WO2008110569A1

WO2008110569A1 - Method for determination of immunomodulatory effect

Info

Publication number: WO2008110569A1
Application number: PCT/EP2008/052921
Authority: WO
Inventors: Simon Skjøde Jensen; Kim HOLMSTRØM
Original assignee: Bioneer A/S
Priority date: 2007-03-12
Filing date: 2008-03-12
Publication date: 2008-09-18

Abstract

The present invention relates to a method for in-vitro determination of the invivo antiinflammatory effect of a test component, such as a compound or a bacterium. The invention also pertains to bifidobacteria having high anti-inflammatory effect.

Description

METHOD FOR DETERMINATION OF IMMUNOMODULATORY EFFECT

FIELD OF THE INVENTION

The present invention relates to the field of inflammatory disease, in particular to a method for determining anti-inflammatory activity of a component such as a compound or a bacterium, as well as to probiotic bacteria.

BACKGROUND OF THE INVENTION

Inflammatory Bowel Disease (IBD) consists of two main forms, ulcerative colitis and Crohn 's disease which are both chronic inflammatory disorders of the gastrointestinal tract. In the western world approximately 1 in 1000 individuals are affected by the disease, yet its etiology is not completely understood. As such, IBD is a chronic disease which severely reduces the quality of life for the patient. IBD is characterized clinically by weight loss, abdominal pain, nausea and diarrhoea which in severe cases can result in death (Lothar Steidler et al., Science, VoI 289, pl352, 2000). Although ulcerative colitis and Crohn 's disease are commonly termed as IBD, the two diseases are histologically, pathologically and immunologically distinct. Studies on patients and animal models of the diseases have concluded that Crohn 's disease affects all layers of the intestinal mucosa, involves dense infiltration of lymphocytes and macrophages, and can occur throughout the GI tract. Ulcerative colitis mainly involves superficial mucosal infiltration with lymphocytes and granulocytes, and occurs mainly in colon and rectum.

Immunologically, recent research results have shown that Crohn 's disease can be considered an autoimmune disease, which is driven by excessive activation of ThI cells. Thl-cells are key effector cells in the development of the pro-inflammatory phenotype characteristic for Crohn 's disease, and causes production of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα). In contrast, ulcerative colitis is characterised by increased T helper 2 (Th2) activation. Therapeutic intervention for treatment of Crohn 's disease can be facilitated through antibody based therapy against IFNγ and TNFα which are key cytokines in a Thl-response (Gerd Bouma and Warren Strober, Nature Reviews Immunology, Vol3, p 521, 2003).

The fact that therapeutic intervention of Crohn 's disease can be facilitated through inhibition of NF-κB, IFNγ and TNFα, clearly demonstrates that these cytokines are key players in the pathogenesis and maintenance of Crohn 's disease. IFNγ and TNFα are positioned in the effector side of the disease, in contrast to the dendritic cell secreted IL12, which acts upstream of the Thl-mediated secretion of IFNγ and TNFα. Therefore, reprogramming the naive antigen presenting cells which are recruited to the intestinal mucosa towards a high ILlO producing phenotype and low IL12 secretion, are believed to be able to suppress the pro-inflammatory condition in IBD, thereby over time improving the inflammatory conditions. Furthermore, the fact that some probiotics have the ability to inhibit NF-κB activity in cells from the GI-tract indicates that these strains may be able to suppress the autoimmune response (Riedel et al., World J Gastroenterol., 12(23), p 3729, 2006).

In ulcerative colitis the Th2 phenotype can also be immunologically reprogrammed by inducing ILlO secretion from dendritic cells. This hypothesis has been proven in a study by L. Steidler (Science, 2000) where a Lactococcus lactis strain was genetically modified to secrete mouse ILlO. This L. lactis strain caused a 50 % reduction of ulcerative colitis in mice with dextran sulfate sodium induced ulcerative colitis, and completely prevented development of ulcerative colitis in mice lacking the ILlO gene, which normally spontaneously develop ulcerative colitis . Although this approach could also be efficient for human treatment, the risk of ILlO gene transfer to human cells or microorganisms in our environment could potentially lead to general suppression of the immune system in healthy humans, which would be detrimental upon infection with a pathogen which requires a proinflammatory response from the human immune system. Hence, selection of naturally occurring probiotic strains which are able to suppress IL12 secretion and thereby the ThI immuneresponse from intestinal mucosal dendritic cells, as well as possibly induce regulatory T-cells by increased ILlO secretion, may yield potent candidates for oral, daily administration to patients suffering from IBD and potentially other inflammatory conditions. Furthermore, such probiotic strains are relatively easier and less expensive to produce than pharmaceuticals, and the risk of side effects and long-term resistance are believed to be lower (Shanahan F, Am J Physiol Gastro intest Liver Physiol 288, G147-21, 2005).

There has been long lasting indications that probiotics may have immunomodulatory and anticarcinogenic effects, as well as other health benefits. There is presently much active research focusing on the development of target-specific probiotics containing well- characterized bacteria that are selected for their health-enhancing characteristics. These new probiotics are entering the marketplace in the form of nutritional supplements and functional foods, such as yogurt functional food products. Thus there is a need for determining and substantiating the claimed health benefits of these probiotics, e.g. the immunostimulatory and anti-inflammatory effects. There is a lack of reliable in-vitro methods for assessing the health effect of probiotics as well as potential pharmaceuticals. Clinical studies in humans are very expensive and thus, it is desirable to reduce these experiments. Likewise, it is desirable to minimize the use of animal studies. Consequently, there is a need for more efficient methods for selecting the microbial strains and the chemical compounds having the most pronounced effects on preventing and/or alleviating specific inflammation based conditions.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned limitations of the known methods for in-vitro determination of the anti-inflammatory effect of a test component, the present invention provides a method for in-vitro determination of the in vivo anti-inflammatory effect of a test component such as a molecule, a mixture of molecules, a microorganism, a fraction of cells or combinations thereof.

It has surprisingly been found that for a given test component an in-vitro method using human dendritic cells gives a result which is closely correlated with the in vivo antiinflammatory effect in mice or humans of this test component.

The present invention provides a method for in-vitro determination of the in vivo antiinflammatory effect of a test component such as a molecule, a mixture of molecules, a microorganism, a fraction of cells or combinations thereof, comprising the steps of a) contacting said test component, mammalian dendritic cells and a pro-inflammatory cocktail, b) quantifying the secretion of at least one pro-inflammatory cytokine and/or chemokine from said mammalian dendritic cells, c) comparing said secretion of at least one pro-inflammatory cytokine and/or chemokine with that of a reference test wherein said mammalian dendritic cells have not been contacted with said test component but have been contacted with said pro-inflammatory cocktail, d) to establish the extent to which said test component reduces secretion of said at least one pro-inflammatory cytokine and/or chemokine.

