Agent for prophylaxis and/or treatment of undesired inflammation activity and cancer prophylaxis

FIELD: biotechnology, in particular utilization of strain Lactobacillus salivarius UCC 118 for prophylaxis and/or treatment of undesired inflammation activity and cancer prophylaxis.

SUBSTANCE: human-original strain Lactobacillus salivarius UCC 118[NCIMB 40829] is isolated from dissected and washed human gastrointestinal tract. Strain application makes it possible to decelerate development of malignant diseases and to reduce undesired inflammation activity such as intestine inflammation or irritable colon.

EFFECT: strain for prophylaxis and/or treatment of undesired inflammation activity and cancer prophylaxis.

42 cl, 29 dwg, 1 tbl, 4 ex

 

This invention relates to the use of Lactobacillus salivarius.

Protective mechanisms that protect the gastrointestinal tract of man from colonization by intestinal bacteria, is very complex and include both immunological and non-immunological aspects (V.J.McCracken and H.R.Gaskins, "Probiotics a critical review", Horizon Scientific Press, UK, 1999, p.278). Natural protective mechanisms include low pH environment of the stomach, bile salts, peristalsis, satinowye layers and antimicrobial compounds such as lysozyme (D.C.Savage, "Microbial Ecology of the Gut", Academic Press, London, 1997, p.278). Immunological mechanisms include specialized lymphoid nodules located under the M-cells, called peyrovani plaques, which are spread all over thin and colon (M.F.Kagnoff, Gastroenterol. 1993, 105, 1275). Luminally antigens presented in these places, leading to stimulation of the relevant subpopulations of T - and b-cells with the formation of cytokine networks and secretion of antibodies in the gastrointestinal tract (M.R.Neutra and J-P Kraehenbuhl, "Essentials of mucosal immunology", Academic Press, San Diego, 1996, p.29; M.E. Lamm, Ann. Rev. Environ. 1997, 51, 311). In addition, the presentation of antigens can occur through the epithelial cells, intraepithelial lymphocytes and the underlying immune cells own plates (S.Raychaudhuri et al. Nat Biotechnol, 1998, 16, 1025). Therefore, the owner makes the value the contributions in the immunological protection of the gastrointestinal tract. However, since the mucous membrane of the gastrointestinal tract is the largest surface through which the host communicates with the external environment must be certain of the control mechanisms for the regulation of immune reactivity by 100 tons of food, which is processed in the gastrointestinal tract during the average lifetime (F. Shanahan. "Discrimination of the gastrointestinal tract", Raven Press, 1994, p.643). In addition, the intestine is inhabited by more than 500 species of bacteria, numbering 1011-1012/g in the colon. Thus, these control mechanisms must be able to distinguish between non-pathogenic attached bacteria from invasive pathogens that could cause significant disruption to the owner. In fact, the intestinal flora contributes to the protection of the owner, competing with new swallowed potentially pathogenic microorganisms.

The absorption of non-pathogenic, or probiotic, bacteria leads to improved immune parameters in healthy volunteers. Examples of such immunomodulatory effects are shown in Table 1.

Table 1.
The effects of the immune improvement after oral absorption of probiotic bacteria.
The observed effectReference/td>
Increased phagocytosis in macrophages10
Increased activity of natural killer cells11
Elevated levels of IFNγ (γ-interferon) in serum12
Increased number of b-cells and NK-cells (natural killers)12
Stimulation lgA-answers11,13-15
Reinforced responses in the form of delayed-type hypersensitivity (DTH)16

Bacteria present in the gastrointestinal tract of man, can stimulate inflammation. Aberrant immune responses to typical microflora involved in some painful conditions, such as inflammatory bowel disease (Brandzeag P. et al., Springer Semin. Immunopathol, 1997, 18, 555). Antigens associated with normal flora, are usually the result of immunological tolerance, and failure to achieve this tolerance is the main mechanism of mucosal inflammation (Stallmach A. et al., Immunol. Today, 1998, 19, 438). Proof of such deterioration tolerance is increased levels of antibodies directed against intestinal flora in patients with IBD (inflammatory bowel disease).

In WO-A-98/35014 described strains of Lactobacillus salivarius isolated from the dissected and washed as docno-intestinal tract of man, which inhibit a wide range of gram-positive and gram-negative microorganisms and which secrete in cell-free supernatant product having antimicrobial activity.

Summary of the invention

The immune system is designed to protect host tissue and destruction of incoming pathogens. After recognition of the presence of bacterial cells of the immune system become active and eliminate bacterial threat. The production of mediators of inflammation stimulates cell activation and destruction of the pathogen.

Unexpectedly, the inventors have found that strains of Lactobacillus salivarius induce anti-inflammatory effects in vitro and in vivo. Discovered that the immune perception of Lactobacillus salivarius leads to the suppression of inflammatory activity. Intentional ingestion of large amounts of Lactobacillus salivarius leads to the suppression of inflammatory activity. Consequently, the invention has considerable potential therapeutic value for the prevention or treatment of undesirable inflammatory responses such as inflammatory bowel disease.

Lactobacillus salivarius is a microorganism-commensal, originally isolated from the microbial flora of the gastrointestinal tract of man. The immune system in the gastrointestinal tract cannot imitate severe reactions to members of this flora, because the resulting inflammatory activity would destroy also the host cells and would disrupt the function of tissues. Therefore, there is some(s) mechanism(s)by which(s) of the immune system can recognize non-pathogenic representatives of commensals of the gastrointestinal flora in contrast to pathogenic microorganisms. This provides a limitation of lesion of tissue of the host, while the protective barrier is still

According to this invention proposed the use of Lactobacillus salivarius in the prevention and/or treatment of undesirable inflammatory activity.

According to the invention of undesirable inflammatory activity may represent an undesirable gastrointestinal inflammatory activity, such as inflammatory bowel disease such as Crohn's disease, ulcerative colitis, irritable bowel syndrome, inflammation of the abdominal pouch (pouchitis) or post-infectious colitis.

Gastrointestinal inflammatory activity may also be a diarrhoeal disease. Diarrhoeal disease may be due to Clostridium difficile, or to be caused by rotavirus (Rotovirus). Diarrhoeal disease may also be a post-infectious diarrhoeal disease.

Inflammatory activity can be the ü due to cancer of the gastrointestinal tract or systemic inflammatory disease, such as rheumatoid arthritis.

In another case of undesirable inflammatory activity may be due to an autoimmune disorder.

In another case of undesirable inflammatory activity may be due to cancer.

As one embodiment of the present invention proposed the use of Lactobacillus salivarius in the prevention of cancer.

As another embodiment of the present invention proposed the use of Lactobacillus salivarius, and Lactobacillus salivarius is contained in the drug.

This product preferably includes another probiotic material. Alternative or additionally, the drug includes a prebiotic material.

In the ideal case, the product includes a carrier for oral administration. Carrier for oral administration can be a pharmaceutically acceptable carrier, such as a tablet, capsule or powder.

Preferably the carrier for oral administration is a protein and/or peptide, in particular proteins and/or peptides that are rich in glutamine/glutamate; lipid; carbohydrate; vitamin, mineral and/or trace element.

Media ingestion is most preferably represents a food product, such as fermented milk, yoghurt, frozen yoghurt, milk powder, concentrated milk, cheeses, dressings Il the drinks.

In one of the embodiments of Lactobacillus salivarius presented in the product in quantities of more than 106colony forming units (CFU) per gram of delivery systems.

In another embodiment of this drug includes adjuvant. This drug can include bacterial component. Alternative or additionally, the drug may include medicinal beginning. This drug can also include biological connection.

As one of the embodiments of the present invention proposed the use of Lactobacillus salivarius, and this strain or the product is intended for administration to animals. Preferably the animal is a mammal, most preferably human.

As another embodiment of the present invention proposed the use of Lactobacillus salivarius, and Lactobacillus salivarius causes changes in immunological marker after the introduction into the system that contains cells that interact with the immune system, and cells of the immune system.

Cells that interact with the immune system, preferably represent epithelial cells. The most preferred immunological marker is a cytokine, especially tumor necrosis factor α (TNFα).

Preferably the cells, which interact with the immune systems of the th, and cells of the immune system preferably have an appropriate background.

In one of the embodiments of the cells, which interact with the immune system, gastrointestinal, respiratory, or genitourinary origin.

