Patent Application
20150283185
The present invention relates to probiotic bacteria strains having a high anti-inflammatory activity. The present invention relates to bacteria strains as strongly inducers of Interleukin-10 (IL-10) production. In particular, the present invention relates to the anti-inflammatory activity shown by said bacteria strains due to its enhancement of IL-10 production in peripheral blood mononuclear cells, with on the other hand a low capability to stimulate the production of the pro-inflammatory Il-12, thus leading to a high IL-10/IL-12 ratio. Further, the present invention relates to the use of at least one bacterium strain for the preparation of a composition for the prevention or treatment of the inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS). Finally, the present invention relates to food products, such as probiotic dietary supplements containing at least one probiotic bacterium strain, as an active ingredient.
It is known that probiotics are live microorganisms which when administered in adequate amounts confer a health benefits on the host. Probiotic lactobacilli and bifidobacteria are increasingly recognized as a way to prevent and/or treat intestinal disorders.
Most of our encounters with antigens or infectious agents occur at mucosal surfaces, which include the surface lining the gastrointestinal, respiratory and genitourinary tracts. Since probiotics are usually absorbed orally, they are thus ideally suited to influence the immune response at the “mucosal frontier” of the gastrointestinal tract, representing more than 300 m2.
The intestinal immune system forms the largest part of the immune system. It interacts with a complex antigenic load in the form of food antigens, commensal bacteria, and occasional pathogens. Dendritic cells (DC) are pivotal in earliest bacterial recognition and in shaping T cell responses. Dendritic cells sense antigen in tissues before migrating to draining lymphonodes, where they have the unique ability to activate and influence functional differentiation of naive Tcells. Signals from DC can determine whether tolerance or an active immune response occurs to a particular antigen and furthermore influence whether a Th1 or Th2 immune response predominates: DC upregulate the co-stimulatory molecules, CD80 and CD86, and produce IL-12 which contributes to a Th1 response. Further, DC may produce IL-10 and IL-4 which promote the generation of a Th2 response or regulatory T cells.
Recognition of hazardous microbes, allergens and toxins as pathogenic agents activates the gastrointestinal immune system. Antigen-specific Treg cells, which mediate oral tolerance to commensal microbes, differentiate between harmless inhabitants of the gut and pathogens. A break in the development or maintenance of oral tolerance may result in an astounding array of detrimental inflammatory disorders, including inflammatory bowel disease (IBD) and colitis. IBD and colitis are conditions in which the immune system of patients reacts excessively to indigenous intestinal bacteria. Treg cell depletion in these disorders effectively breaches tolerance and allows for massive inflammation in the gut. In vivo transfer of Treg cells suppresses the development of the above diseases, through IL-10, TGF-β and CTLA-4-dependent mechanisms.
Probiotic strains can induce pro-inflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-12, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) as well as anti-inflammatory cytokines such as IL-10 and transforming growth factor β. IFN-γ and IL-12 potently augment the functions of macrophages and NK cells, which may be a possible mechanism of their anti-carcinogenic and anti-infectious activity. On the other hand, induction of IL-10 and transforming growth factor β is assumed to participate in the down-regulation of inflammation, since these cytokines can inhibit the functions of macrophages and T cells and promote the development of regulatory T cells. IL-10 is produced by many cells, including Th2 cells, DCs, monocytes, B cells, keratinocytes and regulatory T cells; it has an anti-inflammatory effect and primarily acts to inhibit the Th1 response. IL-10 drives the generation of a CD4+ T-cell subset, designated T regulatory cells 1 (Tr1), suppressing antigen-specific immune responses and actively down-regulates a pathological immune response in vivo.
Several intestinal conditions are under the umbrella of “Inflammatory Bowel Disease (IBD)”, including Crohn's disease, ulcerative colitis and pouchitis.
In inflammatory bowel disease, IL-10 is a cytokine of particular therapeutic interest since it has been shown in animal models that interleukin (IL)-10 (−/−) mice spontaneously develop intestinal inflammation.
It has been shown in animal models that probiotic strains displaying an in vitro potential to induce higher levels of the anti-inflammatory cytokine IL-10 and lower levels of the inflammatory cytokine IL-12, offer the best protection against in vivo colitis in the model.