In another aspect the present invention provides a method for in-vitro determination of the in vivo anti-inflammatory effect of a test component such as a molecule, a mixture of molecules, a microorganism, a fraction of cells or combinations thereof, comprising the steps of a) contacting said test component, mammalian dendritic cells and a pro-inflammatory cocktail, b) quantifying the secretion of at least one pro-inflammatory compound or marker which is not a cytokine and/or chemokine from said mammalian dendritic cells, c) comparing said secretion of at least one pro-inflammatory compound or marker which is not a cytokine and/or chemokine with that of a reference test wherein said mammalian dendritic cells have not been contacted with said test component but have been contacted with said pro-inflammatory cocktail, d) to establish the extent to which said test component reduces secretion of said at least one pro-inflammatory compound or marker which is not a cytokine and/or chemokine.

In another aspect the present invention provides a method for in-vitro determination of the in vivo anti-inflammatory effect of a test component such as a molecule, a mixture of molecules, a microorganism, a fraction of cells or combinations thereof, comprising the steps of a) contacting said test component, mammalian dendritic cells and a pro-inflammatory cocktail, b) quantifying the expression level or activation state of at least one intracellular or membrane bound marker of inflammation from said mammalian dendritic cells, c) comparing said expression level or activation state of at least one intracellular or membrane bound marker of inflammation with that of a reference test wherein said mammalian dendritic cells have not been contacted with said test component but have been contacted with said pro-inflammatory cocktail, d) to establish the extent to which said test component reduces the expression level or activation state of said at least one intracellular marker or membrane bound marker of inflammation.

In another aspect the present invention provides a method for in-vitro determination of the in vivo anti-inflammatory effect of a test component such as a molecule, a mixture of molecules, a microorganism, a fraction of cells or combinations thereof, comprising the steps of a) contacting said test component, mammalian dendritic cells and a pro-inflammatory cocktail, b) determining the functional phenotype of said dendritic cells by applying T-cell cultures and quantifying at least one intracellular or extracellular endpoint or the proliferation of the T-cells that have been contacted with said dendritic cells. c) comparing said at least one intracellular or extracellular endpoint or the proliferation in said T-cell cultures that have been contacted with said dendritic cells with that of a reference test wherein said mammalian dendritic cells have not been contacted with said test component but have been contacted with said pro-inflammatory cocktail, d) to establish the extent to which said test component changes the level of said at least one intracellular or extracellular endpoint or the proliferation in T-cell cultures as a marker of dendritic cell functional phenotype.

In one embodiment of the methods, in step a) said mammalian dendritic cells are contacted with said test component prior to said mammalian dendritic cells being contacted with said pro-inflammatory cocktail. In another embodiment, in step a) said mammalian dendritic cells are contacted with said test component at least one hour, such as at least 2-3 hours after said mammalian dendritic cells having been contacted with said pro-inflammatory cocktail. In yet another embodiment, in step a) said mammalian dendritic cells are contcted with said test component at least one hour, such as at least 2-8 hours after said mammalian dendritic cells having been contacted with said pro-inflammatory cocktail.

This method has been validated to provide an in-vitro determination which has excellent correlation with the in vivo anti-inflammatory effect in mammals, such as mouse and human. Hence, the present invention now provides a method for determination of the in vivo anti-inflammatory effect of a test component that allows further development with limited animal and clinical testing. Hence, it is now possible to identify the components being the most efficacious in relation to anti-inflammatory effect before embarking upon confirmation experiments in humans or in animals.

In one aspect the present invention provides the use of the method for predicting the ability of a test component to prevent or alleviate an inflammatory condition in a human, comprising subjecting the potential test component to said method for in-vitro determination of the anti-inflammatory effect.

In another aspect the present invention provides the use of the method for quantification of the in-vitro effect of a test component on the pro-inflammatory phenotype of said mammalian dendritic cells resulting from contacting said mammalian dendritic cells with said pro-inflammatory cocktail.

In a further aspect the present invention relates to certain strains of Bifidobacterium bifidunπ. In one embodiment the Bifidobacterium bifidum is Bifidobacterium bifidum BI98 (deposited as DSM 19157) or a homolog, descendant or mutant thereof which exhibit probiotic activity. In another embodiment the Bifidobacterium bifidum is Bifidobacterium bifidum BI504 (deposited as DSM 19158) or a homolog, descendant or mutant thereof which exhibit probiotic activity. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. General schematic outline of the in vitro screening model setup.

Figure 2. Depicts a schematic overview of the invention, with the probiotic or antiinflammatory candidate as potentially suppressing inflammatory signals in the dendritic cells, leading to reduced levels of cocktail induced secretion of inflammatory cytokines like IL12, and potential prevention of expression of maturation markers.

Figure 3. Example of cocktail optimization in murine dendritic cells for a cocktail combination resulting in high IL12 levels and low ILlO levels. Multiple dendritic cell maturation/activation components and cytokines are tested like IFNγ (10-40 ng/ml), GA-polymer (10-20 ug/ml), CpG thioate ester nucleotide sequence (1 μM), a probiotic bacteria Lactobacillus acidophilus LX37 (100-200 ug/ml), LPS (100 ng/ml), ILlβ (10 ng/ml), CD40 ligand antibody at 1 μg/ml, lipotheichoic acid (LTA) (200 ng/ml).

Figure 4. FACS-profile of immature (unstimulated) and cocktail stimulated (IFNγ 20ng/ml, CpG 1 μM) (stimulated) dendritic cells show increased expression of maturation markers CD86 (upper two graphs) and CD40 (lower two graphs). The table below shows that CD40 expression increases from 46 to 99 % expression, and CD86 expression increases from 49 to 93 % expression, of the total dendritic cell pool.

Figure 5. A. Dose response experiments with the cocktail containing IFNγ and CpG (optimal concentration at 20 ng/ml IFNγ and 1 μM CpG. The cocktail induces strong IL12 (p70) expression at the highest concentration, and shows a response even when diluted up to 40 fold. B. Changing the concentration of each specific component (range between 1-20 ng/ml IFNγ and 0.1-1 μM CpG).