. In another embodiment of the cells of the immune system have gastrointestinal, respiratory, or genitourinary origin.

As a further embodiment of the present invention proposed the use of Lactobacillus salivarius, where the strain of Lactobacillus salivarius is a Lactobacillus salivarius subspecies salivarius. Preferably Lactobacillus salivarius has a human origin, most preferably from dissected and washed in the gastrointestinal tract of man.

Preferably Lactobacillus salivarius inhibits a wide range of gram-positive and gram-negative microorganisms. Most preferably, it secretes possessing antimicrobial activity of the product in a cell-free supernatant, and the mentioned activity is produced only growing cells and is destroyed by proteinase K and pronasol E.

A particularly preferred strain of Lactobacillus salivarius strain is Lactobacillus salivarius UCC 118 or a mutant or variant.

Lactobacillus salivarius UCC 118 has been deposited at NCIMB on November 27, 1996, and received a registration number NCIMB 40829. The strain of Lactobacillus salivarius described in WO-A-98/35014.

Lactobacillus saliarius may represent a genetically modified mutant or it can be a naturally occurring variant of Lactobacillus salivarius.

Preferably Lactobacillus salivarius presented in the form of viable cells. Or Lactobacillus salivarius may be in the form of viable cells.

A brief description of graphic materials

Figure 1 shows a graph of the levels of C. perfringens in mice, absorbing UCC118, in comparison with the group, absorbing placebo (p<0,05). The results are shown in graph form in the form of averages of logarithms ± standard error for each group.

Figure 2 presents the histogram of the levels of inflammation in mice, absorbing UCC118, in comparison with the control mice. The results are presented as median values ± standard error for each group.

Figure 3 shows a graph of the levels of TNFα (tumor necrosis factor α) in the course of six weeks, during which patients absorb UCC118. The results are presented in the form of a graph of the average level of TNFα (PG/ml) for each time point (n=22).

Figure 4 shows a graph of the performance CDAI (activity index for Crohn's disease) for patients, absorbing UCC118 in the continuation of probiotic food. Indicators CDAI decreased from an average of 180 to 160.

Figure 5 shows a graph of the production of cytokines in vitro after exposure to the impact of UCC118. The results are expressed in PG/ml.

Figure 6 presents a histogram of the levels vneck mocnych TNFα , IL-1RA, IL-6, sIL-6R and IFNα after exposure to the effects of Lactobacillus salivarius UCC118.

Figure 7 presents gene number with specific gene sequences 268 cytokines and related molecules for the study of immune response to UCC118. The bottom panel illustrates the control culture, while the top panel shows the gene expression of cytokines by mononuclear cells of peripheral blood (RVMS) after exposure exposure UCC118; and

on Fig presents the histogram of the levels of TNFα in the presence of different bacterial strains.

Detailed description

The inventors have developed criteria for the selection of in vitro probiotic bacteria, which show a specific action in vivo on his master, such as modulation of the microflora of the digestive tract (gastrointestinal tract) and modulation related to mucosal immune response leading to the production of secretory antibodies specific to the absorbed strain. The inventors have found that Lactobacillus salivarius subspecies salivarius UCC118 not only survives during the passage across the gastrointestinal tract and sticks to cell lines of human intestine, but also, surprisingly, anti-inflammatory effect.

In General, the use of probiotic bacteria is carried out in the form of viable cells. But it can also b is to be extended to non-viable cells, such as dead culture or compositions containing useful factors expressed by the probiotic bacteria. This may include heat-killed microorganisms or microorganisms killed by the impact of altered pH or under pressure. Preparation of the product with non-viable cells is more simple, the cells can easily be incorporated into pharmaceutical preparations and requirements for storage less stringent than for viable cells. Lactobacillus casei YIT 9018 is an example of effective use of heat-killed cells as a method of treatment and/or prevention of tumor growth, as described in U.S. patent No. 4347240.

It is not known whether intact bacteria to manifestations of anti-inflammatory action, or can only be used to separate the active ingredients according to this invention. Identified proinflammatory components of some bacterial strains. Proinflammatory action of gram-negative bacteria is mediated by lipopolysaccharide (LPS). By itself, LPS stimulates proinflammatory network, partly due to the binding of the LPS receptor CD14 on monocytes. It is believed that the components of probiotic bacteria have anti-inflammatory activity due to the actions of whole cells. As the selection of these components is prognoziruete the impact of pharmaceutical quality.

The invention will become more clear from the following examples.

Example 1: detailed description of the detection of in vivo anti-inflammatory action of Lactobacillus salivarius, in particular subspecies salivarius UCC118.

Murine model of inflammation of the gastrointestinal tract

Aberrant immune responses to native microflora involved in some painful conditions, such as inflammatory bowel disease (Brandzeag P. et al. Springer Semin. Immunopathol, 1997, 18, 555). Antigens associated with normal flora, usually lead to immunological tolerance, and failure to achieve this tolerance is the main mechanism of mucosal inflammation (Stallmach A. et al., Immunol. Today, 1998, 19, 438). Proof of such deterioration tolerance is increased levels of antibodies directed against intestinal flora in patients with IBD. In addition, some murine models, predisposed to pathological inflammatory disorders of the gastrointestinal tract, stay healthy when they are placed in sterile conditions or when exposed to antibiotics (R. Kuhn et al. Cell, 1993, 75, 263; Panwala S. M. et al. J. Immunol., 1998, 161,5733).

Mouse C57BL/6 knockout interleukin-10 (IL-10) are predisposed to the development of enterocolitis in the presence of the bacterial flora of the small intestine. When the contents in sterile conditions in mice with knockout of the m IL-10 disease does not develop (R. Kuhn, et al. Cell, 1993, 75, 263). Because the pathogenesis of this disease is associated with the flora of the small intestine, the removal of specific components of this flora can have a beneficial effect on the severity of the disease.

Lactobacillus salivarius subspecies salivarius UCC118 is a probiotic bacterium isolated from the ileum of a healthy person. It is suitable for gastrointestinal colonization, because it satisfies many of the criteria for selection of probiotic strains. They include traits such as tolerance to bile, resistance to acid and adhesion in vitro to cell lines colon of man. Conducted research eating healthy and noted a significant modification of the gastrointestinal flora. In addition, UCC118 was perceived by the immune system of the mucosa, which led to the production and secretion of IgA specific to UCC118.

Thus, UCC118 survive through the gastrointestinal tract, modulates the intestinal flora and is perceived by the immune system of the mucosa. Using a murine model of enterocolitis, a study was conducted of the effects of these probiotic bacteria on the modulation of inflammatory reactions in the gastrointestinal tract. In addition, the inventors have investigated the role of Lactobacillus salivarius subspecies salivarius UCC118 in reducing the speed of plasticheskogo transformation in the gastrointestinal tract.

Within 16 weeks studied twenty-IL-10 KO mice (mouse, knocked out on IL-10) (ten absorbed probiotic microorganisms in milk and 10 absorbed unmodified milk). To determine the number of produced lactic acid bacteria, Clostridium perfringens, bacteroids, coliform, bifidobacteria and enterococci weekly microbial faecal analysis. After killing did microbiological and histological evaluation of the content and small intestine.

The test animals compared to the control were significantly reduced levels of coliform and enterococci in faeces. After killing observed a significant reduction in the number of C. perfringens in the tested mice (Figure 1). In the tested group was not fatal outcomes compared with two deaths from sudden and quickly developed colitis in the control group. Only one of the tested mice developed adenocarcinoma of the colon compared with five in the control group. According assessed inflammation of the mucous membrane at the test animals is lower than in the control mice (Figure 2). Reducing the frequency of tumor development after absorbing UCC118 may be associated with reduced levels of inflammation in the gastrointestinal tract or may be the result of removal of the Pro-carcinogenic elements gastrointestinal flora (umney C.J., et al. Carcinogenesis, 1993, 14, 79; Rowland I.R. (1995). In: Gibson G.R. (ed). Human colonic bacteria: role in nutrition, discrimination and pathology, pp 155-174. Boca Raton CRC Press; Darveau D. Nat. Biotech., 1999, 17, 19).

In conclusion, the absorption of Lactobacillus salivarius UCC118 leads to a significant modulation of the intestinal flora and reduce the rate of mortality, incidence of cancer and indicators of the disease.

