Lactobacillus johnsonii la1 ncc533 (cncm 1-1225) and immune disorders

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates for composition for treatment or prevention of disorders, associated with reduced level of defensins. Composition contains from 0.005 to 1000 mg of Lactobacillus johnsonii Lal (NCC533, No CNCM 1-1225) per a daily dose, with at least 90% of L. johnsonii Lal (NCC533, No CNCM 1-1225) being transferred into state in which they become non-replicating at temperature110-140°C for 5-30 s.

EFFECT: invention provides enhancement of expression of defensin hBD1 mRNA.

5 cl, 2 dwg

 

The present invention generally relates to the prevention and/or treatment of inflammatory and infectious diseases, in particular by boosting the endogenous antimicrobial defenses. One variant of the present invention is the use of nonreplicating L. johnsonii (NCC533 (number CNCM 1-1225) in the treatment or prevention of disorders associated with the immune system including infections.

The environment is polluted a large number of potentially pathogenic microorganisms. The skin keratinocytes, epithelial cells lining the gastrointestinal tract, respiratory tract, urogenital tract provides a physical barrier that protects against invasion of microbes into the body.

In addition, the epithelium contributes to the protection of the host through production and secretion of antimicrobial substances, limiting the access of bacteria and other microorganisms. These antimicrobial molecules are key components of the main line of defense of innate immunity.

One of the most important classes of antimicrobial peptides in humans are defensive. Defensin produced by epithelial cells of the lung, skin, oral cavity, urogenital tract, respiratory tract and gastrointestinal tract. Extras there is a family of β-defensins, including defensin 1 (hBD1) and 2 (hBD2)

hBD1 is expressed on the surfaces of various mucous membranes, such as the mucous membranes of the oral cavity, salivary glands, stomach, small intestine, large intestine, liver and pancreas. hBD2 is also present in the epithelial cells on the numerous surfaces of the mucous membranes, including mucous membrane of the gastro-intestinal tract. Moreover, these two defensin is present in the saliva and in the liquid on the surface of the respiratory tract (Cunliffe, R. N. and Mahida, Y. R. 2004, J Leukoc.Biol. 75:49-58).

hBD1 is expressed constitutive, thus it was not shown that he constitutive activated by bacteria or inflammation (Ou, G., et al., 2009, Scand.J Immunol 69:150-161).

It is well known that probiotics can strengthen the different line of intestinal defense: immune exclusion (elimination), immune clearance and immune regulation. It is also known that probiotics stimulate nonspecific host resistance to microbial pathogens and thus contribute to their destruction.

However, despite this, it was reported that the constitutive expression of hBD1 is unaffected by probiotic bacteria (O'neil, D. A. et al., J Immunol 163:6718-6724) and very weakly activated symbiotic (Escherichia coli) and pathogenic (Salmonella typhimurium) strains (Ou, G., et al., 2009, Scand.J Immunol 69:150-161).

At present, the use of probiotics is aimed at reducing the risk of morbidity�rd, associated with impaired barrier function of the intestine (E. Isolauri, et al, 2002, Gut 2002; 50:54 iii-iii 59). It is believed that probiotics are effective due to survival in the intestine, stability to acid and bile and temporal colonization (settlement) of the mucous surfaces in the intestinal tract.

Accordingly, the vast majority of published literature is devoted to living probiotics. However, some studies have investigated the health benefits derived from nonreplicating bacteria, and most of them showed that inactivation of probiotics, e.g. by a thermal treatment leads to the loss of a perceived health benefit (Rachmilewitz, D., et al., 2004, Gastroenterology 126:520-528; Castagliuolo, et al., 2005, FEMS Immunol. Med. Microbiol. 43:197-204; Gill, H. S. and K. J. Rutherford, 2001, Br.J. Nutr. 86:285-289; Kaila, M., et al., 1995, Arch. Dis.Child 72:51-53).

Currently the use of live bacteria in food products has some drawbacks. Live bacteria are usually not resistant to stress and are therefore difficult to use in industrial scale, while maintaining their viability. In addition, for some categories of products the addition of live microorganisms to the composition is not optimal due to problems of security. Therefore, a need exists in bioactive non-living microorganisms.

The provision of nonreplicating breaking�quarter of microorganisms would allow dilution (reduction) at an elevated temperature, for example, a powdered nutritional composition, which is preferred, at the same time ensuring the preservation of properties that are useful for the health of the patient-consumer. On this basis, it would be desirable to use nonreplicating bacteria instead of their live counterparts, but studies on this aspect are not encouraging.