Figure 6. A. Compared to immature dendritic cells, the pro-inflammatory cocktail (20 ng/ml IFNγ and 1 uM CpG) stimulated dendritic cells show secretion of IL6, IL12, RANTES₁ TNFα, MCPl, MIPIa and KC-chemokine, analyzed using cytokine arrays detecting mouse inflammatory cytokines. B. Using ELISA, the specific cytokine levels of IL6, IL12 and TNFα were shown to be dramatically induced, whereas secretion of ILlO was not affected. C. DCs treated with the pro-inflammatory cocktail was able to induce a Thl-directing phenotype of T-cells by induction of IFNγ -secretion. Addition of purified naive CD4+ T-cells from allogenic mouse spleen were induced to produce the Thl-directing cytokine IFNγ.whereas non-treated dendritic cells did not. Addition of a mixture of allogenic spleen cells, the cocktail treated dendritic cells also caused induction of IFNγ to a level higher than immature dendritic cells. Figure 7. The dendritic cell based screening method as described in claim 1, was validated as a suitable model for screening of anti-inflammatory test components. The known antiinflammatory compounds, the glucocorticoid dex (A), and the anti-inflammatory prostaglandin D2, (B), were shown dose-dependently to reduce the secretion of IL12 to low levels at concentrations that did not cause cell death.

Figure 8. The dendritic cell based screening method as described in claim 1, and the fact that other inflammatory cytokines can be used for end-points in this model is seen by using the anti-inflammatory prostaglandintaglandin D2, which is shown dose-dependently to reduce the secretion of TNFα (A), and IL6 (B) in concentrations that did not cause cell death.

Figure 9. The dendritic cell based screening method as described in claim 1, used to screen for probiotics with anti-inflammatory activity, was correlated to the anti-inflammatory activity seen in mouse models of TNBS induced colitis. The graph shows the preventive effect of three Lactobacillus strains tested in a preventive model of colitis, where the strains were administered orally 5 days prior to induction of colitis. The colitis score was made at day 2 after TNBS administration, and showed a significant (P<0.01) protective effect of Lactobacillus salivarius Ls 33 strain. The Lactobacillus plantarum Lpll5 strain showed occasional protective activity, whereas Lactobacillus acidophilus NCFM showed no protective effect, and even a worsening of the experimental inflammatory condition (data based on results from Foligne et al., World J. Gastroenterology, 2007, 13(2) :236-243). The suppressive ratios were determined for these three strains in the method described here, and showed a significantly suppressive activity of the Lactobacillus salivarius Ls 33 strain, whereas the Lactobacillus plantarum Lpll5 showed no influence on the anti-inflammatory activity, whereas the Lactobacillus acidophilus NCFM also in the present in-vitro screening model showed an additive inflammatory activity, and increased the IL12 secretion above cocktail induced levels alone.

Figure 10. Among a panel of probiotic bacteria, strains showing suppressive and additive effects on IL12 induction together with the pro-inflammatory cocktail, two strongly suppressive strains (BI98 and BI504) were identified (A, B). BI98 and BI504 were both able to suppress the pro-inflammatory cocktail when added after 2 hours (indicated by arrows). A. Lactobacillus strain with non-suppressing activity (suppressive ratio at 0.8) is shown as an example (C). In D, a table showing the span of suppressive ratios determined for 4 Bifidobacteria strains, where the span of suppressive ratios can be seen. A probiotic strain showing a suppressive ratio above 1 is considered to posess anti-inflammatory properties. Figure 11. A. BI98 was able to prevent IL12 (p70) secretion when added to dendritic cells 2 hours prior to addition of pro-inflammatory cocktail (20 ng/ml IFNγ and 1 uM CpG) in a dose dependent manner. The IC50 value for the IL12 suppression of BI98 was below 10 μg/ml, corresponding to the IC50 of approximately 10 nM dex, and approximately 2 μM prostaglandin D2. B. Pulsed field gel electrophoresis (PFGE) of Spel restriction enzyme digests of genomic DNA preparations of BI98 and BI504, respectively. C and D show that cytokine profile of DCs treated with probiotics from 0 to 6 hours before addition of the cocktail. The effect of the cocktail becomes weaker as the timespan increases.

Figure 12. Partial 16S rDNA sequence comprising 549 nucleotides from strain BI98 (query) compared with published sequence of a Bifidobacterium bifidum (accession number: AY694148.1). Sequence alignment performed by BLAST search.

Figure 13. Partial 16S rDNA sequence comprising 564 nucleotides from strain BI504 (query) compared with published sequence of a Bifidobacterium bifidum (accession number: AY694148.1). Sequence alignment performed by BLAST search.

Figure 14. Measurement of Nitric Oxide (NO) in media from murine DCs treated with proinflammatory cocktails, LPS or probiotic strains. The two anti-inflammatory Bifidobacterium bifidum BI98 and BI504 strains slightly induce NO secretion, whereas the strongly IL12 inducing Lactobacillus acidophilus strain LX37 induce high levels of NO. The two Bifidobacterium bifidum BI98 and BI504 strains were also able to reduce the cocktail induced NO secretion to some extent.

Figure 15. Expression of iNOS/NOS2 in murine (A) and human (B) DCs treated as indicated, were analysed by western blot. Control cells showed no iNOS expression, whereas cocktail and LPS showed strong iNOS expression. The probiotic treated DCs showed variable iNOS expression, where DCs treated with the two anti -inflammatory Bifidobacterium bifidum BI98 and BI504 strains showed the weakest iNOS expression. Cocktail F, F2, 6 and 7 showed a clear induction of iNOS in the human DC model (B).

Figure 16. Expression of COX-2 in murine (A) and human (B, C) DCs treated as indicated, were analysed by western blot. Control cells showed low COX-2 expression, whereas cocktails and LPS showed strong COX-2 expression. In C, it is seen that pretreatment with dex for 24 h prevents COX-2 induction induced by LPS, cocktail F2 and 7. In this specific donor, the DCs were less responsive towards cocktail 6.

Figure 17. Shows an example of cocktail optimization based on R848. Human DCs generated from buffy coat derived monocytes were used to screen for pro-inflammatory cocktails with the ability to induce the cytokines TNFα (A) and IL12p70 (B). Cocktail F consists of IFNα, TNFα, Poly I:C, ILlβ and IFNγ. The other components that make up the other cocktails are denoted in the figure and comprises of TLR agonists and cytokines. (C) Is a table of optimized cocktails identified using several rounds of optimization of human DC screening.

Figure 18 and 19. By cocktail optimization in human DCs as in figure 17, 9 cocktails were identified, that could induce TNFα and IL12p70 in human DCs as suitable end-points for the screening model. These cocktails were analysed for their suitability as efficient screening cocktails, by assessment of donorvariation on these 9 cocktails using 15 different human donors. The cytokine level of TNFα and IL12p70 was determined for DCs treated with each of the cocktails, and the level of cytokine was compared to that of cocktail F, which was normalized to 100 %. The variation in cytokine response for IL12p70 is seen in Fig. 18, and for TNFα in Fig. 19. Cocktail compositions can be seen in the table in Fig. 17 C.

Figure 20. The 9 cocktails above were further optimized for end-points which are not a cytokine or chemokine, in this case prostaglandin E2. Cocktails F, F2, J and 10 (R848 and poly I:C) were the most potent in stimulating PGE2 within 24 h incubation in the two donors shown.