Example 2: Study in humans using UCC118 conducted in patients with Crohn's disease in active phase.

Inflammatory bowel disease (IBD) covers a number of inflammatory disorders of the gastrointestinal tract, including Crohn's disease and ulcerative colitis.

Patients suffering from Crohn's disease in the active phase, treated with UCC118. Briefly: 22 patients absorbed UCC118 in dairy product within 6 weeks. Microbiological and immunological measurements were carried out at time 0 weeks, 1 week, 3 weeks and 6 weeks. Studies with placebo control was not performed.

In the course of feeding was measuring systemic levels of cytokines, particularly tumor necrosis factor α (TNFα), a proinflammatory cytokine, which is involved in the pathogenesis of many inflammatory painful conditions, including inflammatory bowel disease. Modern methods of treatment of inflammatory bowel disease are directed about obanno to reduce levels of TNFα Present D.H., et al. New Eng. J. Med., 1999, 340, 1398). In this test, after absorbing UCC118 levels of systemic TNFα were reduced (Figure 3).

In addition, patients were evaluated for their activity index of Crohn's disease (CDAI) in the continuation of the six-week period of the study. This index gives an estimate of the overall health and well-being of each patient (Figure 4). In General, the index of disease activity is slightly improved for the majority of individuals in the study. They represent patients with heart disease in moderately active stage, and would be expected that their CDAI assessment will increase. However, after treatment UCC118 CDAI assessment has not increased, and in fact, they have improved on average from 180 to 160.

Example 3: detailed description of the detection in vitro of the mechanisms underlying the anti-inflammatory action of Lactobacillus salivarius, in particular subspecies salivarius UCC118.

For these studies used a variety of techniques, including ELISA (ELISA analysis) (identification of extracellular proteins), flow cytometry (determination of intracellular proteins) and expression of cDNA (mRNA expression). In particular, it was planned to study the expression of tumor necrosis factor α due to its clinical significance, and when using all three methods noted the suppression of the production of this cytokine after exposure exposure UCC118.

ISOE is isua transluminal (transwell) system analysis with epithelial cells and mononuclear cells of peripheral blood, using ELISA measured the levels of extracellular cytokines. After co-incubation with UCC118 the amount of biogas produced TNFα was significantly reduced compared to control cultures. Moreover, the levels of IL-1RA (receptor antagonist IL-1) and IFNγ (interferon γ) decreased, whereas the levels of IL-6 and soluble receptor of IL-6 (SIL-6R) increased (Figure 5). Intracellular staining against TNFα confirmed the results of ELISA analysis, since the levels of TNFα was lower for UCC118-stimulated sample compared to the control samples.

6 shows taking place Treklyano signal transmission. Joint incubation RVMS and Lactobacillus salivarius UCC118 leads to the stimulation of the production of TNFα. However, joint incubation RVMS, Lactobacillus salivarius UCC118 cells and epithelial cells (SASO-2 cells) leads to a significant inhibition of production of TNFα. Thus, in trehkletevoy model is significantly different scheme of signal transmission compared with only bacteria and RVS.

Gene series determine the amount of mRNA in the cell population. The inventors stimulated mononuclear cells from peripheral blood using UCC118 within 24 hours and examined the effect on gene expression of cytokines (7). Significant modification of the EC is pressie of cytokine genes. For example, genes encoding proinflammatory cytokines IL-1β and TNFα off, while genes encoding cytokines Th2 dysbalance-type, such as IL-6, amplified.

Models in vitro demonstrated that UCC118 capable of inducing cytokines Th2 dysbalance-type (i.e. IL-6 and soluble receptor of IL-6), thus suppressing the production of inflammatory cytokines, such as TNFα and IL-1β. Thus, these results confirm that the absorption UCC118 would be useful for patients suffering from inflammatory diseases such as IBD.

Example 4: Test for anti-inflammatory bacterial strains.

The number of lactic acid bacteria isolated from the gastrointestinal tract of man, was investigated using this new system of analysis anti-inflammatory effect. All bacterial strains were taken from cultures stored under glycerol at -20°S, and incubated anaerobically overnight in MRS-broth and washed in medium containing the antibiotic. Monolayers of epithelial cells were grown for 6 weeks before adding RVMS and bacterial cells.

The results of these stimulations can be observed on Fig. Compared with the control cultures of two bacterial strains inhibited the production of TNFα. Two strains of Lactobacillus salivarius strain UCC118, which inhibited the production of TNFαare subject to the WO-A-9835014. Bifidobacterium longum infants, strain UCC35624 is an object of the PCT application, filed concurrently with the present application.

Inflammation

Inflammation is a term used to describe the local accumulation of fluid, plasma proteins and white blood cells in place which has been subjected to physical damage, infection, or where the immune response. Control of the inflammatory reaction is applied to a number of levels (as a review, see Henderson C. and Wilson, M. 1998, "Bacteria-Cytokine interactions in health and disease". Portland Press, 79-130). Control factors include cytokines, hormones (eg, hydrocortisone), prostaglandins, reactive intermediate compounds and leukotrienes. Cytokines are low-molecular biologically active proteins, which are involved in the generation and control of immune and inflammatory reactions, while also regulating the development, tissue healing and haematopoiesis. They provide a means of communication leukocytes among themselves and with other cell types. Most cytokines are pleiotropic and have multiple biologically overlapping activity. Cytokine cascades and network control inflammatory response, and not the effect of individual cytokines on the individual cell type (Arai Kl, et al., Annu. Rev. Biochem. 1990, 59:783-836). Attenuation of the inflammatory response leads to lower concentrations of sootvetstvujushij activating signals and other mediators of inflammation, leading to the cessation of the inflammatory response. TNFα is a pivotal proinflammatory cytokine, because it initiates a cascade of cytokines and biological activity, leading to an inflammatory state. Therefore, at the present time for the treatment of inflammatory diseases are agents that inhibit TNFαsuch as infliximab.

I believe that proinflammatory cytokines play a major role in the pathogenesis of many inflammatory diseases, including inflammatory bowel disease (IBD). The currently used methods of treating IBD is aimed at reducing the levels of these proinflammatory cytokines, including IL-8 and TNFα. Such treatments may also play a significant role in the treatment of systemic inflammatory diseases such as rheumatoid arthritis.

Due to the detected inventors anti-inflammatory properties of Lactobacillus salivarius these strains may find potential application in the treatment of several inflammatory diseases, especially when used in combination with other anti-inflammatory treatments, such as nonsteroidal anti-inflammatory drugs (NSAID) or Infliximab.

Diarrhoeal disease

The barrier function of the intestinal epithelium can be reduced during secretion, mediated not the main (acetylcholine) and immune (histamine) systems. Some bacterial toxins can also induce CA2+and RKS-dependent secretion and thus can disrupt the epithelial barrier (Ganguly NK and T. Kaur Indian J. Med. Res. 1996, 104:28-37; Groot JA. Vet. Q. 1998, 20(S3):45-9). A number of studies investigated the prevention and treatment of diarrhea with the use of probiotic bacteria. Subsequent work has demonstrated the effectiveness of the introduction of lactic acid bacteria for both prophylactic and therapeutic use against diarrhea in premature babies, infants, children (Isolauri E, et al., Dig. Dis. Sci. 1994 Dec; 39(12):2595-600) and in the treatment of diarrhea associated with antibiotics (Siitonen S, et al., Ann. Med. 1990 Feb; 22(1):57-9), and traveler's diarrhea (Oksanen PJ, et al., Ann. Med. 1990 Feb; 22(1 ):53-6).

Whereas anti-inflammatory effect, the inventors have found that Lactobacillus salivarius may also have Antidiarrhoeal effect may be achieved through drug modulation of camp (cyclic amp). Cl-secretion-dependent cyclic AMP, is a major secretory path in the human intestine (Brzuszczak IM, et al., J. Gastroenterol. Hepatol. 1996; 11(9):804-10). Antidiarrhoeal action cannot be limited to diarrhea, the cause of which is the gastro-intestinal inflammation, and may refer to the General treatment of diarrhoeal diseases.