In the literature reported on the use of live probiotics as a strategy for treatment or prevention of inflammatory bowel disease, and was recently Dotan et al. published review (Dotan, I. and D. Rachmilewitz. 2005; Curr. Opin. Gastroenterol. 21:42 6-25 430). For example, it was shown that a highly concentrated blend of eight live probiotic bacteria (VSL#3) is effective to prevent (Gionchetti, P., et al, 2003, Gastroenterology 124:1202-1209) and the treatment of recurrent or refractory of paucity people (Gionchetti, P., et al., 2000, Gastroenterology 119:305-309; Mimura, T., et al., 2004, Gut 53:108-114). Interestingly, Rachmilewitz et al. (Rachmilewitz, D., et al., 2004, Gastroenterology 12 6:520-528) reported that when using a mouse model of colitis induced DSS, processing viable and γ-irradiated VSL#3, but not killed by heating VSL#3, protected from colitis. Similarly, dead heat L. crispatus was not protective against DSS-induced colitis, while their viable counterparts have reduced weight loss and the activity of MPO in the intestine (Castagliuolo, et a., 2005, FEMS Immunol.Med.Microbiol. 43:197-204). These studies suggest that probiotics are more effective in living form in the case of intestinal inflammation than their nonreplicating counterparts.

It has been shown that inactivated L. reuteri (killed by heating and γ-irradiated) is not able to decrease TNFα-induced production of IL-8 T84 cells, while their live counterparts demonstrated a significant positive effect (MA, D., et al., 2004, Infect.Immun. 72:5308-5314).

Therefore, in the art there is a need for natural compositions that are easy to work in industrial environments that are safe and convenient to use and which will give the possibility of preventing and/or treating inflammatory and infectious disorders, in particular by boosting the endogenous antimicrobial defenses.

In the ideal case of a natural arrangement must be made with probiotic cultures, in particular probiotic microorganisms, which are now generally accepted and recognized by consumers as beneficial to health. Advantageously, the composition should contain nonreplicating bacteria and should be more effective than a composition comprising their live counterparts.

The present inventors paid attention to existence beyond�of such requirements.

Therefore, the objective of the present invention was to improve the prior art and to provide a natural composition, which would make possible the prevention and/or treatment of inflammatory or infectious diseases, in particular by boosting the endogenous antimicrobial defenses, and which would meet the above requirements.

The inventors unexpectedly found that the present invention can be solved by using objects disclosed in the independent claims. The dependent claims further define the preferable embodiments of the present invention.

The objects of the present invention enhance endogenous antimicrobial defenses of mammals by introduction of a product containing microorganisms, such as nonreplicating microorganisms, such as heat-treated microorganisms.

The inventors have reported that L. johnsonii (Lal, NCC 533, No. CNCM 1-1225), in particular nonreplicating L. johnsonii (Lal, NCC 533, No. CNCM 1-1225), for example heat-treated L. johnsonii (Lal, NCC 533, No. CNCM 1-1225), have a surpassing impact on the induction of the expression of antimicrobial peptides in comparison with microorganisms that have been previously established and described in the literature.

For example, it was found that:

- L. johnsonii (al, NCC 533, No. CNCM 1-1225) strongly stimulates constitutive expression of hBD1, and that

- heat-treated L. johnsonii (Lal, NCC 533, No. CNCM 1-1225) activate hBD1 more strongly than their live counterparts.

HBD1 exhibits antibacterial activity against a broad spectrum of bacteria, including E. coli and Pseudomonas aeruginosa, H. pylori (Nuding.S. et al., 2009, Microbes.Infect. 11: 384-393), and also against yeasts, such as Candida albicans (O'neil, D. A. 2003, Mol. Immunol 40:445-450), and viruses (human immunodeficiency virus) (Kota, S. et al., 2008, J. Biol. Chem 283:22417-22429). Thus, these antimicrobial peptides may enhance mucosal barrier and as a result to limit bacterial adhesion and invasion.

More and more facts show that the levels of defensins reduced under certain pathophysiological conditions and that it is a risk factor for the development of infectious and inflammatory diseases and their complications such as (Doss, M. et al., 2010, J Leukoc.Biol. 87: 79-92); Rivas-Santiago, B. et al., 2009, Infect. Immun. 77:4690-4695):

- airway:

cystic fibrosis, reactive airway diseases, pulmonary infections, Smoking, asthma, pneumonia, rhinitis, otitis, sinusitis, tuberculosis;

- in the gastro-intestinal tract:

Crohn's disease (colon and ileum), ulcerative colitis, gastritis and stomach ulcers caused by Helicobacter pylori infection, infectious diarrhea, necrotising e�of caracolito, diarrhea associated with antibiotics, celiac disease, underdevelopment of the bowel;

- in the urogenital tract:

bacterial vaginosis, infection with human immunodeficiency virus (HIV), herpes simplex virus, urinary tract infection;

- on the skin:

atopic dermatitis, chronic ulcer, carcinoma, atopic eczema, burn lesions;

- mouth:

disease in patients with HIV, tonsillitis, gingivitis, dental caries;

- keratitis of the eye.

