic DNA from L.GG L. gasseri, B.b., B.I. and LgasBbBI strains (mixture B L-gasseri. B. And B.1) on the release of IL cytokines -4 and IL-5 using human culture models. The results show that various strains of probiotic bacteria, as well as their genomic DNA, cause significant changes in the profile of cytokines, which are actively isolated in vitro from SEA or Dpt-stimulated PBMCs. In addition, they can modulate the T _n 1 / T _n 2 balance, reducing the production of T _n 2 cytokines and increasing the production of T _n 1 cytokines. Therefore, such probiotic bacteria can have a beneficial effect in allergic diseases as a result of their inhibition effect on the production of T _n 2 cytokines.

To investigate this effect, we first stimulated PBMCs from healthy and allergic test subjects with SEA or Dpt (as the T _n 2 cytokine-producing cell model). Numerous researchers have reported that PBMCs secrete T _n 2 cytokines after excitation with superantigens. When RVMS stimulated SEA or Dpt in this study was preincubated with live probiotic bacteria and their genomic DNA, the production of T _n 2 cytokines was reduced, but not in the presence of E. coli. In addition, this inhibition effect was dose dependent so that, at a concentration of 2 × 10 ⁷ CFU ml ^-1 (corresponding to a ratio of 10: 1 bacterial to PBMC), the effect of inhibition of live bacteria peaked with respect to IL-4 and IL-5 production. This dosage-dependent effect has also been reported in vitro for the production of 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 living probiotic bacteria, but also their genomic DNA can inhibit the production of IL-4 and IL-5 SEA- or Dpt-stimulated PBMCs. The most important point of this study is that, firstly, it presents the beneficial effect of genomic DNA or probiotic bacteria on the cells of allergic patients who are sensitive to house dust mites; that is, microscopic organisms that are found in homes. They are the main cause of dust allergies. All other reports are only related to the beneficial effects of living or killed probiotic bacteria on allergic diseases caused by food allergies or aero allergens. Our data suggest that probiotic bacteria and their genomic DNA act by direct or indirect regulation of the signaling pathway that is required to suppress T _n 2 cytokines, because an inhibition effect was observed even when probiotic bacteria and their genomic DNA were added before or simultaneously (for genomic DNA ) with SEA - or Dpt stimulation. Some experimental studies indicate that modulation of IL-4 and IL-5 production is a multifactorial process, and some cytokines such as IFN-γ are described as possible regulators of IL-4 production. In addition, it is reported that IL-12 is an outstanding cytokine in the challenge of producing IFN-γ by human PBMCs. In our study, probiotic bacteria and their genomic DNA are capable of increasing the production of IL-12 PBMCs (data not shown).

In this regard, bacterial DNA was added to the SEA stimulated PBMC culture and, interestingly, this experiment showed a diverse production of IL-4 and IL-5 cytokines after excitation of bacterial DNA and SEA treatment. Bacterial DNA from probiotic bacteria reduced the secretion of IL-5 much more than from IL-4. Our data indicate that genomic DNA from probiotic bacteria can act as an inhibitor of the production of IL-4a and IL-5 SEA- and Dpt-stimulated PBMCs from healthy and allergic test subjects. In addition, the present study reports that heat-killed probiotic bacteria reduce the production of IL-4 and IL-5 SEA stimulated PBMCs. Considering that the production process, the gastrointestinal tract and high temperatures (70 ° C) affect the viability of bacteria, the results of these studies show a possible effect of inhibiting genomic DANN, which is realized by bacteria killed by heat and added to PBMC. Various articles describe the response of various cytokines to CPG motifs that are present in bacterial DNA, such as genomic DNA from L.GG. It would be interesting to know if the strain-specific cytokine release demonstrated in this study is related to the genomic sequence and CPG motifs of each strain.

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

The observed differences in the rate and magnitude of inhibition of IL-4 and IL-5 as a response to bacterial DNA and SEA or Dpt excitation are interesting information on the effect of living or killed probiotic bacteria and / or bacterial components on the immune response. The growing knowledge of probiotics is exciting, but in the near future it should be established which probiotic DNA (individual strains or combination) has the strongest effect on specific diseases. It is further required that well-organized randomized clinical trials determine the role of genomic DNA from probiotic bacteria as prophylactic and therapeutic agents in the future.

Further details and features of the invention are explained in detail below in relation to examples with the results of a series of experiments. However, they are not intended to limit the invention, but only to explain it. In schematic form

Figure 1

Modulation of PBMC cytokine without (medium) or with stimulation of SEA and Dpt

Figure 2

Dosage-dependent effect of inhibition of live probiotic bacteria on

production of IL-4 (A), IL-5 (B) and (IFN-γ) SEA by stimulation of PBMCs of healthy test subjects.

Figure 3

Canceled

Figure 4

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

Figure 5

The dosage-dependent growth inhibitory effect of genomic DNA on the production of IL-4 (A), IL-5 (B) and IFN-γ (C) PBMCs from healthy test subjects.