Autoimmune disease

Immune is the first system has a large set of specificdate, expressed b - and T-cells. Some of these specificdate aimed at "their" components. The recognition of "his" is normally controlled by the deletion clone and inactivation of lymphocytes activated against "their". However, there is a constant background of autoimmunity with antibodies to many proteins found in serum. Violation in the system of recognition "its not your" leads to autoimmunity. In autoimmune disease resulting from an immune response causes damage to the tissue carrier "guilty" antigen. The deposition of immune complexes, hypersensitivity type II and cell-mediated reactions are the most important mechanisms by which autoimmune damage. Examples of autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, insulin-dependent diabetes mellitus, severe pseudoparalysis myasthenia and pernicious anemia, but are not limited to them. The inventors have found that Lactobacillus salivarius is a bacterium together with immunomodulating. Thus, the absorption either in the form of a single component or in combination with other bacteria in patients suffering from autoimmune disease, may limit organ damage and to help restore normal homeostasis of the body./p>

Inflammation and cancer

Production of multifunctional cytokines with a wide range of tumor types suggests that patients with cancer is constantly undergoing significant inflammatory responses. At present it is not clear what protective effect has this answer against the growth and development of tumor cells in vivo. However, these inflammatory responses can have a harmful effect on the tumor bearing host. Complex interaction of cytokines involved in the regulation of the production of cytokines and cell proliferation in tumor and normal tissues (McGee DW, et at., Immunology Sep 1995, 86(1):6-11; Wu S, et al., Gynecol. Oncol. 1994 Apr, 53(1):59-63). Long considered that the reduction of weight (cachexia) is the single most common cause of death in patients with cancer (Inagaki J, et al., Cancer Feb 1974, 33(2):568-73), and primary malnutrition is a sign of poor prognosis (Van Eys. J. Nutr. Rev. 1982 Dec, 40(12):353-9). The tumor to grow and spread must induce the formation of new blood vessels and destroy the extracellular matrix. The inflammatory response may play a significant role in the above-mentioned mechanisms, thus contributing to the ill health of the owner and progression of tumors. Due to anti-inflammatory properties of Lactobacillus salivarius these bacterial strains can reduce the rate of malignant transformation of the notches. Moreover, intestinal bacteria can produce from dietary compounds substances with genotoxic, carcinogenic and activating tumor development action, as well as intestinal bacteria can activate procarcinogens in DNA-reactive agents (Rowland I.R. (1995). Toxicology of the colon: role of the intestinal microflora. In: Gibson G.R. (ed). Human colonic bacteria: role in nutrition, discrimination and pathology, pp.155-174. Boca Raton CRC Press). Generally, species of Lactobacillus have low activity of enzymes in the metabolism of xenobiotics in comparison with other populations in the intestine, such as bacteroids, eubacteria and Clostridium (Saito Y., et al., Microb. Ecol. Health Dis. 1992, 5, 105-110).

Therefore, the increase in the number of bacteria Lactobacillus in the intestine can favorably modify the levels of these enzymes.

Prebiotics

The introduction of probiotic microorganisms is carried out by the ingestion of a microorganism in a suitable medium. It would be useful to provide an environment that will promote growth of these probiotic strains in the colon. Adding one or more than one oligosaccharide, polysaccharide or other prebiotic promotes the growth of lactic acid bacteria in the gastrointestinal tract (Gibson, GR. Br. J. Nutr. 1998; 80(4):S209-12). Prebiotics call any non-viable food component in a specific way is fermented in the colon inherent bacteria, which is s considered to be useful for example, bifidobacteria, lactobacilli. Types of prebiotics may include prebiotics containing fructose, xylose, soy, galactose, glucose and mannose. The joint introduction of a probiotic strain with one or more prebiotic compound may enhance the growth of introduced probiotic in vivo, bringing more obvious health benefits, and is called symbiotic.

Other active ingredients

Obviously, Lactobacillus salivarius can be administered prophylactically or as a treatment method, either by itself or with other probiotic and/or prebiotic materials described above. In addition, these bacteria can be used as part of the preventive scheme or schemes of treatment with other active materials, such as materials used for the treatment of inflammation or other disorders, particularly of the gastrointestinal tract. Such combinations can be entered in a single drug or in separate preparations, administered at the same or at different times, and using the same or different routes of administration.

The invention is not limited to the above-described embodiments, which may differ in the details.

Subcutaneous administration of probiotic bacteria is associated with attenuation of colitis and arthritis in mice

The aim of this study was: 1) is the definition of the effects of systemic injections L. salivarius 118 on colitis in mice with knockout of Il-10, and 2) determine the effects of subcutaneous administration of L. salivarius 118 on a murine model of arthritis (collagen-induced arthritis.

Methods

Animals

Mice with knockout of interleukin-10 (IL-10 KO mouse)

This study used twenty female mice 129 Ola × C57BL/6-IL 10 CO at the age of 7-9 weeks (&K Universal Ltd, East Yorkshire, UK; The Jackson Laboratory, Bar Harbor, Maine, USA). These mice were maintained on homozygous phenotype and contained in the special conditions of the absence of pathogens, and maintained a temperature of 20±2°and a 12-hour cycle of light/dark. After the start of the study, all mice consumed a standard non-sterile food. Mice had free access to feed and water.

Mouse DBA/1

Used twenty-six male mice DBA/1 at the age of 6-8 weeks (Harlan UK Ltd, Oxon OH ITP, UK). These mice were maintained on homozygous phenotype and contained in the special conditions of the absence of pathogens, and maintained a temperature of 20±2°and a 12-hour cycle of light/dark. After the commencement of this study, mice had free access to feed and water.

Probiotic strains

L. salivarius 118 ssp. salivarius UCC118 was originally isolated from the ileocecal region of the adult person subjected to reconstructive surgery. This probiotic strain is allocated on the basis of what he has to pers is included probiotic properties. Briefly, these properties include: the origin of man, the lack of pathogenicity, resistance to intestinal acid and bile, the ability to adhere to epithelial cells of human rights and the ability to temporarily colonize the gastrointestinal tract of man and to be metabolically active in it. L. salivarius 118 accordingly cultured broth Man, Rogosa, Sharpe (MRS) (Oxoid, UK) at 37°under anaerobic conditions for 24 hours. Prior research has identified a spontaneous rifampicin - resistant variant strain in order to facilitate easy identification of this bacterium from other lactic acid bacteria.

Model II-10 TO colitis

Twenty II-10 KO mice were divided randomly into two groups of ten mice per group. L. salivarius 118) was subcutaneously injected to the study group and the control group was subcutaneously injected with sterile phosphate-saline buffer solution (PBS). L. salivarius 118 first were grown in 10 ml MRS broth by incubating overnight at 37°under anaerobic conditions. Bacteria are washed twice and re-suspended in sterile PBS to a final concentration of 1×109in ml was Subcutaneously injected dose of 1x108bacteria/mouse. These inoculation was performed at weeks 2, 4, 6, 10, 14 and 18, and mice were killed after 19 weeks.

Samples of faeces of mice were collected every week during the PE the iodine research. The study was completed through 19 weeks of feeding, and at this time all surviving mice were killed by displacement of the vertebrae. Blood samples were obtained by puncture of the heart for serological analysis. Blind intestine and the colon were fixed in formaldehyde for histopathological analysis. The spleen was removed from each mouse at killing and splenocytes were isolated for in vitro cultivation.

Histopathology

When the death of the blind intestine and proximal colon (ascending and transverse colon intestine and distal intestine (descending colon the colon, rectum and anal canal) all mice were fixed in 10%formalin and were evaluated by two histopathology. Two of the independent expert, using histological index from 0 to 4, assessed the severity of inflammation in any part of the gastrointestinal tract of mice. This index is based on the degree of erosion of the epithelial layer, the reduction of goblet cells and inflammatory cell infiltration (0 - normal; 1 - minimal evidence of inflammatory infiltrate; 2 - significant evidence of inflammatory infiltrate (cryptic, crypt abscesses); 3 - significant evidence of inflammatory infiltrate with a decrease in goblet cells; 4 - significant evidence of inflammatory infiltrate with erosion of the mucous membrane).