Probiotic-mediated immunomodulation represents an interesting option in the management of IBD and it was shown that both the systemic and mucosal immune systems can be modulated by orally delivered bacteria. However, not all candidate probiotics have been proven equally efficient due to the differences in survival and persistence of the strain in the gastro-intestinal tract, and/or to strain-specific interactions of the probiotic with the host immune system. The selection of a successful protective strain may therefore rely on the proper screening of a large number of candidate strains for their technological and immunomodulatory performance.
Therefore, it remains the need to isolate and select bacteria strains having a marked anti-inflammatory activity. In particular, it remains the need to isolate and select specific bacteria strains as strongly inducers of IL-10 production. Further, it remains the need to isolate and select bacteria strains with a low capability to stimulate the production of the pro-inflammatory Il-12, thus leading to a IL-10/IL-12 ratio at least bigger than one. Finally it remains the need to find out and select bacteria strains which show high persistence in the gastro-intestinal tract due to their resistance to gastric juice, bile salts, pancreatic secretion and to adhesion to gut wall. Last but not least it is important to select bacteria strains without acquired antibiotic resistances.
The Applicant has selected a group of bacteria strains which are able to solve the outstanding problems present in the prior art.
According to a first aspect of the present invention, there is provided a group of bacteria strains or their cellular components having an immunoregulatory function through stimulation of Interleukin-10.
According to a second aspect of the present invention, there is provided a food product containing at least one bacterium strain or its cellular components, as an active ingredient.
According to a third aspect of the present invention, there is provided a composition containing at least one bacterium strain or its cellular components, for use as a medicament.
According to a fourth aspect of the present invention, there is provided a use of at least one bacterium strain or its cellular components for the manufacture of a medicament for the prevention or treatment of inflammatory conditions of the large intestine and small intestine.
According to a fifth aspect of the present invention, there is provided a use of at least one bacterium strain or its cellular components for the manufacture of a medicament for the prevention or treatment of functional bowel disorders.
The Applicant has tested bacteria strains belonging to the following species: L. acidophilus, L. crispatus, L. gasseri, L. delbrueckii, L. salivarius, L. casei, L. paracasei, L. plantarum, L. rhamnosus, L. reuteri, L. brevis, L. buchneri, L. fermentum, B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, and S. thermophilus.
Table 1 shows a group of bacteria strains which find a valid application in the contest of the present invention.
Streptococcus
thermophilus B39
Streptococcus
thermophilus T003
Lactobacillus
pentosus 9/1 ei
Lactobacillus
plantarum 776/1 bi
Lactobacillus
plantarum 476LL 20 bi
Lactobacillus
plantarum PR ci
Lactobacillus
plantarum 776/2 hi
Lactobacillus casei ssp.
paracasei 181A/3 aiai
Lactobacillus belonging
Bifidobacterium
longum 175A/1 aiai
Bifidobacterium
breve 195A/1 aici
Bifidobacterium
lactis 32A/3 aiai
Lactobacillus
plantarum 501/2 gi
Lactococcus lactis
Lactococcus lactis
Lactobacillus
plantarum 501/4 li
Streptococcus
thermophilus GB1
Streptococcus
thermophilus GB5
Bifidobacterium
longum BL 03
Bifidobacterium
breve BR 03
Lactobacillus casei
Lactobacillus
delbrueckii ssp.
bulgaricus LDB 01
Lactobacillus
delbrueckii ssp.
