PL212183B1 - Application of Lactobacillus casei i paracasei - Google Patents

Description

The subject of the invention is the use of new Lactobacillus casei LOCK 0900, Lactobacillus casei LOCK 0908 and Lactobacillus paracasei LOCK 0919 strains in the prevention and treatment of allergic diseases.

It is known from US patent US 2004/0029127 to use bacterial strains of the genus Lactobacillus casei to prepare a composition that can be used orally in order to enhance the reaction of a specific immune system to a pathogenic agent. Said pathogenic agent is a pathogen of the respiratory system, in particular a pathogenic virus belonging to rhinoviruses, respiratory scyntial viruses and myxoviruses.

The mentioned Lactobacillus casei strain is the CNCM 1-1518 strain, which is an ingredient of food or dietary supplement.

Lactobacillus casei and paracasei strains are known, the characteristics of which are presented in Polish patent applications P-382760 Lactobacillus casei strain no. LOCK 0900, P-382761 Lactobacillus casei no. LOCK 0908, P- 382762 Lactobacillus paracasei no. LOCK 0919.

It was surprisingly found that the strains of Lactobacillus casei and paracasei presented in these applications have an influence on the production of cytokines, i.e. proteins that play a fundamental role in the pathogenesis of allergic diseases. In IgE-dependent allergy (in atopic diseases), the cytokine balance is disturbed towards activation of the so-called proallergic cytokines (IL-4, IL-5) produced by TH2 lymphocytes. The condition is due to the lack of stimulation of the production of pro-inflammatory cytokines such as IFN-gamma, TNF-α, IL-12. Proallergic cytokines are responsible for the production of IgE and the activation of eosinophils directly responsible for atopic reactions.

The essence of the invention is the use of Lactobacillus casei strains LOCK deposit No. 0900, Lactobacillus casei deposit No. LOCK 0908 and Lactobacillus paracasei deposit No. LOCK 0919 showing the ability to activate the pro-inflammatory cytokine profile and the ability to inhibit the pro-allergic cytokine IL-5 for the production of a nutritional or pharmaceutical composition for prophylaxis and / or treatment of allergic diseases.

I Study of the translocation of Lactobacillus casei ŁOCK 0908 and ŁOCK 0900 and Lactobacillus paracasei ŁOCK 0919 strains into the blood and internal organs

Experimental mouse model

The mixture of Lactobacillus casei and paracasei bacteria was administered to 2-month-old Balb / c mice (n = 5) for 7 days. Bacteria diluted in pH 7.4 phosphate buffer were administered via the gavage intragastrically twice a day in 0.5 ml in a daily dose of 10 ⁹ cfu. Control mice (n = 5) received the vehicle in which the bacteria were suspended.

Assessment of translocation across the intestinal barrier

In order to investigate the translocation of bacteria across the intestinal barrier, the presence of Lactobacillus bacteria in the mesenteric lymph nodes, blood and internal organs (spleen, liver) was assessed. Blood was collected via intracardiac puncture and then plated on MRS agar. Internal organs (whole liver, half spleen from each mouse) and all combined lymph nodes isolated from each group of mice were weighed, homogenate made, diluted 1: 5 in phosphate buffer and plated on MRS agar. The plates were incubated for 72 h at 37 ° C and then the number of colony forming bacteria was counted.

Lymphocyte cultures

Lymphocytes were isolated from the spleen and crushed in RPMI medium (Sigma). The homogenate was filtered through nylon mesh, the cells were washed 3 times in RPMI medium and resuspended at 5x10 ⁷ cfu / ml complete.

RPMI medium with 10% fetal calf serum (Sigma) and penicillin with streptomycin (Sigma). Cell viability as assessed by trypan blue (Sigma) was> 90%. Cultures were performed in 96-well plates (Nunc). To 100 ml of the cells, 100 ml of a mixture of heat-inactivated bacteria (incubated for 60 min. At 96 ° C) at a concentration of 10 ⁷ ml and 100 ml of medium. Concentration 5 µg / ml Concanavalin A (Con A) (Sigma) was used as a positive control, and a culture medium as a negative control. 10 cultures were set up for the mixture, ConA and the culture medium alone. Cultures were incubated in a humid containing atmosphere

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5% (v / v) CO2 for 72h at 37 ^° C. After this period, culture supernatants were collected, duplicate cultures were mixed and frozen at -56 ^° C for further studies.

Detection of cytokine levels in culture supernatants

The levels of interleukin (IL) -5, tumor necrosis factor (TNF) -alpha, and interferon (IFN) -gamma were assessed in the supernatants. The level of individual cytokines was determined by enzyme immunoassay (ELISA) method using commercial DuoSet kits from R&D Systems according to the manufacturer's instructions.