Figure 21. PGE2 secretion from human DCs was during the optimization seen in Fig. 20 shown to be high for cocktail J and 10. Two known COX inhibitors, indomethacin and NS398 were used to demonstrate that the model can be used for screening of anti-inflammatory small molecule drugs or drug candidates. 6 h prior to addition of cocktail F or 10, these two drugs were added to the cells to allow inhibition of COX enzymes, responsible for PGE2 generation. After 24 h PGE2 levels were determined and shown to be blocked by both compounds.

Figure 22. The two probiotic strains BI98 and BI504 can dose-dependtly suppress the IL12 secretion from human DCs when added to the DCs 6 h prior to addition of cocktails. Cocktail 6 consists of LPS and IFNγ. Cocktail 10 consists of R848 and poly I:C. Probiotics were added as freeze dried bacteria.

Figure 23. Selected human DC cocktails were used to validate the screening model with the known anti-inflammatory drug dex. Dex was added to immature DCs 6 or 24 h prior to addition of cocktail 5, 6, 8, 9 or 10. Dex was able to suppress both IL12p70 (A and B) and TNFα (C and D) in a dose dependent manner for all cocktails used. Cocktails consists of: cocktails 5 (=cocktail J), cocktail 6 consists of LPS and IFNγ, cocktail 8 consists of R848, cocktail 9 consists of R848 and IFNγ, and cocktail 10 consists of R848 and poly I:C. Figure 24. Surface staining by flow cytometric analysis of immature (unstimulated), LPS and cocktails (see table in Fig. 17 C for details) stimulated human dendritic cells from three donors. Immature dendritic cells were differentiated from CD14 monocytes cultured with GM-CSF and IL-4. The surface expression of relevant activation markers was analyzed on day 7. All cocktails except cocktail 8 induced a high increase in expression of the activation markers CD80 and CD86, whereas the increase in expression of CD40 , CD83 and HLR-DR was more moderate. (A) Flow cytomeric analysis of one representative donor (donor 1) out of three. A total of 5000 events were collected by gating hDC defined by forward (FSC) and side-scatter (SSC) characteristics. All histograms were gated for CDIa cells (70-95 %). Appropriate isotype antibodies were used and > 96 % of stained cells were included by the P2 gate. (B) Mean florescence intensity (MFI) values for relevant activation markers on immature and cocktail treated hDCs from three donors.

Figure 25. Phenotypic surface analysis of the suppressive effect that dex has on cocktail treated human DCs. Dex was able to lower the expression of activation markers CD80, CD86 and CD40 induced by all the shown cocktails after 6h pre-treatment. Pre-treatment of the immature DC with dex before addition of cocktail 10 prevented the expression of activation markers below the immature state. The experiment was performed on two donors. (A) Flow cytomeric analysis of one representative donor (donor 1) out of two. A total of 5000 events were collected by gating hDC defined by forward (FSC) and side-scatter (SSC) characteristics. All histograms were gated for CDIa cells (70-95 %). Appropriate isotype antibodies were used and > 96 % of stained cells were included by the P2 gate. (B) Mean florescence intensity (MFI) values for relevant activation markers on immature, dex and cocktail treated hDCs from two donors.

Figure 26. Mixed lymphocyte culture (MLC) performed on CD4+ T cells and allogeneic DCs from humans. Mature cocktail stimulated (cocktail 6 or 10) dendritic cells were more potent inducers of T cell proliferation in the MLC than immature dendritic cells (A and B). (A)

Pretreatment of DC with dex for 6 h before adding cocktail 10, significantly prevent CD4+T cell proliferation to a level similar to that of immature DCs. Similar results are shown for two donors. (B) After 24 h pre-treatment with dex, vitamin D3 or a combination of these, cocktail 6 and 10 induced proliferation was lowered or prevented. CD4+T cells was purified by Dynal beads from buffycoats and cultured with graded doses of allogeneic DCs for 5 days. DCs were mitomycin C treated in order to inhibit their proliferation. Proliferation of

CD4+ T cells was determined by thymidine incorporation in the last 18-24h of cell culture.

(C) Flow cytomic analysis of naturally occurring CD25+/FoxP3 regulatory T (Treg) cells induced by dex in a MLC reaction. T Cells were isolated from MLCs from two donors and

Flow cytomeric analysis was performed. A total of 5000 events were collected by gating lymphocytes, defined by forward (FSC) and side-scatter (SSC) characteristics. A gate for CD4+ T cells was set. There was a 2,5% and 4,6% incensement of CD25/Foxp3 positive cells for each of the two donors respectively, which presumably reflects an induction of Treg cells. However, as CD25/FoxP3 expression is up-regulated transiently following T cell activation, a maturation of dendritic cells with cocktail 10 also induced a high CD25/FoxP3 expression in the T cells. Pretreatment with dex for 6 h before addition of cocktail increased the CD25/FoxP3 expression further, properly reflecting an additive effect of T cell activation induced and Treg induced expression of CD25/FoxP3. A validation of the Treg induction must be investigated by its functionality.

Figure 27. Measurements of the ThI induced cytokine IFNγ and the Th2-induced cytokine IL- 5 in the mixed lymphocyte culture on day 5. Cocktail 6 induce secretion of both IL-5 and IFNγ in the MLC whereas cocktail 10 only induce IFNγ. Dex and Vitamin D3 prevent the cocktail 6 induced secretion of IL-5 and IFNγ cocktail 10 induced IFNγ secretion is only markedly inhibited.

Figure 28. As an example of an end-point where the activation state measured by phosphorylation state of an intracellular protein is quantified as an inflammatory marker is shown on the phosphorylation of Iκβa (upper panel) and the total level of this enzyme (lower panel). The quantification was made using antibodies towards Ser 32 on Iκβa, and total amount of Iκβa analysed on DC lysates using Western blot.

Figure 29. Surface staining by flow cytometric analysis (FACS) of immature, cocktail (CpG and IFN-γ) treated and dex-treated murine DCs. The cocktail induced a very high expression of CD86 and CD40 shown as mean florescence intensity (MFI) values in the FACS diagrams.

Dex was able to lower the expression of the activation marker CD86 and CD40 induced by the cocktail after 2 h pretreatment. Immature dendritic cells were bone marrow derived and differentiated in culture with GM-CSF and IL-4. A total of 5000 events were collected by gating mDC defined by forward (FSC) and side-scatter (SSC) characteristics (Pl). All histograms were gated for CDlIc cells (70-95 %). Appropriate isotype antibodies were used and > 96 % of stained cells were included by the P2 gate.