bulgaricus LDB 02
Streptococcus
thermophilus Y02
Streptococcus
thermophilus Y03
Streptococcus
thermophilus Y04
Streptococcus
thermophilus Y05
Bifidobacterium
adolescentis BA 03
Bifidobacterium
adolescentis BA 04
Bifidobacterium
breve BR 04
Bifidobacterium
pseudocatenulatum
Bifidobacterium
pseudocatenulatum
Staphylococcus
xylosus SX 01
Bifidobacterium
adolescentis BA 02
Lactobacillus
plantarum LP 07
Streptococcus
thermophilus YO8
Streptococcus
thermophilus YO9
Streptococcus
thermophilus YO100
Lactobacillus
fermentum LF06
Lactobacillus
fermentum LF07
Lactobacillus
fermentum LF08
Lactobacillus
fermentum LF09
Lactobacillus
gasseri LGS01
Lactobacillus
gasseri LGS02
Lactobacillus
gasseri LGS03
Lactobacillus
gasseri LGS04
Bifidobacterium
adolescentis EI-3
Bifidobacterium
adolescentis EI-15
Bifidobacterium
adolescentis EI-18
Bifidobacterium
catenulatum EI-20
Streptococcus
thermophilus FRai
Streptococcus
thermophilus LB2bi
Streptococcus
thermophilus LRci
Streptococcus
thermophilus FP4
Streptococcus
thermophilus ZZ5F8
Streptococcus
thermophilus TEO4
Streptococcus
thermophilus S1ci
Streptococcus
thermophilus 641bi
Streptococcus
thermophilus 277A/1ai
Streptococcus
thermophilus 277A/2ai
Streptococcus
thermophilus IDC11
Streptococcus
thermophilus ML3di
Streptococcus
thermophilus TEO3
Streptococcus
thermophilus G62
Streptococcus
thermophilus G1192
Streptococcus
thermophilus GB18
Streptococcus
thermophilus CCR21
Streptococcus
thermophilus G92
Streptococcus
thermophilus G69
Streptococcus
thermophilus YO 10
Streptococcus
thermophilus YO 11
Streptococcus
thermophilus YO 12
Streptococcus
thermophilus YO 13
Weissella ssp. WSP 01
Weissella ssp. WSP 02
Weissella ssp. WSP 03
Lactobacillus
plantarum LP 09
Lactococcus lactis
Lactobacillus
plantarum LP 10
Lactobacillus
fermentum LF 10
Lactobacillus
fermentum LF 11
Lactobacillus casei
Bifidobacterium
bifidum BB01
Lactobacillus
delbrueckii LD 01
Lactobacillus
delbrueckii LD 02
Lactobacillus
delbrueckii LD 03
Lactobacillus
delbrueckii LD 04
Lactobacillus
delbrueckii LD 05
Lactobacillus
acidophilus LA 02
Lactobacillus
paracasei LPC 08
Lactobacillus
pentosus LPS 01
Lactobacillus
rhamnosus LR 06
All strains have been deposited in accordance with the Treaty of Budapest and are accessible to the public on request from the competent Depositing Authority. Such Depositing Authorities include BCCM LMG (Belgian Coordinated Collections of Microorganisms, Laboratorium voor Microbiologie—Bactärienverzameling Universiteit Gent, Belgium) and DMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; Inhoffenstr. 7B, D-38124 Braunschweig, Germany).
The bacteria strains or their cellular components, according to the present invention, contribute to the prevention or treatment of immune diseases including autoimmune diseases such as inflammatory bowel diseases, and contribute to maintenance of the immunological homeostasis (health maintenance) of mammals such as human beings, domestic animals, and pet animals.
In other words, the bacteria strains or their components according to the present invention are high in safety and can be orally administered. Thus, the above microorganisms and the cellular components thereof are useful in that immunoregulatory cells can efficiently induced in the body by making use of the microorganism or the cellular components thereof as an active ingredient of pharmaceutical products, a food product, and the animal feeding stuff.
Other aspects and features of the invention will be more fully apparent from the following disclosure and appended claims.
The invention will be fully described by means of the following description without any limiting effects.
In a preferred embodiment a bacterium strain is selected from the group consisting of L. paracasei LMG P 21380, L. plantarum LMG P-21021, Bifidobacterium lactis LMG P-21384, Bifidobacterium breve DSM 16604 or its cellular components, which induces the production of Interleukin-10. Further, said bacteria strains exhibit a IL-10/IL-12 ratio comprised from bigger than 1 and less than 150, preferably comprised from 10 and 100, more preferably comprised from 30 and 60.
Advantageously, the bacteria strain is Bifidobacterium breve DSM 16604 which induces the production of Interleukin-10 and exhibits a IL-10/Il-12 ratio which is comprised from 50 and 100, preferably from 70 and 80.
The bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components.
In another preferred embodiment a food product comprises at least one bacterium strain which is selected from the group consisting of L. paracasei LMG P-21380, L. plantarum LMG P-21021, Bifidobacterium lactis LMG P-21384, and Bifidobacterium breve DSM 16604, as an active ingredient. Said bacteria strains induce the production of Interleukin-10. Further, said bacteria strains exhibit a IL-10/IL-12 ratio comprised from bigger than 1 and less than 150, preferably comprised from 10 and 100, more preferably comprised from 30 and 60. Advantageously, the bacteria strain is Bifidobacterium breve DSM 16604 which induces the production of Interleukin-10 and exhibits a IL-10/Il-12 ratio which is comprised from 50 and 100, preferably from 70 and 80.
The bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components.