Presented in this description, the results show that L. Johnsonii Lal (NCC533, NO. CNCM 1-1225) have a stronger ability to increase endogenous antibacterial protection than previously identified probiotic bacteria, and thus can be more effective for the prevention and treatment of bacterial overgrowth in the small intestine (SIBO), inflammatory and infectious diseases.

In addition to this data, the inventors show (in contrast to what might be expected based on the prior art) that thermal treatment does not reduce, and further increases the strong antibacterial effect of L. johnsonii Lal (NCC533, NO. CNCM 1-1225).

One embodiment of the invention is a composition comprising L. johnsonii Lal (NCC533, NO. CNCM 1-1225), for use in the treatment or prevention of disorders associated with immune�th system, including infections.

According to the present invention disorders associated with the immune system, can be treated or prevented by increasing the endogenous expression of hBD1.

The present invention also relates to compositions containing L. johnsonii Lal (NCC533, NO. CNCM 1-1225), for the treatment or prevention of disorders associated with decreased expression of hBD1, for example, microbial infections.

The present invention also relates to the use of L. Johnsonii Lal (NCC533, NO. CNCM 1-1225) for the preparation of a composition for the treatment or prevention of disorders associated with the immune system.

Can be used, at least partially, nonreplicating L. johnsonii Lal (NCC533, NO. CNCM 1-1225). Nonreplicating, in particular heat-treated L. johnsonii Lal (NCC533, NO. CNCM 1-1225) have the advantage of being even more effective than their live counterparts.

The use of nonreplicating microorganisms, such as heat-treated L. johnsonii Lal (NCC533, NO. CNCM 1-1225), instead of their live counterparts has additional advantages:

- reducing the potential risk of sepsis associated with live probiotics, in sensitive populations,

- providing a safe alternative for patients with weakened immune systems and

- reduce problems during processing can result in storage stable liquid products�you with a long shelf life.

Thus, in one embodiment of the present invention, at least 90%, e.g. at least 95%, preferably at least 98%, most preferably at least 99%, ideally at least 99.9% of or all of L. johnsonii Lal (NCC533, NO. CNCM 1-1225) are nonreplicating.

The present invention also relates to compositions containing L. johnsonii Lal (NCC533, NO. CNCM 1-1225) in which at least 95%, preferably at least 98%, most preferably at least 99%, ideally at least 99.9% of or 100% of L. johnsonii Lal (NCC533, NO. CNCM 1-1225) are nonreplicating.

Thus, the present invention also relates to bioactive, nonreplicating, for example, heat-treated L. johnsonii Lal (NCC533, NO. CNCM 1-1225).

"Nonreplicating" L. johnsonii Lal (NCC533, NO. CNCM 1-1225) include L. johnsonii Lal, which were thermally treated. Also included are L. johnsonii Lal (NCC533, NO. CNCM 1-1225), which is inactivated, dead, non-viable and/or present as fragments such as DNA, metabolites, cytoplasmic compounds and/or materials of the cell wall.

"Nonreplicating" means that with the help of classical methods of sowing is impossible to detect viable cells and/or colony forming units. Such classical methods of planting are summarized in the book by m�crobiology James Monroe Jay, Martin J. Loessner, David A. Golden. 2005. Modern food microbiology. 7th edition, Springer Science, New York, N. Y. 790 p. In most cases, the absence of viable cells can be detected, as specified below, in the absence of viable colonies on the agar plates or in liquid turbidity of the growth medium, which does not increase after inoculation with different concentrations of bacterial preparations ("nonreplicating samples) and incubation under appropriate conditions (aerobic and/or anaerobic atmosphere for at least 24 hours).

L. johnsonii Lal (NCC533, NO. CNCM 1-1225) may be translated into a condition in which they will not replicate (i.e. make them nonreplicating), by heat inactivation. Inactivation by heating may occur at least about 70°C.

For inactivation of probiotics may be any heat treatment is carried out long enough to achieve inactivation. For example, such heat treatment may be performed at least within 10 seconds.

Usually at a higher temperature requires a shorter heating time, while at lower temperatures require longer heating.

For example, L. johnsonii Lal (NCC5 33, NO. CNCM 1-1225) may be translated into a condition in which they will not replicate at a temperature of from 110 to 140°C for 1-30 seconds,�reamer 10-20 seconds.

These established time frames refer to the time during which L. johnsonii Lal exposed to a predetermined temperature. Note that the heat treatment time may vary depending on the nature and quantity of the provided composition L. johnsonii Lal and depending on the configuration used for heating the device. Heat treatment may be conducted at atmospheric pressure, however it can also be carried out at high pressure. The usual limits of the pressure range from 1 to 50 bar, preferably 1-10 bar, preferably from 2 to 5 bar. Optimum pressure depends on the nature of containing microorganisms of the composition, and temperature.

If tracks are available Lal (NCC533, NO. CNCM 1-1225), are subjected to any heat treatment, for example, to packaging and distribution, it is preferable to use this stage of heat treatment for inactivation Lal NCC533.