Figure 30. Mixed lymphocyte culture (MLC) performed on CD4+ T cells (C57/bl6) and allogeneic DCs (BALB/c) from mice. Mature cocktail stimulated DCs were more potent inducers of T cell proliferation in the MLC than immature dendritic cells. (A) Pretreatment of the immature mDCs with dex for 24 h before adding cocktail, significantly prevent CD4+T cell proliferation, although not to a level of immature DCs. B) After 24 h pre-treatment with dex and Vitamin D3 together the cocktail induced proliferation of allogeneic T cell is completely prevented. CD4+T cells was purified by Dynal beads from spleen and cultured with graded doses of allogeneic dendritic cells for 5 days. Dendritic cells were mitomycin C treated in order to inhibit their proliferation. Proliferation of CD4+ T cells was determined by thymidine incorporation in the last 18-24 h of cell culture.

Figure 31. Measurements of the Th2-induced cytokine IL-4 in a murine mixed lymphocyte culture on day 5. It was possible to measure IL-4 secretion in the culture supernatant from the MLC, containing immature as well as cocktail (CpG and IFN-γ) treated dendritic cells. A 24 h pretreatment of the murine dendritic cells before cocktail addition significantly prevents the IL-4 secretion in the allogeneic MLC reation.

DEFINITIONS

In the present context, the term "dendritic cells" (DC) is intended to mean non-lymphocyte antigen presenting cells (APC), distinct from macrophages, that are able to initiate immune responses after endocytosis or contact with exogeneous proteins and microorganisms. The endocytosed proteins are processed by the dendritic cells and exposed as peptides on MHC class II molecules. Dendritic cells mainly induce activation of T- and B-cells. In this specific invention, dendritic cells can be derived from mammalian tissues, specifically derived from bone marrow, spleen, thymus, blood, monocytes, cell lines, stem cells and stem cell lines. Dendritic cell subtypes can comprise plasmacytoid, myeloid, etc (see e.g. Shortman and Yong-Jun, Nature Reviews Immunology, 2(2002)151-161). In one embodiment the dendritic cells used in the present invention are myeloid dendritic cells. In another embodiment the dendritic cells used in the present invention are plasmacytoid dendritic cells.

In the present context, the term "immature dendritic cells" is intended to mean non- matured dendritic cells with endocytic and phagocytic properties characterized by a set of species-specific surface markers. Immature dendritic cells of murine origin usually express high levels of CCRl, 5 and 6, and low levels of CCR7, CD40, CD54, CD58, CD80, CD83, CD86 and DC-LAMP. The Immature dendritic cells show low levels of secretion of inflammatory cytokines like IFNα/γ, ILlα/β, IL6, IL12, TNFα, and have a poor T-cell activating capacity.

In the present context, the term "differentiation inducing composition" is intended to mean a composition comprising one or more proteins inducing differentiation of immature DCs. Non-limiting examples of such polypeptides are cytokines (e.g. IL4, ILlO, IL12, granulocyte macrophage colony stimulating factor (GM-SCF), Macrophage stimulating factor (M-CSF), Flt-3 ligand, stem cell factor (SCF), IL3, TGFβ, TNFα,.hormones like glucocorticoids, Vitamin A, D3 and analogues thereof. The proteins mentioned above are species compatible. The differentiation inducing composition is designed according to the species of origin of the dendritic cells.

In the present context, the term "pro-inflammatory cocktail" is intended to mean a combination of cytokines and chemokines e.g. RANTES, MCPl, MIPIa, IFNα/γ, ILlα/β, IL6, IL12, TNFα, TGFβ, PRR-ligands, TLR-ligands [e.g. lipopolysaccharide (LPS), zymosan and peptidoglycan], prostaglandins and in particular PGE2, sphingolipids, phospholipids, lipoteichoic acid (LTA), natural or synthetic nucleic acid sequences [Polyinosinic-polycytidylic acid (Poly I:C) which is a synthetic double-stranded RNA (dsRNA), CpG thioate oligonucelotides], extracts or microorganisms with immunostimulatory properties and antibodies with agonist activity towards dendritic cell receptors, in particular to the CD40 receptor. A cocktail can consist of a single or multiple of the aforementioned components that are able to activate dendritic cells and induce expression of maturation markers and/or pro-inflammatory cytokines and chemokines. The pro-inflammatory cocktail is designed according to the species of origin of the dendritic cells.

In the present context, the term "pro-inflammatory compound which is not a cytokine and/or chemokine" is intended to mean a compound that is secreted from dendritic cells but which compound is not a cytokine and/or chemokine. The "pro-inflammatory compound which is not a cytokine and/or chemokine" includes but are not limited to comprise eicosanoids, prostaglandins, leukotrienes, thromboxanes, fatty acids and Nitric oxide.

In the present context, the term "activation state" is intended to mean assessment of the activation of a protein through posttranslational changes, e.g. by determining the degree of phosphorylation of a protein, where the phosphorylation of the protein is linked to the function of the protein. In this context, activation of a given protein can correlate with either increased or decreased phosphorylation at a specific site within the protein.

In the present context, the term "intracellular marker of inflammation" is intended to mean a molecule and typically but not limited to a protein whose expression level, function or activation state is altered upon treatment of a dendritic cell with a pro-inflammatory compound. "Intracellular markers of inflammation" typically comprises proteins that are involved in signaling pathways activated by cytokines, TLR and PRR agonists, receptor ligands to lipid derivatives like eicosanoids, prostaglandins, leukotrienes, thromboxanes, fatty acids.

In the present context, the term "test component" is intended to mean the component or substance which is subjected to the method for determining its anti-inflammatory effect. Non-limiting examples of test component are a compound, a small chemical entity, a macromolecule such as a peptide, a protein, a polysaccharide, an oligonucleotide, a nucleic acid, a lipid, a sugar, a natural extract from e.g. plants, fruits etc., a cell fragment, a microorganism, a bacterium and mixtures and combinations thereof. Thus, it is to be understood that the test component may be in any degree of purity, e.g. in crude, partly purified or in highly purified form.

In the present context, the term "cytokines" is intended to mean proteins derived from the immune system that exerts biological responses. Cytokines are involved in signaling between components of immune system. Cytokines are mainly produced by macrophages, dendritic cells, T and B cells and natural killer cells. Cytokines consists of monokines (e.g. IL6, GM-CSF), interferons (e.g. Interferon α/β/γ), Tumor necrosis factor family (TNFα/β).

In the present context, the term "chemokines" is intended to mean the family of proteins involved in cell migration, activation and chemotaxis. Non-limiting examples are MCP-I, MlP-lα/β and RANTES. Chemokines are mainly produced by macrophages, dendritic cells, T and B cells, monocytes and natural killer cells.