In a further preferred embodiment a composition comprises at least one bacterium strain which is selected from the group consisting of L. paracasei LMG P-21380, L. plantarum LMG P-21021, Bifidobacterium lactis LMG P-21384, and Bifidobacterium breve DSM 16604 or its cellular components, as producer of Interleukin-10, for use as a medicament for the prevention or treatment of inflammatory conditions of the large intestine and small intestine or for the prevention or treatment of functional bowel disorders. The inflammatory conditions are selected from the group comprising Crohn's disease and ulcerative colitis while the functional bowel disorders are selected from the group comprising diarrhea and constipation.
Said bacteria strains induce the production of Interleukin-10. Further, said bacteria strains exhibit a IL-10/IL-12 ratio comprised from bigger than 1 and less than 150, preferably comprised from 10 and 100, more preferably comprised from 30 and 60. Advantageously, the bacteria strain is Bifidobacterium breve DSM 16604 which induces the production of Interleukin-10 and exhibits an IL-10/Il-12 ratio which is comprised from 50 and 100, preferably from 70 and 80.
The bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components.
In a preferred embodiment, the composition contains bacteria strains and/or their cellular components, as an active ingredients, in an amount comprised from 1×106 to 1×1011 CFU/g, respect to the weight of the composition, preferably from 1×108 to 1×1011 CFU/g.
In a preferred embodiment, the composition contains bacteria strains and/or their cellular components, as an active ingredient, in an amount comprised 1×106 to 1×1011 CFU/dose, preferably from 1×108 to 1×1010 CFU/dose.
The dose may be of 1 g, 3 g, 5 g, and 10 g.
The composition may further comprise additives and co-formulates pharmaceutically acceptable.
The composition of the present invention may include vitamins (for example folic acid, riboflavin, vitamine E, ascorbic acid), antioxidants compounds (for example polphenols, flavonoids and proanthocyanidines), aminoacid (for example glutamin, metionin) and also mineral (for example selenium and zinc).
In another particularly preferred embodiment, the composition of the present invention further includes at least a substance having prebiotic properties in an amount comprised from 1 to 30% by weight, respect to the total weight composition, preferably from 5 to 20% by weight.
Said prebiotic substance preferably includes carbohydrates which are not digested and absorbed by the organism. Said carbohydrates are preferably selected from: fructo-oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan-o-ligosaccharides (or COS), beta-glucans, arabic gum modified and re-sistant starches, polydextrose, D-tagatose, acacia fibers, bambu', carob, oats, and citrus fibers. Particularly preferred prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein-below as FOSs-c.c); said FOSs-c.c. are not digestable glucides, generally obtained by the conversion of the beet sugar and including a saccharose molecule to which three glucose molecules are bonded.
In a preferred embodiment the bacteria strain Bifidobacterium breve DSM 16604 is in combination with at least one bacteria strains selected from the group consisting of L. paracasei LMG P-21380, L. plantarum LMG P-21021, and Bifidobacterium lactic LMG P-21384. The bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components.
The following bacteria strains have been tested. Three Lactobacillus strains: L. rhamnosus (LR04) DSM 16605, L. paracasei (LPC 00) LMG P-21380, L. plantarum (LP 01) LMG P-21021, and two Bifidobacterium strains: B. lactis (BS 01) LMG P-21384, and B. breve (BR 03) DSM 16604 belonging to the most representative species of probiotic bacteria, were selected based on their resistance to acid, digestive enzyme, and bile and other characteristics such as antibiotic resistance and safety of use.
Living (viable) and dead (killed) bacteria samples were prepared starting from frozen stocks collection as follows. Pure Lactobacillus strains were cultured in de Man, Rogosa and Sharpe broth (MRS, DeMan et al. 1960) while Bifidobacterium strains, were cultured in MRS or Tryptone Phytone Yeast broth (TPY, Scardovi 1986), supplemented with 0.05% L-cysteine-hydrochloride. The cultures were prepared at 37° C. under anaerobic conditions for 16-22 hours. All bacteria were harvested by centrifugation (3000 g for 15 min) during exponential and/or stationary growth phase in order to collect cells. Pelleted bacteria, were then washed in phosphate buffered saline (PBS) and concentration was determined by means of colony-forming unit (CPU) counting. With reference to the preparation of living (viable) bacteria samples, washed pelleted bacteria were diluted to a final working concentration of 1×109 CFU/mL in PBS containing 20% glycerol and stored at −80° C. until used for assay. Alternatively bacteria could be diluted in RPMI-1640 and the suspension aliquoted and stored at −20° C.