Culturespecific

Spleens from mice were removed at the time of the killing. Each spleen were immediately placed serum-free medium Needle, modified by way of Dulbecco (DMEM). The spleen was sifted through a sterile metal filter in 5 ml of 0.87%ammonium chloride for lysis of erythrocytes. Cell suspension was twice centrifuged at 100 g for 10 minutes. Cells re-suspended in DMEM (10% fetal calf serum) at a concentration of 1×106cells/ml for in vitro cultivation. Selected cells were cultured together with proinflammatory bacterium Salmonella typhimurium (1×106cells/ml) for 72 hours at 37°C. Cellular supernatant were isolated and stored at -80°C. Analysis of cytokines was performed on supernatant using enzyme-linked immunosorbent assay (ELISA) (BD Pharmingen, Oxford, UK). Analyzed the cytokines represented the tumor necrosis factor α (TNF-α), interleukin-12 (IL-12) and transforming growth factor - β (TGF-β).

Microbial analysis

Samples of faeces were collected every week, weighed and dispersible in 10 ml of PBS. Microbial analysis of faecal samples included the calculation of L. salivarius 118 ssp. salivarius UCC118, the total number of lactic acid bacteria, total number of bifidobacteria, coliform and Clostridia perfringens. This analysis was carried out by seeding by pouring and races is ostranenie on MRS-agar (pH 5.5) plus rifampicin; MRS-agar with the addition of 0.05% cysteine hydrochloride (Sigma, St. Louis, Missouri, USA) plus rifampicin; MRS-agar; MRS-agar with 5% sheep blood, 0.2% lithium chloride (BDH Laboratory Supplies, Poole, UK), 0.3% sodium propionate (Sigma) and 0.05% cysteine hydrochloride (Sigma), on agar with bile and purple red, environment, OPSP, selective for Clostridia perfringens, with the addition of A SR 76 and SR 77, respectively (all from Oxoid, UK, unless otherwise stated). Cups with agar with bile and purple red incubated under aerobic conditions for 24 hours, and all the other cups were incubated anaerobically for 48 hours at 37°C. Anaerobic conditions were created using sets, generating CO2, (Anaerocult A, Merck Darmstadt, Germany) in a pressurized gas vessels (BD BBL, Dublin, Ireland).

Collagen-induced arthritis

Twenty-six mice DBA/I was randomly split into three groups. Mice in the first group were injected subcutaneously L. salivarius UCC118 dose of 1×108bacteria/mouse (preparation is the same as for IL-10 TO study). The second group was subcutaneously injected equal volume of sterile PBS. Inoculation was carried out in weeks 1, 4 and 8. The last group (n=6) did not receive any of these treatments.

At week 6 the arthritis was induced as follows: bovine collagen type II (Chondrex) was dissolved in 0.05 M acetic acid to a concentration of 2 mg/ml by stirring at 4°With those who tell of the night. Then this mixture was emulsiable in equal volumes of complete adjuvant's adjuvant (2 mg/ml of the strain M. tuberculosis H37Ra (Difco BD, Dublin, Ireland). Groups 1 and 2 were immunized subcutaneously in the tail, 100 µl per week 6. On week 9 in all three groups was carried out by booster immunization with 50 μl of collagen emulsified in incomplete Freund's adjuvant (Difco). From week 10 onwards mice every day was estimated on the subject of visual appearance of arthritis in peripheral joints. Visual symptoms were assessed using the following index: 0 - normal; 1 - weak, but definite redness and swelling of ankle or wrist, or apparent redness and swelling limited to individual digits, regardless of the number of affected fingers; 2 - mild redness or swelling of ankle or wrist; 3 - severe redness and swelling of the entire paw, including the fingers; 4 - maximum inflamed paw with involvement of multiple joints. The study was completed in 12 weeks, and all mice were killed by displacement of the vertebrae. When the killing was measured thickness of each of the legs using a Vernier caliper.

Histopathology

When the death of the limbs were removed and fixed in formaldehyde for histopathological analysis, the independent expert. After signing in paraffin, limb surface decalzinirute using Calbonex (KB Scientific, Togher,Cork, Ireland). The tissue sections (7 μm) were stained with hemolysin and eosin. Histological analysis of cartilage destruction and bone through the formation of pannus and infiltration of mononuclear cells in synovial tissues was performed using the following metrics: the destruction of cartilage and bone through the formation of pannus: 0 - no changes; 1 - minor change (formation of pannus in cartilage); 2 - moderate change (invasion of pannus in cartilage/subchondral bone plate); 3 - strong change (invasion of pannus in the subchondral bone plate); infiltration of mononuclear cells: 0, no infiltration; 1 - slight infiltration: 2-moderate infiltration; 3 - strong infiltration.

Statistical analysis

Analysis of variance for distinguishing groups of inflammatory activity was performed using ANOVA analysis, and the difference between groups on microbial number was assessed using the area under the curve.

Results

Model II-10 KO colitis

Histopathology

After killing assessment 0-4 awarded each studied intestine: the caecum, proximal colon (ascending and transverse colon) and distal intestine (descending colon, rectum and anal canal), and the total possible score 12 must be awarded each the mu Department. After examination of all slices was calculated an average for each group. Average score for inflammation control group was 8.25 (0,94), and the average score of inflammation for the group treated with L. salivarius UCC118, made 4.35 (0,9). Thus, the "probiotic" group showed a significant reduction in the assessment of inflammation compared to the control group (p<0,05) (Figure 1'). In addition, it was found that four mice in the PBS control group have severe pancholi with the same degree of lesions of the proximal and distal colon. In the probiotic group was not identified case pancolitis.

Microbial analysis

The faeces samples from all mice were analyzed to assess changes in the microflora and definition of transit probiotic strain. No significant differences cultivated bacteria counting in samples of faeces total number of lactic acid bacteria, total number of bifidobacteria, coliform and Clostridia perfringens (data not shown). The strain of L. salivarius 118 was not isolated from any of the mouse in the control or "probiotic" group (data not shown).

Immunological assessment

Analysis of cytokines was performed on supernatant of splenocytes by ELISA after stimulation in vitro proinflammatory bacterium Salmonella typhimurium. Analyzed the cytokines represented TNF-α, IL-12 and TGF-β .

The levels of TNF-α were significantly reduced in the group, which was introduced L. salivarius 118, after stimulation of proinflammatory stimulus (the levels of TNF-α in the control group was 1522,4 (to 112.2), the levels of TNF-α in the second group, which was introduced L. salivarius 118, amounted 872,8 (143,8); (p<0,05) (Fig). The levels of TNF-α not detected in unstimulated cultures.

The levels of IL-12 after stimulation Salmonella typhimurium showed a significant reduction in the probiotic group (p<0,05). The levels of IL-12 in the control group was 2471,2 (256) compared to 1386,4 (347,2) in the group which was administered L. salivarius 118 (p<0,05) (Fig B). The levels of IL-12 detected in supernatant of unstimulated splenocytes, were lower in the probiotic group compared with the control group.

In the absence of stimulation, the levels of TGF-β were increased in the probiotic group compared with the control group (levels of TGF-β in the control group amounted to 114.4 (71,6); the levels of TGF-β in the second group, which was introduced L. salivarius 118, amounted 362,3 (79,6); p<0,05) (Fig). No statistical significant difference between the levels of TGF-β in the two groups after stimulation with Salmonella.

The levels of cytokines in serum samples were below detectable limits in all groups in the analysis by ELISA.

Collagen-induced arthritis

Daily clinical score of mice on the subject of visual signs of arthritis/p>

Daily macroscopic quantitative assessment of visual signs of arthritis was performed from week 10 until the killing (week 12) using the accepted evaluation system.

L. salivarius 118 had a vast effect on the development of the disease. Since week 10, day 3, week 11, day 12, observed a significant reduction in the evaluation of arthritis in the group, which was introduced by L. salivarius 118 (p<0,05). Reducing the effects of arthritis was also significant at week 12, day 15 (p=0.05) (Figure 3').

The thickness of the legs

The thickness of each paw was measured using a caliper at the time of the killing. The results were described as the thickness of the legs are more of the norm (the average thickness for the unaffected/negative control group was of 1.62 (0,051 mm). In the group, which was introduced L. salivarius 118, observed a significant reduction in thickness compared with the control group (p=0.0002) (in the group, which was introduced L. salivarius 118, the thickness of the paw was 0,292 (0,071); in the control group, the thickness of the paw was 0,677 (0,071 )(Fig A).