In the present context, the term "maturation markers" is intended to mean markers which are expressed by the mature/activated dendritic cells and non-limiting examples of maturation markers are CD40, CD54, CD58, CD80, CD86, CD83, CCR7, MHCII and DC- LAMP. The maturation markers to be assayed are selected based upon the species of origin of the dendritic cells.

In the present context, the term "suppressive ratio" is calculated from the level in growth media of pro-inflammatory cocktail induced chemokines and/or cytokines IL12 (subunit p40 or p70), IL6 or TNFα in the pro-inflammatory cocktail treated dendritic cells (DC) and the level of the same chemokines and/or cytokines when the test component is added before, after or simultaneously with the cocktail. The suppressive ratio is calculated using the formula:

Suppressive ration = Concentration in growth medium from DC w cocktail /

Concentration in growth medium from DC treated with test component and cocktail

In the present context, the term "PRR-ligands" is intended to mean a compound, peptide, polysaccharide, biological response modifiers, organism (in particular microorganism of bacterial, fungal or viral origin), natural extract or fractions thereof, all in crude, partly purified or purified form, that are able to interact with PRR-receptors on antigen presenting cells.

In the present context, the term "functional endpoint" is intended to mean the ability of dendritic cells to modulate cells of the immune system to a functional phenotype that is indicative for the regulation of the adaptive immune response. The most relevant functional endpoint for DCs in the present invention is the stimulation of T-cells towards either ThI, Th2, Thl7 or Treg cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for in-vitro determination of the in vivo anti- inflammatory effect of a test component such as a molecule, a mixture of molecules, a microorganism, a fraction of cells or combinations thereof, comprising the steps of a) contacting said test component, mammalian dendritic cells and a pro-inflammatory cocktail, b) quantifying the secretion of at least one pro-inflammatory cytokine and/or chemokine from said mammalian dendritic cells, c) comparing said secretion of at least one pro-inflammatory cytokine and/or chemokine with that of a reference test wherein said mammalian dendritic cells have not been contacted with said test component but have been contacted with said pro-inflammatory cocktail, d) to establish the extent to which said test component reduces secretion of said at least one pro-inflammatory cytokine and/or chemokine.

In one embodiment of the methods, in step a) said mammalian dendritic cells are contacted with said test component prior to said mammalian dendritic cells being contacted with said pro-inflammatory cocktail. In another embodiment, in step a) said mammalian dendritic cells are contacted with said test component at least one hour, such as at least 2-3 hours after said mammalian dendritic cells having been contacted with said pro-inflammatory cocktail. In yet another embodiment, in step a) said mammalian dendritic cells are contcted with said test component at least one hour, such as at least 2-8 hours after said mammalian dendritic cells having been contacted with said pro-inflammatory cocktail. In yet another embodiment, in step a) said mammalian dendritic cells are contcted with said test component at 1 to 24 hours after said mammalian dendritic cells having been contacted with said pro-inflammatory cocktail. In yet another embodiment, in step a) said mammalian dendritic cells are contcted with said test component at 1 to 36 hours after said mammalian dendritic cells having been contacted with said pro-inflammatory cocktail.

The screening model makes use of the fact that dendritic cells are key regulators of the immune system and thus determinants of the inflammatory response upon pathogen challenge or their presence in inflammatory environments. The ability of DCs to responod to these foreign pathogens and cytokines from inflammatory sites is used as a model to screen for new compounds and microbial cells with potential anti-inflammatory activity. The general theoretical outline of the model involves isolating dendritic cell precursors from a mammalian source, growing them and obtaining immature dendritic cells, stimulating the immature dendritic cells with a test component together with a cocktail of biological compounds capable of inducing a pro-inflammatory phenotype, the nature of which can vary depending on the purpose of the specific experiment being conducted, and then analyzing the output in terms of changes in dendritic cell end-popints such as for cytokines and maturation markers from the mature dendritic cells to determine the anti-inflammatory capacity of the test component (figure 1). In an embodiment of the invention dendritic cell of non-human origin are used, such as murine dendritic cells.

In one embodiment of the invention said at least one pro-inflammatory cytokine and/or chemokine is selected from the group consisting of IL12 (p40/p70), TNFα, IFNy, IFNα, IL6, RANTES, IPlO, MCPl/2, MIPIa and mixtures thereof. In another embodiment of the invention the pro-inflammatory cocktail has been designed to increase secretion of at least one factor selected from IL12 (p40/p70), TNFα, IL6, RANTES, MCPl/2 and MIPIa, while concomitantly decreasing ILlO secretion. In another embodiment the ratio between secreted IL12 (p40/p70) and ILlO is at least 3 mg IL12 (p40/p70) per mg ILlO, such as at least 6 mg IL12 (p40/p70) per mg ILlO, or such as at least 9 mg IL12 (p40/p70) per mg ILlO.

In another embodiment of the invention said pro-inflammatory cocktail has been designed to increase the expression level of at least one intracellular endpoint as said at least one intracellular marker of inflammation. In another embodiment of the invention said proinflammatory cocktail has been designed to increase the expression level of at least one extracellular endpoint as said at least one pro-inflammatory compound which is not a cytokine and/or chemokine. In another embodiment of the invention said pro-inflammatory cocktail has been designed to increase the expression level of at least one intracellular or extracellular endpoint. In yet another embodiment said pro-inflammatory cocktail comprises at least one compound selected from the group consisting of ILlβ, IL6, TNFα, IFNy, IFNα, RANTES, IPlO, PGE2, TSLP, IL4, IL5, ILlO, IL13, TGFβ, MCPl/2, MIPIa, Poly I:C, CpG-or ODN-oligonucleotides, LPS, R848, peptidoglycan, LTA, Pam3Cys (and its derivatives), MALP- 2, paclitaxel, flagellin, gardiquimod, R837, loxoribine, lipomannan, FSL-I, profilin and zymosan. In yet another embodiment said pro-inflammatory cocktail comprises at least one compound selected from the group consisting of ILlβ, IL6, IFNy, IFNα, RANTES, IPlO,