Results

Translocation of bacteria across the intestinal barrier

No pathological symptoms were observed after administration of the Lactobacillus bacterial mixture; mice gained weight comparably in both study groups. Bacteria have not been grown from the blood and internal organs (liver and spleen). In contrast, in the mesenteric lymph nodes, 250 colony forming units (CFU) of Lactobacillus were found in the group receiving the bacteria. Bacteria were not isolated from the lymph nodes isolated from the control group.

Conclusions

Intestinal bacteria are safe and do not translocate into the blood and internal organs. Lactobacillus casei and paracasei bacteria selectively pass to the mesenteric lymph nodes where they constitute an antigenic reservoir that specifically activates the immune system.

II. Study of the influence of Lactobacillus casei and paracasei strains on the production of cytokines.

Production of cytokines

ConA and Lactobacillus casei and paracasei bacteria stimulated the production of cytokines in cell cultures. No differences in IL-5, TNF-alpha and IFN-gamma production were observed between groups when the ConA stimulated cultures were assessed. On the other hand, Lactobacillus bacteria stimulated statistically significant production of pro-inflammatory cytokines TNF-alpha and IFN-gamma in cultures of lymphocytes isolated from mice inhabited with a mixture of Lactobacillus bacteria compared to cultures obtained from control mice. In contrast to the pro-inflammatory cytokines, proallergic IL-5 after stimulation with Lactobacillus bacteria was produced in much lower amounts in the group receiving the bacteria compared to the control group (Table 1).

T a b e l a 1

Production of cytokines in cultures of lymphocytes stimulated with a mixture of Lactobacillus casei and paracasei (Bm) bacteria and concanavalin A (ConA) in the group of mice inhabited with bacteria and in the control group

Mice colonized with bacteria Control mice TNF-alpha Bm 1012 ± 223 * 881 ± 132 What for 449 ± 85 444 ± 93 INF-gamma Bm 442 ± 98 * 151 ± 23 What for 4054 ± 1002 4001 ± 987 IL-5 Bm 29 ± 3 # 37 ± 3 What for 1146 + 289 1285 ± 298

* statistically significantly higher production of pro-inflammatory cytokines in the group of settled mice as compared to control mice for TNF-alpha p = 0.04, for IFN-gamma p = 0.01.

^# statistically significantly lower production of proallergic IL-5 in the group of settled mice compared to control mice (p = 0.04).

Conclusions

Lactobacillus casei and paracasei bacteria activate the pro-inflammatory cytokine profile, while they inhibit the pro-allergic cytokine IL-5.

The obtained results confirm the probiotic features of the mixture of Lactobacillus bacteria, with the possibility of using the strains in the prevention and / or treatment of atopic diseases.

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III The influence of Lactobacillus casei and paracasei bacteria on the cytokine profile of blood cells isolated from children with IgE-dependent and IgE-independent allergies

In order to investigate the effect of the mixture of Lactobacillus casei and paracasei bacteria strains on cytokine production in allergic children, an in vitro model using whole blood cell cultures was used. The activation of the cytokine profile was investigated by assessing the level of pro-inflammatory cytokines (interleukin (IL) -12, IL-18, interferon (IFN) -gamma, tumor necrosis factor (TNF) -alpha), proallergic (IL-5) and immune response regulators (transforming factor growth (TGF) -beta1) in the culture supernatants.

The study included 22 children with symptoms of atopic eczema due to allergy to cow's milk proteins, aged 3-15 months (mean age 9 months). On the basis of serum IgE level and determination of specific IgE antibodies against the panel of inhaled and food allergens, children were divided into 2 groups: with IgE-dependent eczema (presence of specific antibodies and increased IgE level) and IgE-independent (IgE level normal) , no specific antibodies). IgE-dependent allergy was found in 12th, IgE-independent allergy in the remaining 10th.

Cell cultures were prepared from whole blood. Heparin blood was diluted 1: 5 in RPMI-1640 culture medium (Sigma) free of calf serum containing streptomycin / penicillin (Sigma). 150 mg of diluted blood was applied to a 96-well culture dish (Nunc), followed by addition of 150 μΐ heat-inactivated bacteria in a final dilution of 2.5 x 10 ⁶ ml. As a positive control, a polyclonal activator of cytokine production - phytohaemagglutinin (PHA) (Disco Laboratories, Detroit, USA) was used at 150 μΐ / well at a final dose of 10 μg / mL. As a negative control, 150 µΐ / well of culture medium (unstimulated cells) was used. For each child, 5 cultures were established for the mixture of strains, PHA and the culture medium itself. Cultures were incubated in a humid atmosphere containing 5% (v / v) CO2 for 72h at 37 ° C. After this period, the culture supernatants were harvested, duplicate cultures were mixed and frozen at -56 ^° C for further testing.