Generally, compositions containing Lal (NCC 533, No. CNCM 1-1225) may be subject to short-term treatment at high temperature (HTST), instant pasteurization or ultra-high temperature processing (UHT).

UHT processing is ultra-high temperature process or ultra-high temperature processing (both denoted abbreviated UHT), intended�ment, at least partial sterilization of the composition by heating within a short time of about 1-10 seconds, at a temperature exceeding 135°C (275°F), this temperature is required to kill bacterial spores in milk. For example, the processing of milk using temperatures exceeding 135°C, allows to reduce the bacterial load within the required holding time (2-5 sec) that allows for a continuous flow process.

There are two main types of UHT systems - direct and indirect system. In a direct system products are processed by steam injection or steam flooding, whereas in the indirect system, the products are subjected to heat treatment using a plate heat exchanger, tubular heat exchanger, or scraping of the heat exchanger. In the process of obtaining product on any stage or in multiple stages can be applied to the combination of UHT systems.

HTST processing is defined as follows (high temperature/ short time): this method of pasteurization designed to achieve a 5-fold log reduction in the number of microorganisms that kills 99.9999% of the number of viable organisms in milk. He is considered adequate for destroying almost all yeasts, moulds and bacteria that cause spoilage, as well as for guaranteed�enough about the destruction of the normal pathogenic, stable to heat organisms. In the HTST process milk is heated to of 71.7°C (161°F) for 15-20 seconds.

Instant pasteurization is a method of heat pasteurization of perishable beverages like fruit and vegetable juices, beer and dairy products. Pasteurization is used to fill in the containers in order to destroy causing spoilage microorganisms to make products safer and to increase their shelf life. At the time when the liquid moves in a controlled continuous flow, it is exposed to temperatures of 71.5°C (160°F) to 74°C (165°F) for approximately 15 to 30 seconds.

For the purpose of the present invention, the term "high-temperature short-time processing" includes short-time treatment at a high temperature (HTST), UHT processing and instant pasteurization.

The compositions of the present invention may contain Lal in an amount sufficient for at least partial treatment of infections and disorders associated with the immune system, and/or their complications. A quantity sufficient to achieve this goal, is defined as "therapeutically effective dose". Quantities effective to achieve this goal will depend on a number of factors known to those skilled in the art, such as severity of disease, weight and General condition�Oia consumer health, and from the effect of the food matrix (food matrix).

When prophylactic use of a composition according to the invention are introduced to the consumer, subject to or otherwise associated with the risk of developing diseases associated with the immune system, in an amount sufficient at least to partially reduce the risk of such disorders. This amount is defined as a "prophylactically effective dose". And in this case, the exact number will depend on several factors, for individual patient, such as the condition and weight of the patient, and from the effect of the food matrix (food matrix).

Specialists in the art can appropriately adjust a therapeutically effective dose and/or prophylactically effective dose.

In most cases, the composition of the present invention contains Lal (NCC533, NO. CNCM 1-1225) in a therapeutically effective dose and/or prophylactically effective dose.

Typically, a therapeutically effective dose and/or prophylactically effective dose is in the range of from about 0.005 mg to 1000 mg Lal in the daily dose.

In quantitative terms, Lal (NCC533, NO. CNCM 1-1225) may be represented in the composition in an amount corresponding to from 104up to 1012CFU/g of dry composition. Obviously, nonreplicating microorganisms do not form colonies, �which is why this term should be understood as the number of nonreplicating microorganisms which is derived from 104-1012CFU/g reproducing bacteria. This includes microorganisms that are inactivated, non-viable or dead or present as fragments such as DNA or cell wall or cytoplasmic connections. In other words, the number of microorganisms contained in the composition, expressed in terms of colony forming ability (CFU) of such number of microorganisms, as if all organisms were alive, regardless of whether they are actually nonreplicating, for example, inactivated or dead, fragmented or a mixture of any or all of these States.

For example, the composition in accordance with the present invention may contain an amount of L. johnsonii Lal (NCC533, NO. CNCM 1-1225), corresponding to approximately 104up to 1012CFU per daily dose.

The composition of the present invention may contain about 0.005 mg - 1000 mg L. johnsonii Lal (NCC533, NO. CNCM 1-1225) in a daily dose.

The composition of the present invention can be a composition of any type. The composition may be intended for oral, enteral, parenteral (subcutaneous or intramuscular), or local ocular administration, or administration by inhalation, inside the rectal and intravaginal administration.

In with�yahzee with this composition of the present invention can be selected from the group consisting of food compositions, food products including pet food, beverages, compositions for a balanced diet, nutritional supplements, nutraceuticals, food additives, pharmaceutical compositions, cosmetic compositions, medicaments and compositions for local application.

Can be added prebiotics. Prebiotics can support the growth of probiotics before they will be transferred to the state "nonreplicating". Prebiotics can also act synergistically with viable probiotic bacteria that are present in the composition and/or to be added.