In detail, the figures show:

Figure 1

Modulation of the PBMC cytokine without (medium) or with stimulation of SEA and Dpt.

RVMS from a healthy (A, n = 8) and allergic (B, n = 8) test subject were preincubated for 3 hours (at 2 × 10 ⁵ cells / ml) with medium without (RVMS) or with four strains of probiotic bacteria, Lgb.b. B.1 (a mixture of probiotic bacteria and gram-negative control bacteria (E-coli TG1) with a 10: 1 bacterial cell ratio before stimulation with SEA-superantigen (2 μg ml ^-1 ), Dpt (2 μg ml ^-1 ) or not.

IL-4, IL-5 and IFN-γ were quantified after hours of incubation in supernatants with a specific ELISA. Asterisks indicate specific suppression or stimulation (* p <0.05, ** p <0.01) of the production of T _n 1 and T _n 2 cytokines compared to the control.

Figure 2

The dosage-dependent effect of the inhibition of live probiotic bacteria on the production of IL-4 (A), IL-5 (B) and (IFN-γ) by SEA-stimulation of PBMCs in healthy test subjects. RVMS from four healthy donors (2 × 10 ⁶ ml- ¹ cells) were cultured with four strains of probiotic bacteria, L. GG, L. gasseri, Bb, BI at concentrations of 5 × 10 ⁴ , 5 × 10 ⁵ , 2 × 106, 5 × 10 ⁶ , 2 × 10 ⁷ , 5 × 10 ⁷ , corresponding to 0.025, 0.25, 1, 2.5, 5, 10 and 25 ratios of bacteria-to-cells for 3 hours before stimulation with 2 μg ml ^{- 1} SEA. After 48 hours of incubation, IL-4, IL-5 and IFN-γ were measured by a specific ELISA. The error message line shows the standard errors of the mean.

Figure 3 is canceled.

Figure 4

The growth inhibitory effect of genomic DNA from probiotic bacteria on the production of IL-4, IL-5 and IFN-γ SEA- or Dpt-stimulated PBMC from healthy (A, n = 5) and allergic (B, n = 5) test subjects. PBMCs from five healthy and five allergic test subjects were cultured for 48 hours (at 2 × 10 ⁶ ml ^-1 cells) with genomic DNA from 4 strains of probiotic bacteria, L.GG, L. gasseri, Bb, BI and LgBbBI (mixture L . gasseri, Bb and BI). At a concentration of 75 μg ml ^-1 before stimulation with SEA (2 μg ml ^-1 ) or Dpt (2000 SQ -EML ^-1 corresponding to the introduction of 2 μg per ml ^-1 ). Medium and LPS (100 ng ml ^-1 ) were used as a control. IL-4, IL-5 and IFN-γ were quantified after 24 hours of incubation in supernatants with a specific ELISA.

Asterisks indicate outstanding inhibition (* p <0.05, ** p <0.01) of cytokine production compared to the control (medium). Data are expressed as mean +/- SEM.

Figure 5

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

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

Claims (6)

1. A pharmaceutical preparation for the prevention, suppression or elimination of allergic reactions in humans, characterized in that the genomic DNA of probiotic, gram-positive bacteria and / or bacterial strains Lactobacillus gasseri PA 16/8, and / or Bifidobacterium bifidum MG 20 / 5, and / or Bifidobacterium longum SP 07/3 as viable and / or inactivated bacteria. 2. A pharmaceutical preparation for shifting the balance of Tn1-Tn2 in the human body in the direction of increasing Tn1 and / or decreasing Tn2, characterized in that the genomic DNA of probiotic, gram-positive bacteria and / or bacterial strains Lactobacillus gasseri PA 16/8 are present, and / or Bifidobacterium bifidum MG 20/5, and / or Bifidobacterium longum SP 07/3 as viable and / or inactivated bacteria. 3. A food product or an additive to a food product, characterized in that the genomic DNA of probiotic, gram-positive bacteria and / or bacterial strains of Lactobacillus gasseri PA 16/8 and / or Bifidobacterium bifidum MG 20/5 and / or Bifidobacterium longum SP 07/3 as viable and / or inactivated bacteria. 4. The use of genomic DNA of probiotic, gram-positive bacteria and / or strains of bacteria Lactobacillus gasseri PA 16/8, and / or Bifidobacterium bifidum MG 20/5, and / or Bifidobacterium longum SP 07/3 as viable and / or inactivated bacteria for the treatment of and / or prevention and / or reduction of the risk of allergic reactions of a person. 5. The use of genomic DNA of probiotic, gram-positive bacteria and / or strains of bacteria Lactobacillus gasseri PA 16/8, and / or Bifidobacterium bifidum MG 20/5, and / or Bifidobacterium longum SP 07/3 as viable and / or inactivated bacteria for the treatment of and / or prophylaxis and / or reduction of the risk of Tn1-Tn2 shift in the human body in the direction of increasing Tn1 and / or decreasing Tn2. 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 for subcutaneous injection, or for intravenous injection, or as a liquid for inhalation.

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