PGE2, TSLP, IL4, IL5, ILlO, IL13, TGFβ, MCPl/2, MIPIa, Poly I:C, R848, LTA, Pam3Cys (and its derivatives), MALP-2, paclitaxel, flagellin, gardiquimod, R837, loxoribine, lipomannan, FSL-I, profilin and zymosan. In another embodiment said pro-inflammatory cocktail does not contain any of the compounds LPS, TNFα, peptidoglycan and CpG-or ODN- oligonucleotides. In another embodiment said pro-inflammatory cocktail comprises at least two compounds selected from the group consisting of ILlβ, IL6, TNFα, IFNy, IFNα, RANTES, IPlO, PGE2, TSLP, IL4, IL5, ILlO, IL13, TGFb, MCPl/2, MIPIa, Poly I:C, CpG- oligonucleotides, LPS, R848, peptidoglycan, LTA, Pam3Cys (and its derivatives), MALP-2, paclitaxel, flagellin, gardiquimod, R837, loxoribine, profilin and zymosan. In another embodiment said pro-inflammatory cocktail has been designed to change said activation of said at least one intracellular or extracellular endpoint. In another embodiment said proinflammatory cocktail has been designed to increase secretion of at least one factor from the dendritic cells or T-cells selected from IL12 (p40/p70), TNFα, IL6, RANTES, MCPl/2 and MIPIa, MDC, TARC, IL2, IL4, IL5, IL13, IL17, IL23, IL15, IL18, TGFβ, prostaglandins, leukotrienes, thromboxanes, nitric oxide (NO) and fatty acids. In another embodiment said pro-inflammatory cocktail has been designed to increase secretion of at least one factor from the dendritic cells or T-cells selected from IL4, IL5, IL13, IL17, IL23, IL15, IL18, TGFβ, prostaglandins, leukotrienes, thromboxanes, nitric oxide (NO) and fatty acids. In another embodiment the ratio between secreted IL12 (p40/p70) and ILlO is at least 3 mg IL12 (p40/p70) per mg ILlO, such as at least 6 mg IL12 (p40/p70) per mg ILlO, or such as at least 9 mg IL12 (p40/p70) per mg ILlO.

In yet another embodiment of the invention the pro-inflammatory cocktail comprises IFN-γ. In another embodiment the pro-inflammatory cocktail comprises IFN-γ in a concentration to contact the dendritic cells with an IFN-γ concentration in the range from about 1 ng/ml to about 200 ng/ml, such as in the range from about 5 ng/ml to about 50 ng/ml. In another embodiment the pro-inflammatory cocktail comprises CpG oligonucleotide. In another embodiment the pro-inflammatory cocktail comprises at least one member of the group consisting of IL6, TNFα, poly I:C and ILlβ. In another embodiment the pro-inflammatory cocktail comprises IFN-γ in a concentration to contact the dendritic cells with about 20 ng/ml IFN-γ and CpG-oligonucleotide in a concentration to contact the dendritic cells with about 1 μM CpG-oligonucleotide.

In one embodiment of the invention the test component is selected from the group consisting of a compound, a small chemical entity, a macromolecule such as a peptide, a protein, a polysaccharide, an oligonucleotide, a nucleic acid, a lipid, a sugar, a natural extract, a cell fragment, a microorganism, and mixtures and combinations thereof.

In another embodiment the intracellular marker is selected from the group consisting of intracellular enzymes and proteins which are not cytokines or chemokines, comprising intracellular nucleotides such as RNA or mRNA, intracellular organic or inorganic molecules such as nitric oxide (NO) and lipid derivatives. In another embodiment said at least one intracellular marker, preferably a protein, is involved in signaling processes downstream of Toll like receptor activation. In another embodiment said at least one intracellular marker, preferably protein, is involved in signaling processes downstream of pattern recognition receptor activation. In another embodiment said at least one intracellular marker, preferably protein, is involved in signaling processes downstream of cytokine or chemokine receptor activation. In another embodiment said at least one intracellular marker, preferably protein, is involved in signaling processes responsible for synthesis and/or generation of eicosanoids. In another embodiment said at least one intracellular marker and preferably protein is involved in signaling processes responsible for synthesis and/or generation of prostaglandins. In another embodiment said at least one intracellular marker, preferably protein, is involved in signaling processes responsible for synthesis and/or generation of leukotrienes. In another embodiment said at least one intracellular marker, preferably protein, is involved in signaling processes responsible for synthesis and/or generation of thromboxanes.

In another embodiment the at least one pro-inflammatory compound is selected from the group consisting of lipid derivatives such as eicosanoids, sphingolipids, prostaglandins, phospholipids, bioactive lipids, fatty acids (poly or monounsaturated and even saturated fatty acids), leukotrienes, oxylipins, thromboxanes, and inorganic molecules such as nitric oxide (NO).

In another embodiment said cytokines, chemokines, markers or substances are selected from the group consisting of IL2, IL4, IFNγ, ILlO, TGFβ, IL5 and IL13, IL17, IL6. In another embodiment said secreted substances comprising lipid derivatives of prostaglandins, leukotrienes, eicosanoids, bioactive lipids or fatty acid derivatives. Extracellular endpoints:

Inflammatory diseases like e.g. rheumatoid arthritis and Crohn 's disease, are associated with induction of inflammatory mediators. Some of the newly discovered mediators are derived from lipids within the diseased area or secreted from invading immune cells, mainly by phospholipase catalyzed release of arachidonic acid (AA) that is further converted to various eicosanoids that work to promote the inflammatory reaction by recruitment of relevant immune cells and initiate the subsequent events in the inflammatory pathway. Other eicosanoids are released later in the inflammatory process and are believed to participate in resolving the inflammation. Inflammation associated lipid mediators include e.g. prostaglandins, leukotrienes and thromboxanes. It is well established that AA is the substrate for the cyclooxygenase 1 and 2, (COX-I and COX-2), and that COX-2 is a well known target for treatment of inflammation. Elevated COX-2 expression in inflammatory disease induces the conversion from AA to prostaglandins, which possess potent biological effects for induction and later resolving of the inflammation (Funk C. D., Science, 294, 1871-1875, 2001). Typically, prostaglandin E2 (PGE2) is produced in the early stages of inflammation, whereas prostaglandin D2 (PGD2) is present in the later stages of inflammation (Tillery S. L. et al., the Journal of clinical investigation, 108, 15-23, 2001). Examination of inflamed tissue has revealed increased expression of both COX-2 and several prostaglandin synthases, leukotriene synthases and thromboxane synthases, which are responsible for the increased synthesis of their respective lipid mediators. The secretion of anti-inflammatory test components is linked to the ability of dendritic cells to induce these enzymes and generate their respective lipid mediators. Hence, the secreted lipid mediators may be useful as endpoints in the screening procedure.

Non-lipid associated organic or inorganic molecules are also shown to be involved in the development of inflammatory diseases. One classical example is the increased expression in DCs of the inducible nitric oxide synthase (iNOS or NOS2), which is responsible for producing nitric oxide in inflammatory tissue. It has been suggested that DCs in autoimmune disease like psoriasis are potential targets for treatment using pharmaceuticals able to suppress NO and TNFα produced from these cells (Guttman-Yassky E et al., J Allergy Clin Immunol. 119(5), 1210-7, 2007). Hence, non-lipid secreted mediators may be useful as endpoints in the screening procedure.