Violation associated with the immune system, may be selected from the group consisting of infections, in particular bacterial, viral, fungal and/or parasitic infections; inflammations; deficiency of phagocytes; defects of the epithelial barrier, the immaturity of the immune system, SIBO, and combinations thereof.

In one embodiment of the present invention a composition comprising L. johnsonii Lal (NCC533, NO. CNCM I-122), can be used to treat or prevent microbial infections, such as viral, fungal and/or parasitic infections.

Violation associated with the immune system, can also be selected from the group of disorders associated with reduced levels of defensins, in particular hBD1. Such violations can bitwinrar from the group consisting of cystic fibrosis, reactive airway diseases, pulmonary infections, Smoking, asthma, pneumonia, rhinitis, otitis, sinusitis, tuberculosis, Crohn's disease (colon and ileum), ulcerative colitis, celiac disease, underdevelopment of the bowel, gastritis and stomach ulcers caused by Helicobacter pylori infection, infectious diarrhea, necrotising enterocolitis, diarrhoea associated with antibiotics, bacterial vaginosis, infection with human immunodeficiency virus (HIV), herpes simplex virus, urinary tract infections, atopic dermatitis, chronic ulcer, carcinoma, atopic eczema, burn injury, tonsillitis, gingivitis, dental caries, keratitis of the eye and their combinations.

The composition of the present invention may be used to increase endogenous antimicrobial defenses.

This can be achieved, for example, by stimulating endogenous expression of hBD1.

The present inventors found that L. johnsonii Lal (NCC533, NO. CNCM 1-1225) strongly stimulate constitutive expression of hBD1 and that nonreplicating, for example, heat-treated L. johnsonii Lal (NCC533, NO. CNCM 1-1225), stimulate the expression of hBD1 even more than their live counterparts.

Therefore, an object of the present invention also includes a method of increasing the efficiency of L. johnsonii Lal (NC533, No. CNCM 1-1225) in the treatment or prevention of disorders associated with the immune system comprising the step of converting the L. johnsonii Lal (NCC533, NO. CNCM 1-1225) in a state wherein they are nonreplicating, for example by means of heat treatment.

Violation associated with the immune system, may be, for example, one of the violations listed above.

In one embodiment of the present invention, the method includes a step of heat treatment at least at a temperature of about 70°C for at least about 10 seconds.

Specialists in the art it is clear that they can freely combine all features of the present invention described in the document, without leaving the scope of the described invention. In particular, features described in relation to compositions of the present invention can be applied to use cases and/or method of the present invention and Vice versa.

Additional advantages and features of the present invention will become apparent from the following examples and figures.

Figure 1 shows that heat-treated at 120°C for 15 h Lal (NCC533, NO. CNCM 1-1225) strongly influence hBD1 mRNA in epithelial intestinal cells in vitro compared with other strains, subjected to heat treatment. T84 cells were incubated for 4 h � heat-treated strains. HBD1 gene expression was analyzed by PCR in real time. Bars represent mean values ± standard error of the mean, normalized relative to the expression of cells not subjected to stimulation.

Figure 2 shows that the high temperature and short time processing Lal (NCC533, NO. CNCM 1-1225) were the best conditions for stimulating the expression of hBD1 mRNA. T84 cells were stimulated for 4 hours with live and heat-treated Lal (NCC533, NO. CNCM 1-1225) at 120°C for 15 h or at 85°C for 20 min. hBD1 gene Expression was analyzed by PCR in real time. Bars represent mean values ± standard error of the mean, normalized relative to the expression of cells not subjected to stimulation.

Examples

The Protocol of the experiment:

Used T84 cells from passage 30-40 and were cultured in basic medium by a modified Dulbecco/F-12 (Sigma D 6421) containing 5% fetal calf serum (PCS) (Amined BioConcept) and 2 mm glutamine. Cells were seeded at a concentration of 2×106cells/well in 6-well culture plates and were grown as a monolayer at 37°C in atmosphere of 5% CO2- 95% air. Cells were grown for 1 week after confluence and were incubated with serum and the environment, not containing antibiotics, at least within 12 hours�. This stage was necessary to exclude the expression of defensin-induced serum, and to prevent any influence of antibiotics on probiotics and cellular immune response. The cells are further incubated with probiotics or heat-treated strains within 4 hours. At the end of the incubation time cells were washed with PBS and collected with the reagent for the isolation TriPure according to the Protocol of the supplier. The expression of human genes hBD1 and hBD2 in the treated cells was assessed using quantitative PCR.

In this experiment we used bacterial strains B. longum (NCC 2705, No. CNCM 1-2618), B. lactis NCC 2818, No. CNCM 1-3446), L. johnsonii (Lal, NCC533, NO. CNCM 1-1225), L. paracasei (ST11, NCC 24 61, No. CNCM 1-2116). Studies were conducted on live or heat-treated strains for 15 seconds at 120°C or for 20 minutes at 85°C.