Survival of bacteria upon freezing and thawing was determined by amount of live bacteria by means of colony-forming unit (CFU) counting and/or with staining for cFDA (live) and PI (dead). For all strains tested, >80% was alive upon thawing. The percentage of viability was not dependent on the time of storage. One fresh aliquot was thawed for every new experiment to avoid variability in the cultures between experiments.
With reference to the preparation of the dead bacteria samples, one of the following procedures may be Heatkilled bacterial cultures were prepared by heating the above washed pelleted bacteria resuspended in distilled water at 100° C. for 30 min. Alternatively bacteria, can be γ-irradiated or sonicated. Apart from one of the above procedures used for having a dead bacteria sample, the above sample may be treated in a liquid form or in a freeze-dried one.
The bacteria strains of the present invention were co-cultured with PBMCs (Peripheral Blood Mononuclear Cells) in order to study the specific capability to induce cytokine production by immunopotent cells.
PBMCs were isolated from peripheral blood of healthy donor as described. Briefly, after Ficoll gradient centrifugation, mononuclear cells were collected, washed in PBS and adjusted to 2×106 cells/mL in a complete medium consisting of RPMI 1640 supplemented with L-glutamin (300 mg/l), penicillium (100 U/ml), streptomycin (64 U/ml and 10% heat inactivated FCS (Fetal Calf Serum).
Alternatively a RPMI complete medium can also be obtained by RPMI-1640 supplemented with L-glutamin (300 mg/gentamicin (500 μg/mL), penicillin (100 U/mL), streptomycin (64 U/ml) and 20% heat-inactivated human AB serum or 10% FCS.
Monocytes can be purified from PBMCs negative magnetic cell sorting. The positively selected cells can be used as source of peripheral blood lymphocytes (PBLs). Monocytes as well as PBLs can be counted and resuspended at concentration of 5×106 cells/mL in complete RPMI medium. For mononuclear cells (PBMCs, Monocytes and PBLs) cryopreservation in liquid nitrogen, that cells, collected after Ficoll gradient centrifugation, were resuspended at concentration of 1×106 cells/mL in a complete medium consisting of RPMI 1640 supplemented with 10% DMSO (Dimethyl sulfoxide).
PBMCs cultures were set up in duplicate or triplicate in 96-well flat or round-bottom polystyrene microtitre plates. All cultures contained 0.1-0.5×106 PBMCs (or monocytes or PBLs) in complete medium. PBMCs were cultured in medium only or stimulated with phytoemoglutinine (PHA) at a final concentration of 50 μg/mL or lipopolisaccharides (LPS) at a final concentration of 0.5-1 μg/mL. The co-cultures with the live bacteria samples were obtained by adding a thawed aliquot of live bacteria sample to the PBMCs cultures having a cell:bacteria ratio of 1:1, 1:10 or 1:200.
The above bacteria-cell optimal concentration can be determined after proliferation test with different relative concentration (for example varying concentrations of bacterial cell fractions from 106 to 109 CFU/ml).
With reference to the co-cultures test with dead bacteria samples, PBMCs were cultured with 5-20 μg/mL (preferably μg/mL) of dead bacteria samples (heatkilled, γ-irradiated or sonicated) in freezed-dried form or with dead bacteria samples in the liquid form having a bacteria:cell ratio from 50:1 to 250:1 (preferably 200:1).
Control cultures contained unstimulated PBMCs, PHA-stimulated PBMCs, monocytes, PBLs all without bacteria strains or live bacteria sample only.
The plates were incubated at 37° C. in 5% CO2. The supernatants of cultures were collected at 24, 48, 72 hours and 5 days, clarified by centrifugation and stored at −20° C. until cytokine analysis. Neither medium acidification nor bacterial proliferation was observed.
Cytokines IL-10 and IL-12 levels were measured by standard Enzyme-Linked Immunosorbent Assay (ELISA) using commercial kits (like Quantikine Kits, R&D Systems Minneapolis, Minn.), as instructed by the manufacturer, as well known at the skilled person in the art.
Briefly, standards and samples (supernatants from the above co-cultured) were added into the plates and incubated for 2 h at room temperature. The specific horseradish peroxidase-conjugated antibody was added to all wells after they were washed 4 times, and the plates were incubated for 1 hour at room temperature. The plates were then washed and incubated for 30 minutes with 3-3′,5,5′-tetramethylbenzidine substrate reagent solution. The reaction was stopped by the addition of 1.8 M H2SO4. The absorbency of all ELISAs was read at 450 nm with a microtiter plate reader. Standard curves for the cytokines were constructed.