Histopathology

After killing histopathological changes were evaluated using a scale of 0-3 based on the destruction of cartilage and bone through the formation of pannus (Figure 5'). Infiltration of inflammatory cells was also assessed on a scale of 0-3, ranging from a lack of infiltration of cells to a strong infiltration. After the research is all slices was calculated an average for each group, moreover, the maximum possible score was 6. In the group vaccinated with L. salivarius 118, observed a significant decrease in infiltration of inflammatory cells compared with the control group (average rating of inflammatory cells in the control group was 1,18 (0,11), and the group vaccinated with L. salivahus 118, this estimate of 0.85 (0,11); p=0.04) (Fig B). Found a significant decrease in the destruction of cartilage and bone, as well as infiltration of inflammatory cells in the anterior limbs of mice that were administered probiotic bacteria (average histological score for the control group amounted to 1.30 (0,46), and in the group which was administered L. salivarius 118, this assessment was 0,46 (0,19); p=0.03) (Fig).

Discussion

The results of this study confirm that systemic injection of L. salivarius 118 anti-inflammatory effect. Dose and frequency of injection were arbitrary. The systemic nature of probiotic activity is reflected in the reduction of Pro-inflammatory cytokines and increasing regulatory cytokine TGF-β.

In General, the mechanism of action of probiotics is not fully understood. It has been shown that the intestinal flora influences the development and functioning of the immune system, and believe that the microflora is influenced by, among other intestinal function, immune response of the mucous membrane through which peredachi signal to the intestinal epithelium. I believe that the interaction between Toll-like receptors and dendritic cells in the gut are involved in this relationship. Dendritic cells inhabiting the intestinal mucosa are mainly immature and potentially susceptible to modulation by the environment containing microorganisms.

Model II-10 TO colitis and collagen-induced arthritis is associated with abnormal regulation of proinflammatory Th1 cytokines. In II-10 TO model IL-12 is a key mediator responsible for the induction of colitis, and is required to maintain proliferation chronically activated Th1 cells. It was shown that anti-IL2 and anti-TNF-treatment reduces joint damage in collagen-induced arthritis. This confirms the role of IL-12 and TNF-α arthritis, and modulation of these cytokines provides a therapeutic target in cancer. Associated negative regulation of levels of IL-12 and THF-αdetected in this study means that the probiotic effect may be mediated by changes in these cytokines.

The impact of probiotics on the microbial flora, inflammation and cancer development in il-10 ko mice

The aim of this study was to evaluate the ability of Lactobacillus salivarius ssp. salivarius UCC118 to modulate the severity of enterocolitis in IL-10 KO mice in a controlled pilot study.

Methods

This study used ten male mice C57BL/6 J-10 and IL ten male mice C57BL/10 J-IL 10 ages 4-8 weeks (Jackson Laboratories, Maine, USA). These mice were maintained on homozygous phenotype and contained in the special conditions of absence of pathogens. In mice maintained a 24-hour cycle of light/dark. After the start of the study, all mice consumed a standard non-sterile food.

Probiotic strain

L. salivarius ssp. salivarius UCC118 (NCIMB 40829) was originally isolated from the ileocecal region of the adult person subjected to reconstructive surgery. This probiotic strain is allocated based on the fact that he has suitable probiotic properties. Briefly, these properties include: the origin of man, the lack of pathogenicity, resistance to intestinal acid and bile, the ability to adhere to epithelial cells of human rights and the ability to temporarily colonize the gastrointestinal tract of man and to be metabolically active in it. L. salivarius 118 accordingly cultured broth Man, Rogosa, Sharpe (MRS) at 37°under anaerobic conditions for 24 hours. Prior research has identified a spontaneous rifampicin - resistant variant strain UCC118 in order to facilitate easy identification of this bacterium from other lactic acid bacteria.

Study feeding

Two is cat IL-10 KO mice were divided randomly into one of two groups. "Probiotic" group every day gave 1×109Lactobacillus salivarius ssp. UCC118 in pasteurized milk, and the control group were given only unmodified pasteurized milk. L. salivarius 118 first were grown in 10 ml MRS broth (Oxoid, UK) by incubation overnight at 37°under anaerobic conditions. 1% inoculum (about./about.) was transferred into 400 ml of fresh MRS broth and incubated as described above. UCC118 was besieged by centrifugation and re-suspended at a concentration of 1×109cells/ml in 10% pasteurized skimmed milk. UCC118 was given IL-10 KO mice in the water vessels and mice had free access to this mixture.

Samples of faeces of mice were collected before feeding (week 0) and every week throughout the study period. The study was completed after 16 weeks of feeding, and at this time all surviving mice were killed by displacement of the vertebrae. Blood samples were obtained by puncture of the heart for serological analysis. Samples of intraluminal contents were removed under sterile conditions from the ileum, cecum and colon for microbiological analysis, and pieces of ileum, cecum and colon were fixed in formaldehyde for histopathological analysis.

Serology

Levels specific to UCC118 antibodies in serum was measured by the BL is using standard analysis of agglutination. Briefly, serum from IL-10 KO mice were diluted 1/10, 1/20, 1/40, 1/80 and 1/160 sterile phosphate-saline buffer solution (PBS). Serum incubated overnight with probiotic UCC118 and analyzed for the presence of agglutination. The reciprocal of the lowest dilution to the result of the agglutination was used as an indicator of levels of antibodies specific to UCC118.

Microbial analysis

Samples of faeces were collected every week, weighed and dispersible in 10 ml of PBS. Microbial analysis of faecal samples included the calculation of L. salivarius ssp. salivarius UCC118, the total number of lactic acid bacteria, total number of bifidobacteria, enterococci, bacteroids and coliform. This analysis was carried out by seeding by pouring and spreading on MRS-agar plus rifampicin; MRS-arape; MRS-arape with the addition of 5% sheep blood, and 0.2% LiCl2, 0.3% sodium propionate and 0.05% cysteine, on agar Slanetz and Bartley, on agar Wilkins Chalgren with the addition of additives SR108 and 5% horse blood and agar with blood red and purple (Oxoid, UK), respectively. In addition samples of faeces and content samples from the ileum, cecum and colon were evaluated for the presence of the same bacteria as described above, plus Clostridium perfringens, as measured by the OPSP agar with the addition of A SR 76 and SR 77 (Oxoid, UK). Cups with agar with blood and red and purple agar Slanetz and Bartley encubierta and in aerobic conditions for 24 hours and 48 hours, and all the other cups were incubated anaerobically for 48 hours at 37°C. Anaerobic conditions were created using sets, generating CO2, (Anaerocult A, Merck) in a pressurized gas vessels (BBL).

Histopathology

When the death of the pieces of the small intestine, cecum and colon were fixed in 10%formalin and were evaluated histologically. Two of the independent expert, using histological index from 0 to 5, assessed the severity of damage due to inflammatory activity in the gastrointestinal tract of mice. This index is based on the degree of erosion of the epithelial layer, the reduction of goblet cells and inflammatory cell infiltration. In addition, these fabrics were investigated for the presence of neoplastic cells.

Statistical analysis

Analysis of variance for distinguishing groups of inflammatory activity was performed using analysis, ANOVA, and differences between groups on microbial number was assessed using the area under the curve. Fisher exact test was used to determine statistical differences in the development of tumors and mortality in mice between the group, which was given probiotic strain and the control group.

Results

Serology

After consumption of probiotic bacteria UCC118 for 16 weeks in the peripheral blood is determined the levels of antibodies, specific to the bacteria. No differences in the levels of antibodies to mice consuming UCC118, compared to mice consuming only placebo (10,1+4,1 compared to 9.7+2,5 respectively). This suggests that UCC118 no system is perceived by the immune system IL-10 KO mice.

Microbiology

During weeks 1 feeding in faeces from all mice in the probiotic group was identified probiotic UCC118. Within 16 weeks of feeding UCC118 was allocated in the amount of approximately 1×106per gram of faeces (1"). From mice in the placebo group UCC118 not cultivated. Levels of coliform and enterococci in faeces were reduced in mice consuming UCC118, compared to mice consuming only pasteurized milk (2"). Total levels of lactic acid bacteria, bifidobacteria and bacteroids has not changed in both groups (Figure 2").

In addition to weekly microbiological analysis after killing took samples from the ileum, cecum and colon. The number of C. perfringens was significantly reduced in mice consuming UCC118, especially in the colon (P<0.05) as compared to mice consuming only product placebo (3"). Found no significant differences in the total number of lactic acid bacteria, bifidobacteria, coliform, enterococci or bacteroids.