Results

Heat-treated at 120°C for 15 h Lal (NCC533, NO. CNCM 1-1225) cause strong hBD1 mRNA expression after 4 hours of incubation (Figure 1) as opposed to any other approved heat-treated strains. These results are unique, because at the present time in the scientific community it is considered that the constitutive expressed HBD1 in fact, is not subject to modulation by microbes, products of vital activity of microorganisms or inflammation.

And live termicheski treated Lal (NCC533, No. CNCM I-1225) strongly influence the expression of hBD1 mRNA, but the highest stimulation of hBD1 was caused by a heat-treated Lal (high temperature and short time treatment) (Figure 2).

1. Composition for treating or preventing disorders associated with reduced levels of defensins containing from 0.005 mg to 1000 mg L. johnsonii La1 (NCC533, NO. CNCM 1-1225) the daily dose, where at least 90%, preferably at least 95% or most preferably 100% of L. johnsonii La1 (NCC533, NO. CNCM 1-1225) are nonreplicating, and L. johnsonii La1 (NCC533, NO. CNCM 1-1225), translated into a state wherein they are nonreplicating, at a temperature of from 110 to 140°C for 5-30 seconds.

2. A composition according to claim 1, where defensin is a hBDl.

3. A composition according to claim 1, which is selected from the group consisting of food compositions, food products including pet food, beverages, compositions for a balanced diet, nutritional supplements, nutraceuticals, food additives, pharmaceutical compositions, cosmetic compositions, compositions for topical application and medicines.

4. A composition according to any one of claims. 1-3, where the violation is associated with a reduced level of defensins, selected from the group consisting of cystic fibrosis, reactive airway diseases, pulmonary infections, Smoking, asthma, pneumonia, renie�and, otitis, sinusitis, tuberculosis, Crohn's disease (colon and ileum), celiac disease, underdevelopment of the intestine, ulcerative colitis, gastritis and stomach ulcers caused by Helicobacter pylori infection, infectious diarrhea, necrotising enterocolitis, diarrhoea associated with antibiotics, bacterial vaginosis, infection with human immunodeficiency virus (HIV), herpes simplex virus, urinary tract infections, atopic dermatitis, chronic ulcer, carcinoma, atopic eczema, burn injury, tonsillitis, gingivitis, dental caries, keratitis eye.

5. A composition according to any one of claims. 1-3, intended to boost the endogenous antimicrobial defenses and/or endogenous expression hBDl.



 

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3 tbl, 4 ex

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Pseudomonas aeruginosa No.1 KVL-DEP is deposited in the collection of FSBI "VGNKI". When using the vaccine based on the proposed strain, the titre of antibodies to somatic antigen O3 Pseudomonas aeruginosa in blood serum of rabbits and pigs, 14 days after the last immunisation is 1:768 and 1:1024, respectively.

EFFECT: strain has high immunogenic activity and is intended for production of a vaccine against pseudomonosis of pigs.

3 tbl, 4 ex

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Pseudomonas aeruginosa No.25-DEP is deposited in the collection of FSBI "VGNKI". When using the vaccine based on the proposed strain, the titre of antibodies to somatic antigen O19 Pseudomonas aeruginosa in blood serum of rabbits and pigs, 14 days after the last immunisation is 1:512 and 1:716.8, respectively.

EFFECT: strain has high immunogenic activity and is intended for production of a vaccine against pseudomonosis of pigs.

3 tbl, 4 ex

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Pseudomonas aeruginosa No.34-DEP is deposited in the collection of FSBI "VGNKI". When using the vaccine based on the proposed strain, the titre of antibodies to somatic antigen O1 Pseudomonas aeruginosa in blood serum of rabbits and pigs, 14 days after the last immunisation is 1:819.2 and 1:1024, respectively.

EFFECT: strain has high immunogenic activity and is intended for production of a vaccine against pseudomonosis of pigs.

3 tbl, 4 ex

FIELD: biotechnology.

SUBSTANCE: urogenital mycoplasmosis is diagnosed by detecting Mycoplasma hominis in the clinical material, as well as for semi-quantitative determining the titre of the pathogen. The nutrient medium comprises PPLO broth, phenol red, brilliant blue, L-arginine hydrochloride, yeast extract, ceftriaxone, amoxiclav (sodium salt of amoxicillin/clavulanic acid potassium salt), vancomycin hydrochloride, clarithromycin, fluconazole and distilled water in a predetermined ratio of components.

EFFECT: invention enables to improve the accuracy and to reduce the time of detection of Mycoplasma hominis.