The minimum detectable dose of IL-10 and Il-12 was typically less than 3.9 pg/ml and 5.0 pg/ml, respectively.
Statistical analyses were performed with the Wilcoxon Mann-Whitney test to reveal significant differences between cytokine production in response to different strains of bacteria. Differences were considered to be significant at P<0.05.
The in vitro immune-stimulation by 5 live bacterial strains of PBMCs collected from healthy donors, revealed distinct capability of the strains to induce IL-10 and IL-12, so that IL-10 and IL-12 levels displayed a strain-specific pattern, as shown in
The
Variations of IL-10 concentrations were substantial with values ranging between 200 and 1700 pg/mL depending on the bacterial strain. For the IL-12 production, we also observed significant variations between strains, covering a range of cytokine levels of 10 to 1200 pg/mL.
Bifidobacterium breve BR 03 is able to module the immune responses by inducing the production of IL-10 by in vitro cultured mononuclear cells. Bifidobacterium breve BR 03 strongly induced IL-10 production (1688 pg/ml). On the contrary, it has a low capability to stimulate the production of the pro-inflammatory IL-12 (22 pg/ml).
The capacity of the probiotic strain B. breve BR 03 to boost the production of IL-10 differed considerably between other strains studied, among which can be considered the most potent inducers, see
In addition to a high IL-10 induction potential, it is important to minimize the IL-12 induction by the probiotic bacteria, when considering selecting a strain for an anti-inflammatory application. The pro-inflammatory cytokine IL-12, is mainly produced by phagocytic and antigen-presenting cells (APCs) as a quick reaction against bacteria, intracellular parasites or other infectious agents. In addition to an important role in the first line of defence against infection, IL-12 will limit or inhibit differentiation of Th2 T cells, itself acting as an immunoregulatory molecule in the Th1 response. IL-12 will induce IFN-γ and directly or indirectly activate natural killer cells, thus enhance further release of pro-inflammatory cytokines which promote an antigen-specific immune response.
This IL-12 production enhancing feedback mechanism, mediated by IFN-γ, is potentially leading to uncontrolled cytokine production. Fortunately, IL-10, as a regulatory cytokine, is a potent inhibitor of IL-12 production by these phagocytic cells and may suppress the emergence of an unbalanced Th1 response, such as the one seen in the gastrointestinal tract of IBD patients in a acute phase of inflammation; hence the importance in selecting probiotic strains with a favorable IL-10/IL-12 ratio.
It is possible to use the IL-10/IL-12 ratio to distinguish between strains exhibiting a “pro-” versus “anti-inflammatory” profile (low versus high IL-10/IL-12 ratio, respectively). This approach was found to be useful to identify strains with marked opposite profiles and can be used as a standardized in vitro test, allowing preliminary classification of candidate probiotic strains according to their immune modulation capacity that would be predictive of their in vivo effect. The importance of the ratio between these two cytokines was also recently demonstrated by Peran et al. In the study, administration of a specific strain of Lactobacillus salivarius ssp. salivarius facilitates the recovery of the inflamed tissue in the TNBS model of rat colitis. This beneficial effect was partly associated to the ability of the strain to modify the cytokine profile in macrophages, reducing the amount of inflammatory cytokine IL-12, while increasing the amount of the anti-inflammatory cytokine IL-10.
The use of PBMC from a diversity of healthy human donors to screen the immunomodulatory activity of candidate probiotic strains by direct stimulation appears to be a good predictive indicator of in vivo anti-inflammatory strains. Despite the fact that this assay does not clarify the physiological mechanism(s) involved, it seems to mimic how the immune system may sense the bacterial strain and consequently polarise the immune response. Strains leading to a high IL-10/IL-12 ratio would more easily slow down an early Th1 response.
In this context, assessing effects of 5 different probiotic bacteria, we found that Bifidobacterium breve BR 03 is the most potent “anti-inflammatory” strain eliciting the best IL-10/IL-12 ratio, as illustrated in
The
Taking into account the above, all the bacteria strains identified in the present invention show:
This application is a continuation of U.S. application Ser. No. 13/254,730, filed on Nov. 28, 2011, which is a 35 U.S.C. §371 national stage filing of International Application No. PCT/EP2009/052591, filed on Mar. 5, 2009. The entire contents of these applications are explicitly incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13254730 | Nov 2011 | US |
| Child | 14535068 | US |