Histopatholo the Oia

After killing both groups of mice were evaluated inflammatory activity of the caecum, ileum and colon (Figure 4"). Observed a strong trend to reduced inflammatory activity in all sites (table 2). The most significant difference inflammatory activity between the two groups was observed in the colon (P=0,09). Observed a significant reduction of cancer between the two groups. Gastrointestinal neoplastic transformation in mice in the placebo group was 50%, while only 10% of mice in the group consuming UCC118, watched neoplastic change (P=0.07). Moreover, 20% of mice in the placebo group died before the end of the study, and in the group consuming UCC118, all mice survived.

Discussion

The results of this study demonstrate the survival of probiotic Lactobacillus salivarius ssp.salivarius UCC118 after transit through the gastro-intestinal tract, and significant change in the gastro-intestinal flora associated with probiotic. Consumption of probiotics was associated with a tendency to decrease tumor growth and attenuation of inflammation of the gastrointestinal tract. These results justify a large-scale study using probiotics for reliable estimates of the statistical significance of these studies.

For modulation of gastro-Kish is Noah flora of this probiotic lactic acid bacterium proposed a number of mechanisms. UCC118 produces in vitro antimicrobial factor, which is antagonistic in relation to a broad range of gram-positive and gram-negative microorganisms. The production of this antimicrobial factors in vivo may eliminate competing microorganisms, providing the advantage of survival of new bacteria. In addition, UCC118 strongly adheres to the epithelial cells of the gastrointestinal tract in vitro. Thus, the competitive removal of other microorganisms from this niche will have an impact on the composition of the faecal flora.

Consumption UCC118 reduces inflammation of the gastrointestinal tract in the mouse model. Colonization of the gastrointestinal tract that probiotic bacteria also leads to a modification of bowel flora with possible elimination of Pro-inflammatory species. Thus, removal of inflammatory lesions represents a possible mechanism by which probiotic bacteria can modulate the severity of the disease. In addition, probiotic bacteria may have a more direct impact on inflammatory responses in the gastrointestinal tract through interaction with the immune system of the mucosa. The right balance of Th1/Th2 responses in the gut is critical to maintain the integrity of the intestine. Consumption Poo is the number of some types of bacteria can help restore the proper Th1/Th2 balance in mice with knockout of Il-10. Direct interaction UCC118 and the immune system of the mucosa can induce Th2 responses type, leading to the restoration of the Th1/Th2 balance and reduce inflammation in the model.

In addition, the authors investigated the role of probiotic UCC118 in reducing the rate of neoplastic changes in the gastrointestinal tract. Inflammation of the gastrointestinal tract has a strong influence on the integrity of the mucous membrane and its ability to resist damage induced by transient factors, thus increasing the risk of developing a neoplastic disease. In addition, some inflammatory mediators can stimulate the growth of tumor cells in the gastrointestinal tract. Intestinal bacterial flora is involved in the pathogenesis of malignant diseases of the gastrointestinal tract. Sterile rats treated with the carcinogen 1,2-dimethylhydrazine, have a lower incidence of tumors of the colon than processed similarly rats with normal microflora. Intestinal bacteria can produce from food substances with genotoxic, carcinogenic and tumor-stimulating activity, and intestinal bacteria can activate procarcinogen. In General, lactic acid bacteria are not involved as causal factors in these painful conditions and deistvitel is but can anlagenservice harmful components of the gastro-intestinal flora. Thus, probiotic UCC118 can slow the progression of malignant diseases of the gastrointestinal tract by reducing inflammatory activity in it or by modification of the bacterial flora.

Some probiotic bacteria are very attractive biotherapeutic agents for the treatment of inflammation of the gastrointestinal tract due to their impact on the composition of the intestinal flora and immune system activity. Consumption of probiotic UCC118 successfully changed the gastro-intestinal flora, reduced inflammation and reduced neoplastic lesions in this mouse model. Intriguing and clinically significant opening of the authors was a reduced incidence of colon cancer in animals consuming probiotic bacteria. In this respect, the reported beneficial effects of probiotics in people with ulcerative colitis may have special application for long-term reduction dysplasia/neoplasia in these patients.

The ileum
Table 2.
Assessment of inflammation of the gastrointestinal tract. Compared assessment of inflammation of the ileum, cecum and colon in mice consuming probiotic UCC118 and product placebo.
CecumThe colonIntestinal tract
"Probiotic" group0,83±0,402,70±0,402,33±0,402,08±0,28
The placebo group1,43±0,533,33±0,293,33±0,412,81±0,27

1. The use of Lactobacillus salivarius UCC118[NCIMB 40829] for prevention and/or treatment of undesirable inflammatory activity.

2. The use according to claim 1, where undesirable inflammatory activity is undesirable gastrointestinal inflammatory activity.

3. The use according to claim 2, where gastrointestinal inflammatory activity is an inflammatory disease of the intestines.

4. The use according to claim 3, where gastrointestinal inflammatory activity is Crohn's disease.

5. The use according to claim 3, where the gastro-intestinal activity is ulcerative colitis.

6. The use according to claim 2, where gastrointestinal inflammatory activity represents the irritable bowel syndrome.

7. The use according to claim 2, where gastrointestinal inflammatory activity is an inflammation of the abdominal pouch (pouchitis).

8. The use according to claim 2, where the gastro-to the muscle inflammatory activity is a post-infectious colitis.

9. The use according to claim 2, where gastrointestinal inflammatory activity is a diarrheal disease.

10. The use according to claim 9, where diarrhoeal disease due to Clostridium difficile.

11. The use according to claim 9, where diarrheal disease caused by rotavirus.

12. The use according to claim 9, where diarrhoeal disease is a post-infectious diarrhoeal disease.

13. The use according to claim 2, where inflammatory activity caused by cancer of the gastrointestinal tract.

14. The use according to claim 1, where the inflammatory activity is a systemic inflammatory disease.

15. The application 14, where systemic inflammatory disease is a rheumatoid arthritis.

16. The use according to claim 1 or 2, where undesirable inflammatory activity is due to an autoimmune disorder.

17. The use according to claim 1, where undesirable inflammatory activity due to cancer.

18. The use of Lactobacillus salivarius UCC118[NCIMB 40829] for cancer prevention.

19. The use according to any one of claims 1 to 18, where Lactobacillus salivarius UCC118[NCIMB 40829] contained in the product.

20. The application of claim 19, where the product includes a different probiotic.

21. The application of claim 19 or 20, where the product comprises a prebiotic.

22. The use according to any one of p-21, where the product includes a carrier for oral administration.

23. The application of article 22 where a carrier for oral administration is a pharmaceutically acceptable carrier in the form of tablets, capsules or powder.

24. The application of article 22 where a carrier for oral administration is a protein and/or peptide, in particular proteins and/or peptides that are rich in glutamine/glutamate; lipid; carbohydrate; vitamin, mineral and/or trace element.

25. The application of article 22 where a carrier for oral administration is a food product, such as fermented milk, yoghurt, frozen yoghurt, milk powder, concentrated milk, processed cheese, salad or drinks.

26. The use according to any one of p-25, where Lactobacillus salivarius UCC118[NCIMB 40829] use more than 106CFU/g product.

27. The use according to any one of p-26, where the product includes an adjuvant.

28. The use according to any one of p-27, where the preparation additionally comprises bacterial components.

29. The use according to any one of PP-28, where the product additionally includes medicinal beginning.

30. The use according to any one of p-29, where the preparation additionally comprises a biological connection.

31. The use according to any one of claims 1 to 30, where the specified strain or preparation intended for administration to animals.

32. Use p, where the animal is a mammal.

33. Use p where Miu is apitude is a person.

34. The use according to any one of claims 1 to 33, where Lactobacillus salivarius UCC118[NCIMB 40829] causes changes in immunological marker after the introduction into the system that contains cells that interact with the immune system, and cells of the immune system.

35. The application 34, where cells that interact with the immune system, are epithelial cells.

36. The application 34 or 35, where immunological marker is a cytokine.

37. Use p, where the cytokine is a tumor necrosis factor α (TNFα).

38. The use according to any one of PP 37, where cells that interact with the immune system, and cells of the immune system have an appropriate background.