4 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel N-containing heteroaryl derivatives of formula I or II or their pharmaceutically acceptable salts, which possess properties of JAK kinase, in particular JAK3, and can be applied for treating such diseases as asthma and chronic obstructive pulmonary disease (COPD). In formulae A represents carbon and B represents nitrogen or A represents nitrogen and B represents carbon; W represents CH or N; R1 and R2, independently represent hydrogen, C1-4alkyl, halogenC1-4alkyl, -CN; R3 represents C1-4alkyl, R9-C1-4alkyl, Cy1, where Cy1 is optionally substituted with one or several substituents R10; R4 represents hydrogen, C1-4alkyl, R12R7N-C0alkyl, where one of R7 and R12 represents hydrogen, and the other represents C1-4alkyl or group R13, which is selected from C1-5alkyl, Cy2-C0alkyl; R5 represents hydrogen; R6 represents hydrogen, C1-4alkyl, C1-4alkoxyC1-4alkyl, hydroxyC1-4alkyl, R12R7N-C1-4alkyl, R16CO-C0alkyl, Cy1; R7 represents hydrogen or C1-4alkyl; R9 represents halogen, -CN, -CONR7R12, -COR13, CO2R12, -OR12, -SO2R13, -SO2NR7R12, -NR7R12, -NR7COR12; R10 represents C1-4alkyl or R9-C0-4alkyl; R11 represents C1-4alkyl, halogen, -CN, -NR7R14; R12 represents hydrogen or R13; R13 represents C1-5alkyl, hydroxyC1-4alkyl, cyanoC1-4alkyl, Cy2-C0alkyl or R14R7N-C1-4alkyl; where Cy2 is optionally substituted with one or several constituents R11; R14 represents hydrogen or C1-4alkyl; R16 represents C1-4alkyl, halogenC1-4alkyl, C1-4alkoxyC1-4alkyl, hydroxyC1-4alkyl or cyanoC1-4alkyl; Cy1 represents monocyclic carbocyclic unsaturated or saturated ring, selected from C3-C6cycloalkyl, phenyl, or saturated monocyclic 4-6-membered heterocyclic ring, containing from 1 to 2 heteroatoms, selected from N and S, or partially unsaturated 10-membered bicyclic heterocyclic ring, containing oxygen atom as heteroatom, which can be substituted with group R11, where said ring is bound with the remaining part of molecule via any available C atom, and where one or several ring C or S atoms are optionally oxidised with formation of CO or SO2; and Cy2 represents monocyclic carbocyclic unsaturated ring, selected from C3-C6cycloalkyl, or aromatic monocyclic 4-6-membered heterocyclic ring, containing from 1 to 2 heteroatoms, selected from N and S, or unsaturated 10-membered bicyclic heterocyclic ring, containing oxygen atom as heteroatom, which can be substituted with group R11, where said ring is bound with the remaining part of molecule via any available atom C or N.

EFFECT: obtaining novel heteroaryl derivatives.

27 cl, 41 ex

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry, particularly to affinity-matured CRIg versions. Declared is the CRIg versions, which is an alternative complement pathway inhibitor at least twice stronger than human CRIg with a native sequence, and optionally possesses C3b-bindingaffinity at least twice as much. There are also declared a chimeric molecule and a pharmaceutical composition, both containing the above CRIg version. The CRIg version can be used for preparing a therapeutic agent for treating a complement-associated disease or condition.

EFFECT: invention enables improving the therapeutic effectiveness of CRIg polypeptides.

17 cl, 17 dwg, 4 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to quinolines substituted by phosphorus-containing group of formula and applicable in medicine, wherein Z represents V1 and V2 are independently specified in hydrogen or halogen; one of R and R` represent phosphorus-containing substitute Q; the other one is specified in hydrogen or methoxyl; wherein the phosphorus-containing substitute Q represents A represents O; L represents C1-6alkyl; J represents NH or C3-6heterocycloalkyl and J is optionally substituted by G3; X is absent or represents -C(=O)-; X is absent or represents C1-6alkyl; each of R1 and R2 are independently specified in C1-6alkyl or C1-6alkoxy; G3 represents C1-6alkyl, R3S(=O)m-, R5C(=O)- or R3R4NC(=O)-; R3, R4 and R5 are independently specified in 3 or C1-6alkyl; m is equal to 0-2.

EFFECT: there are presented new protein kinase inhibitors effective for treating the diseases associated with abnormal protein kinase activity.

20 cl, 42 ex, 8 tbl, 3 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biotechnology, namely to novel IL-17-inhibiting polypeptides, corresponding to fused proteins, to compositions and their application for medicinal purposes. Polypeptide contains amino acid sequence, which is selected from group, consisting of GVTLFVALYD YKAFWPGDLS FHKGEKFQIL RTSDGDWWEA RSLTTGETGY IPSNYVAPVD SIQ (SEQ ID NO: 39), GVTLFVALYD YKAFWPGDIS FHKGEKFQIL RTSDGEWWVA RSLTTGEEGY IPSNYVAPVD SIQ (SEQ ID NO: 57) or GVTLFVALYD YKAFWPGDIS FHKGEKFQIL RTSDGEWWIA RSLTTGEEGY IPSNYVAPVD SIQ (SEQ ID NO: 107); amino acid sequence, which has, at least, 80%, preferably, at least, 90%, more preferably, at least, 95% identity of amino acid sequence with SEQ ID NO: 39, SEQ ID NO:57 or SEQ ID NO: 107; fragment or functional derivative of SEQ ID NO: 39, SEQ ID NO: 57 or SEQ ID NO: 107, obtained due to substitution, addition and/or removal of not more than 5 amino acids.