Determination of cytokines in supernatants

The levels of individual cytokines in the supernatants were determined by enzyme immunoassay (ELISA) method using commercial DuoSet kits from R&D Systems according to the manufacturer's instructions. The sensitivity of the DuoSet test for individual cytokines is presented in Table 2. IL-18 levels were assessed using the MBL reagent kit (Nagoya) according to the manufacturer's instructions. The sensitivity of the test was> 12.5 pg / ml.

T a b e ΐ a 2

The sensitivity of the DuoSet test for individual cytokines

Type of test Sensitivity (pg / ml) IL-5 > 11.7 IL-12 > 15.5 IFN-gamma > 7.8 TNF-alpha > 7.8 TGF-beta1 > 15.5

Statistical analysis

Statistical analysis was performed using the Mann-Whitney test, p <0.05 was considered statistically significant.

Results

Production of proallergic cytokines

The level of IL-5 in the cultures activated with the mixture of bacteria remained below the sensitivity of the assay, while PHA stimulated the secretion of this cytokine in both IgE-dependent and IgE-independent allergy children (Table 3).

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T a b e l a 3

Level of IL-5 in supernatants obtained from cultures of blood cells stimulated with Lactobacillus cesei and paracasei (Bm) bacteria and phytohaemagglutinin (PHA) in children with IgE-dependent and IgE-independent allergy

Activator IgE allergy-dependent (n = 12) IgE-independent allergy (n = 10) PHA 2617 ± 787 1279 369 Bm * N.D. N.D. The breeding medium N.D. N.D.

The results are presented as the arithmetic mean ± standard deviation. * Level of IL-5 not measurable (below the sensitivity of the test).

Production of pro-inflammatory cytokines

Lactobacillus casei and paracasei bacteria activated the production of all pro-inflammatory cytokines in children with IgE-dependent and IgE-independent allergy, except for IL-18 in children with IgE-independent allergy, when compared to the negative control.

No statistically significant differences were found between the IgE-dependent and IgE-independent groups in the activation of TNF-alpha (Table 4) and IFN-gamma (Table 5), although in both groups the mixture of bacteria stimulated the production of these cytokines much more strongly than PHA (without obtaining significance statistical).

Contrary to the above mentioned cytokines, the bacteria activated the production of IL-12 more significantly than PHA, mainly in the group of children with IgE-dependent allergy (p <0.0001), and to a lesser extent with IgE-independent allergy (p = 0.0426 ) (Table 6).

T a b e l a 4

TNF-alpha level in supernatants obtained from cultures of blood cells stimulated with Lactobacillus casei and paracasei (Bm) bacteria and phytohaemagglutinin (PHA) in children with IgE-dependent and IgE-independent allergies

Activator IgE allergy-dependent (n = 12) IgE allergy - independent (n = 10) Bm 2559 ± 603 1951 ± 846 PHA 1508 ± 312 1410 ± 597 The breeding medium * N.D. N.D.

The results are presented as the arithmetic mean ± standard deviation.

* TNF-alpha level not measurable (below test sensitivity).

T a b e l a 5

IFN-gamma level in supernatants obtained from cultures of blood cells stimulated with Lactobacillus casei and paracasei (Bm) bacteria and phytohaemagglutinin (PHA) in children with IgE-dependent and IgE-independent allergies

Activator IgE allergy-dependent (n = 12) IgE-independent allergy (n = 10) Bm 1752 ± 367 1583 ± 283 PHA 632 ± 198 990 ± 223 The breeding medium * N.D. N.D.

The results are presented as the arithmetic mean ± standard deviation. * INF-gamma level not measurable (below the sensitivity of the test).

T a b e l a 6

Level of IL-12 in supernatants obtained from cultures of blood cells stimulated with Lactobacillus casei and paracasei (Bm) bacteria and phytohaemagglutinin (PHA) in children with IgE-dependent and IgE-independent allergies.

Activator IgE-mediated allergy (n = 12) IgE-independent allergy (n = 10) Bm 1337 ± 228 # 1605 ± 716 # PHA 182 ± 23 173 ± 18 The breeding medium N.D. * N.D.

The results are presented as the arithmetic mean ± standard deviation.

* Level of IL-12 not measurable (below the sensitivity of the test).

^# statistical significance of Bm versus PHA; p <0.0001 for IgE-mediated allergy, p = 0.0426 for IgE-independent allergy.