39. The use according to any one of p-38, where cells that interact with the immune system, gastrointestinal, respiratory, or genitourinary origin.

40. The use according to any one of p-39, where cells of the immune system have gastrointestinal, respiratory, or genitourinary origin.

41. The use according to any one of claims 1 to 40, where Lactobacillus salivarius UCC118[NCIMB 40829] presented in the form of viable cells.

42. The use according to any one of claims 1 to 40, where Lactobacillus salivarius UCC118[NCIMB 40829] presented in the form of viable cells.

Priority items:

15.01.1999 according to claims 1-20, 22, 31-33, 41;

20.09.1999 on PP, 34-40;

17.01.2000 on p is .21, 23-24, 26-30, 42.



 

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3 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to producing nutrient media used in microbiology. Medium for culturing heterotrophic microorganisms comprises the following components, g: red California worms as a protein basis, 90-100; peptone, 9-11; sodium chloride, 4-5; agar-agar, 18-20, and distilled water, up to 1 l. Invention provides simple method for preparing medium, availability of substrate with high-nutrient properties and low cost.

EFFECT: improved and valuable properties of nutrient medium.

2 cl, 3 tbl, 5 ex

FIELD: biotechnology, biochemistry.

SUBSTANCE: invention relates to a method for enzymatic synthesis of L-phosphinotricine by transamination reaction from 4-(hydroxymethylphosphinyl)-2-oxobutyric acid and using aspartic acid as a donor of amino-group. The quantitative conversion can be carried out by using suitable technology and equimolar amounts of donor and amino-group acceptors that results to the complete utilization of donor amino acid, i. e. aspartic acid. Using thermostable aspartate aminotransferases provides high rate of reaction and high volume output, respectively.

EFFECT: improved preparing method.

9 cl, 3 tbl, 4 ex

FIELD: biotechnology, in particular meat product manufacturing.

SUBSTANCE: claimed method includes deep cultivation of Paracoccus denitrificants K-3 denitrification strain under aeration condition in nutrient medium containing organic amine nitrogen source, yeast-based nitrogen source, glucose, magnesium sulfate, manganese sulfate, and water; cell mass separation, concentration, blending with protective medium, freeze drying, and blending of bacterial concentrate with filler. Nutrient medium additionally contains sodium chloride, monosubstituted sodium phosphate, disubstituted potassium phosphate, and potassium nitrite; alkaline hydrolyzate of cattle blood, and redox potential reducing component. Pancreatic casein hydrolyzate containing 600 mg% of amine nitrogen or pancreatic defatted milk hydrolyzate containing 600 mg% of amine nitrogen are used as amine nitrogen source, and baking yeast autolyzate is used as yeast-based nitrogen source in specific component ratio. Preparation of present invention holds denitrification activity during long time (8-9 months) and assures low sodium nitrite concentration in finished meat products.

EFFECT: convenient bacterial preparation; meat products of improved characteristics.

2 cl, 1 dwg

FIELD: veterinary microbiology.

SUBSTANCE: the suggested method deals with reinoculation of mycobacterial L-forms onto dense nutritive medium Fast-3L for it additionally contains growth factor - native cattle or equine blood serum at the dosage of about 0.1-0.2 ml/5 ml medium. The innovation enables to increase sensitivity of the method and shorten the terms for obtaining revertant cultures.

EFFECT: higher efficiency.

2 ex, 2 tbl

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a nutrient medium containing components chosen in the following ratio, g/1 l: casaminic acids, 10-12; yeast extract, 3-5; potassium hydrogen phosphate, 7-8; magnesium sulfate, 0.1-0.2; sodium citrate dihydrate, 0.5-0.6; ammonium sulfate, 2-2.5, and glucose, 1-2. Using a liquid nutrient medium provides the intensive growth of staphylococci since first hours of cultivation and allows attainment of maximal yield of antibacterial compound after 8 h growth of culture. Invention is designated for culturing staphylococci in aim for preparing antibacterial peptide compounds.

EFFECT: improved and valuable properties of nutrient medium.

1 dwg, 2 tbl, 3 ex

FIELD: biotechnology, petroleum-extracting industry.

SUBSTANCE: invention relates to microbiological methods for extraction of residual oil from flooded pools at the late stage of the deposit exploitation. Method involves the successive pumping in an aqueous solution of mixture of active silt with a stabilizing agent into forcing stratum wherein starch and hydrolysis sugar are used in the following ratio of components, wt.-%: active silt, 0.1-5.0; starch, 0.1-1.0; hydrolysis sugar, 0.5-3.5, and water, the balance. Using the method in petroleum-extracting industry allows stabilization active silt particles in volume of the pumped solution and more penetration into stratum, to enhance effectiveness of petroleum extraction from heterogeneous collectors by 12.8%, to diminish flooding degree of extracted production and nonproductive water pumping in.

EFFECT: improved method for displacement.

2 tbl, 3 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to producing ferments, probiotics, biologically active supplements and foodstuffs. The consortium comprises Enterococcus durans, the strain RSH (VKPM B-8717) and Leuconostoc mesenteroides subsp. mesenteroides, the strain RSH (VKPM B-8716). Invention provides preparing the consortium with the stable ratio of cultures, high titer of microorganisms, enhancing organoleptic and rheological properties of prepared products and to prolong fitness period of product.

EFFECT: improved and valuable properties of consortium.

1 tbl

FIELD: biotechnology, microbiology.

SUBSTANCE: method involves mixing the following components, g/l: powdered fish pancreatic hydrolyzate, 22.0-27.0; sodium chloride, 4.0-6.0; agar, 14.5-15.5; sorbitol, 9.0-11.0, and distilled water, balance, up to 1 l. After heating and melting agar pH value is brought about to 7.2 ± 0.2, and 18-22 ml of indicator is added to the prepared medium consisting, g/l: acid fuchsine, 4.5-5.5; bromothymol blue, 3.5-4.5; sodium hydroxide (1% solution), 150.0-170.0 ml, and distilled water, balance, up to 1l. Acid fuchsine is dissolved preliminary in 1% sodium hydroxide solution and bromothymol blue and distilled water are added to the prepared solution. Invention provides enhancing clearness of change colonies color in differential medium, decreasing incubation time and prolonged storage of medium ready for using. Invention can be used in clinical and epidemiological studies.

EFFECT: improved preparing method, improved and valuable properties of medium.

1 ex

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to a compound and to all its enantiomeric and diastereomeric forms and pharmaceutically acceptable salts that are able to prevent extracellular release of inflammatory cytokines. Proposed compounds have the formula (I): wherein R represents: (a) -OR3 or (b) -NR4aR4b; R3 represents unsubstituted or substituted phenyl wherein substitutes are: (i) halogen atom; (ii) (C1-C6)-alkyl; (iii) trifluoromethyl; (iv) trichloromethyl; (v) tribromomethyl; (vi) cyano-group, and (vii) (C1-C6)-alkoxy-group; each R4a and Rb represents independently: (a) hydrogen atom or (b) -[C(R5aR5b)]xR6 wherein index x = 0-5; each R5a and R5b represents independently hydrogen atom, linear or branched (C1-C4)-alkyl, (C3-C7)-cyclic alkyl; R6 represents -OR7 or (C1-C4)-alkyl; R7 represents hydrogen atom or (C1-C4)-alkyl; R1 represents halogen-substituted phenyl; each among links R2a and R2b is chosen independently from the groups consisting of: (a) hydrogen atom; (b) -O(CH2)jR8; (c) -(CH2)jCO2R10; (d) -(CH2)jCON(R10)2; (e) a double bond when R2a and one R2b are chosen with formation of a double bond; (f) a ring when one R2a and one R2b are chosen with formation a ring and indicated ring is chosen from the group consisting of: (i) benzene and (ii) dioxalane; each R8 and R10 represents independently hydrogen atom or (C1-C4)-alkyl; j represents index from 0 to 5; m represents index from 1 to 3; n represents index from 1 to 3, and m + n = 4. Also, invention relates to a pharmaceutical composition based on abovementioned compounds that inhibits extracellular release of inflammatory cytokines, and a method for regulation of extracellular release of inflammatory cytokines.

EFFECT: valuable medicinal properties of compounds.

10 cl, 9 tbl, 11 ex

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