EFFECT: invention makes it possible to bind IL-17 with high specificity and affinity.

33 cl, 17 dwg, 3 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention refers to immunology. What is disclosed is using a peptide with an amino acid sequence GLAGGSAQSQRAPDRVL for selecting CεmX-specific antibodies and antigen-binding fragments of these antibodies. What is presented is the CεmX-specific antibody, as well as a method providing selecting the antibodies specifically binding the peptide according to the invention, and a method of treating IgE-mediated diseases involving administering the antibody into an individual according to the invention.

EFFECT: it has been disclosed that the monoclonal antibodies specifically binding the CεmX segment GLAGGSAQSQRAPDRVL can effectively bind to mIgE in human B-cells and are applicable for targeting to these B cells for treating IgE-mediated diseases.

14 cl, 5 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of immunology. Claimed is isolated antibody to ICOS protein of people with increased effector function. Also described are cell and method of obtaining antibody in accordance with claimed invention, pharmaceutical composition, method of treating autoimmune disease or disorder, transplant rejection and malignancy of human T-cells, as well as method of depletion of ICOS-expressing T-cells, method of destroying germ centre structure in secondary lymphoid organ of primates, methods of depleting B-cells of germ centre of secondary lymphoid organ and circulating B-cells, which have undergone class switching, in primates.

EFFECT: invention can be further applied in therapy of diseases, mediated by T-cells.

33 cl, 21 dwg, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to the strain Lactobacillus rhamnosus CNCM I-3690 and to a dairy food product containing the above strain. The presented strain possesses the mannose-specific adhesive properties. The strain possesses the antimicrobial properties in relation to, e.g. Escherichia coli, Salmonella enteritidis and Lysteria monocytogenes.

EFFECT: strain possesses the immunomodulatory properties, particularly possesses an ability to inhibit an inflammatory reaction of HT-29 epithelial cells.

2 cl, 2 dwg, 5 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. There are disclosed versions of a dimer compound for forming a multimer capable to reproduce the effector function of aggregated IgG with identical monomers. Each monomer of the dimer comprises: a monomer of IgG2 link region or a monomer of isoleucine zipper, dimerising each of which forms a multimerising region, and at least Fc-domain monomer containing a link region, CH2 domain and CH3 domain of IgG1. What is described is a multimer compound capable to reproduce the effector function of aggregated IgG and containing two or more dimers. There are disclosed a method for changing the immune response using the dimer or multimer, as well as a multimer-based method of treating an inflammatory disease.

EFFECT: using the invention provides the new compounds capable to bind at least one FcR specified in a group consisting of: FcγRI, FcγRII, FcγRIII, FcγRIV, or their non-human version that can find application in medicine for IVIG substitution for treating a wide range of diseases, including the inflammatory and autoimmune diseases.

7 cl, 25 dwg, 5 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology. Presented are variants of anti-CD20 modified antibody or its antigen-binding fragment. Each of the variants is characterised by the fact that it contains a variable light and heavy chain domain, and induces a higher apoptosis level as compared to anti-B-Ly1 chimeric antibody. There are presented: a mixture of antibodies, wherein at least 20% of oligosaccharides in Fc domain have a branched chain and are not fucosylated, as well as a pharmaceutical composition for producing a therapeutic agent for a malignant haematological or autoimmune disease by using the antibodies or the mixture of antibodies. Described are: an expression vector, a based host cell, variants of coding polynucleotides, as well as a method for producing the antibody in the cell.

EFFECT: using these inventions provides the new antibodies with the improved therapeutic properties, including with increased binding of Fc receptor, and with the increased effector function that can find application for treating the malignant haematological or autoimmune disease.

32 cl, 3 ex, 9 tbl, 26 dwg

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to field of pharmaceutics and medicine and deals with composition, possessing property to stimulate antimicrobial defence by increasing expression of endogenous β-defensins, containing strain B. longum NCC 2705, depositary number CNCM 1-2618, with said strain being brought, at least, partially into non-replicable condition by thermal inactivation, processing , at least, about 70°C. Method of stimulating endogenous antimicrobial activity of mammal by increasing expression of endogenous β-defensins, includes introduction of claimed composition.

EFFECT: group of inventions provides increase of efficiency in comparison with live strains.

12 cl, 2 dwg

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