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The bacteria also statistically stimulated the production of IL-18, but only in the group of children with IgE-dependent allergy, both compared to PHA (p = 0.03) and unstimulated cells (p = 0.02). It seems that children with IgE-dependent allergy have a significantly reduced ability to produce IL-18, because polyclonal PHA stimulation leads to statistically significantly lower IL-18 secretion compared to unstimulated control cells. Such an effect was not observed in the group of children with IgE-independent allergy. In the group of children with IgE-independent allergy, bacteria caused increased production of IL-18, but the difference compared to the unstimulated control did not reach statistical significance (Table 7).

T a b e l a 7

Level of IL-18 in supernatants obtained from cultures of blood cells stimulated with Lactobacillus casei and paracasei (Bm) bacteria and phytohaemagglutinin (PHA) in children with IgE-dependent and IgE-independent allergies.

Activator IgE-mediated allergy (n = 12) IgE-independent allergy (n = 10) Bm 78 ± 25.6 * # 65 ± 22.5 PHA 47.8 ± 13.3 57.8 ± 15.2 The breeding medium 49.8 ± 12.4 53.3 ± 14.6

The results are presented as the arithmetic mean ± standard deviation.

* statistical significance of Bm versus PHA only in IgE-dependent allergy, p = 0.03;

^# statistical significance of Bm versus culture medium (control) only in IgE-mediated allergy, p = 0.02.

Regulatory cytokine production

Lactobcillus casei and paracasei bacteria activated the production of TGF-beta1 in children with IgE-dependent and IgE-independent allergies in a statistically significant way compared to the negative control. The level of TGF-beta1 was statistically significantly higher in cultures stimulated with bacteria than

PHA (Table 8).

T a b e l a 8

TGF-betal level in supernatants obtained from cultures of blood cells stimulated with Lactobacillus casei and paracasei (Bm) bacteria and phytohaemagglutinin (PHA) in children with IgE-dependent and IgE-independent allergies

Activator IgE allergy-dependent (n = 12) IgE-independent allergy (n = 10) Bm 3711 ± 803 * # 3535 ± 1200 * # PHA 1834 ± 325 1729 ± 691 The breeding medium 1145 ± 242 757 ± 155

The results are presented as the arithmetic mean ± standard deviation.

* statistical significance of Bm versus PHA in IgE-dependent (p = 0.04) and IgE-independent (p = 0.04) allergy.

^# statistical significance of Bm versus culture medium (control) in IgE-dependent allergy (p = 0.01) and in IgE-independent allergy (p = 0.001).

Results

The mixture of bacteria, regardless of the type of allergy (IgE-dependent and independent), stimulates blood cells to produce mainly pro-inflammatory cytokines and the factor regulating the immune response, TGF-beta1.

Only in the group of children with IgE-dependent allergy the mixture of bacteria activates the production of pro-inflammatory IL-18.

The mixture of bacteria does not activate proallergic IL-5 regardless of the type of allergy.

Conclusions

The mixture of Lactobacillus casei and paracasei bacteria has features that activate the cytokine profile of the immune system towards a pro-inflammatory response in children with IgE-dependent and IgE-independent allergies, which indicates the possibility of using the above-mentioned bacteria in the treatment and / or prevention of allergic diseases. Taking into account the pathomechanism of allergy, the mixture of strains may have a clinical effect mainly in children with IgE-dependent (atopy) allergy.

Due to the ability to activate the cytokine regulating the immune response, bacterial strains can be used both in people with IgE-dependent and IgE-independent allergies.

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Execution examples.

P r z k ł a d I

The composition contains a lyophilized suspension of 10 ⁹ Lactobacillus casei ŁOCK 0900, Lactobacillus casei ŁOCK 0908, Lactobacillus paracasei ŁOCK 0919, maltodextrin and ascorbic acid. The composition is packaged in sachets or capsules.

P r z x l a d II

The composition contains a lyophilized suspension of 10 ⁹ Lactobacillus casei ŁOCK 0900, Lactobacillus casei ŁOCK 0908, Lactobacillus paracasei ŁOCK 0919, maltodextrin, inositol and ascorbic acid. The composition is packaged in sachets or capsules.

The composition is intended for use in infants and children with an increased risk of allergic reactions, including atopic dermatitis.

Claims (1)

The use of Lactobacillus casei deposit number LOCK 0900, Lactobacillus casei deposit number LOCK 0908 and Lactobacillus paracasei deposit number LOCK 0919 showing the ability to activate the pro-inflammatory cytokine profile and the ability to inhibit the proallergic cytokine IL-5 for the production of a nutritional or pharmaceutical composition for the prevention and / or treatment of allergic diseases.