Particularly interesting extracellular endpoints comprise the following:

HMGBl : Human mature DCs actively secrete High mobility group box 1 (HMGBl) in response to inflammatory stimuli which acts as a primary pro-inflammatory signal. The secretion of HMGBl by DCs is necessary for up-regulation of co-stimulatory molecules such as IL12 production and subsequent activation of a ThI polarization of T cells (Dumitriu, I. et al. journal of immunol. 174: 7506-7515, 2005). HMGBl is released by inflammatory cells including DCs in inflammatory conditions in critically ill patients, and has been proposed as a target for treatment (Fink M. P., Crit Care, 11(5) :229, 2007).

Delta 4 Notch-like ligand : Toll like receptor (TLR)-dependent ThI responses may occur in the absence of IL-12. It was found that LPS activates MyD88-dependent Delta 4 Notch-like ligand expression on DCs and these cells direct ThI differentiation by an IL12-independent and Notch-dependent mechanism in vitro and in vivo (Skokos D. et al. J exp med. 204 (7) : 1525-31, 2007).

OX40L: OX40L is a costimulatory molecule which has been reported to be present on DCs after activation. It interacts with OX40 which is expressed transiently on activated CD4+ T cells leading to enhanced clonal expansion and secretion of cytokines. It has been shown that the expression of OX40L by activated DCs may drive the pathogenic response of colitis (Malmstrόm, V. j immunol. 166: 6972-6981, 2001). Additionally, Thymic stromal lymphopoietin (TSLP) induce human DCs to express OX40L which subsequently can induce TNFα producing inflammatory Th2 cell responses (Ito, T., J exp med. 202 (9) : 1213-23, 2005). OX40L expression as a marker on DCs is thought to be involved in the initial steps of development of allergic disease, and is therefore a critical marker expressed on DCs (Hoshino A. et al., Eur J Immunol. 33(4), 861-9, 2003).

5S-hydroxyeicosatetraenoic acid (5S-HETE) : 5S-HETE is a major eicosanoid produced by DCs in the absence of IL4, and might be a useful endpoint in the present screening model (Spanbroek R, et al., Proc. Nat. Acad. Sci. 24;98(9) :5152-7, 2001).

Prostglandin E2 (PGE2) : PGE2 is a well-known prostaglandin involved in inflammation and autoimmune disease. PGE2 is induced in DCs treated with LPS, and shown to increase the disease severity in models of Crohn 's disease (Teloni R., et al., Immunology.

Jan;120(l) :83-9, 2007, Sheibanie AF., J Immunol. 178(12), 8138-47, 2007). PGE2 is increased in various clinical inflammatory conditions, including e.g. chronic periodontitis and Type 1 diabetes mellitus (Araya AV. et al., Eur Cytokine Netw. 2003, 14(3): 128-33).

Leukotriene B4 (LTB4) : LTB4 is a major eicosanoid produced by DCs in the absence of IL4, (Spanbroek R, et al., Proc. Nat. Acad. Sci. 24;98(9) :5152-7, 2001). LTB4 is secreted from DCs after certain stimuli but is also a potent stimulator of migration of mature DCs, and might be a useful endpoint in the present screening model (Jozefowski S., et al., Immunology. 116(4) :418-28, 2005). 15S-hydroxyeicosatetraenoic acid (15S-HETE) and 5S-15S-hydroxyeicosatetraenoic acid (5S-15S-diHETE) are two major eicosanoids produced from IL-4-treated DCs, and might be a useful endpoint in the present screening model. (Spanbroek R, et al., Proc. Nat. Acad. Sci. 24;98(9) :5152-7, 2001).

Osteoprotegerin (OPG) : OPG is a marker of DC maturation, and is expressed as a function of treatment with maturation inducing reagents like e.g. LPS (Schoppet M. et al., J Cell Biochem. 15;100(6) : 1430-9, 2007). Furthermore, patients suffering from rheumatoid arthritis as well as Crohn 's disease showed higher OPG concentrations (Schulte CM, Aliment Pharmacol Ther. 20, 4:43-9, 2004).

TNFα: Tumor necrosis factor α is a hallmark cytokine with key roles in development of inflammation in autoimmune disease like IBD and arthritis. TNFα is a target for pharmaceutical treatment with antibody based therapy with several clinically proven antibodies. The ability of a compound to suppress TNFα-secretion from DCs is therefore an interesting end-point in the present screening method (M Karin., Proc Am Thorac Soc. 2(4) :386-90; discussion 394-5. 2005).

Other extracellular endpoints not mentioned in detail could include secreted matrix metalloproteinases (MMPs) which are induced in clinical inflammatory conditions or Th2 directing cytokines MDC and TARC, secreted from DCs in inflammatory diseases of Ezcema.

Intracellular endpoints:

Whereas the above mentioned enzymes like iNOS, COX2 and various synthases act on the effector function of dendritic cells, and cause secretion of inflammatory mediators, these enzymes are themselves potential endpoints in the present DC-screening model. Many other inflammatory related intracellular enzymes and proteins are highly relevant target endpoints for assessment of anti-inflammatory test components. These potential endpoints are associated with the signaling mechanisms leading from TLR, Pattern recognition receptors (PRR), cytokine or chemokine receptor activation to transcriptional regulation of the effector enzymes described above. Hence, using these signaling proteins or their mRNAs as endpoints in the claimed DC-based screening model one can assess the anti-inflammatory properties of test components. Receptor activated endpoints of interest for assessment of anti-inflammatory activity involves the level of protein (or mRNA encoding this) as well as the level of protein activation e.g. by phosphorylation of such proteins; AKTl, AKT2, AKT3, BTK, ERK, FADD, IKKα, IKKβ, IKKε, IKKγ, IRAKI, IRAK4, IRAK-M, IRFl, IRF3, IRF5, IRF7, JNK, MAL, MAPKAP, MAPkinases (p38), MEKK, MKK3, MKK6, MKK7, MLK3, MyD88, NFkB (subunit p50, p65, cREL, RELA), PI3K, RIPI, RIP2, RIP3, SARM, SOCS, STATl, STAT2, STAT3, SYK, TABl, TAB2, TAKl, TBKl, TIRAP, TOLLIP, TRAF3, TRAF6, TRAM and TRIF.

Intracellular endpoints involved in DC or immune cell function, in inflammatory diseases, are particularly interesting. Such endpoints comprise:

Nuclear factor K beta (NFkB) : is a transcription factor involved in receptor mediated signaling in DCs. NFkB consists of 3 subunits, p50 and cRel which are crucial for IL-12 and IL-18 gene expression and ReIA which is crucial for expression of the inflammatory cytokines like TNFα, IL-lα and IL-6 (Wang, J. et al. J. Immunol, 178 (ll) :6777-88, 2007). Inhibitors of the kinase responsible for NFkB activation, IkappaB kinase, are expected to have potent anti-inflammatory activity (Karin M. Proc Am Thorac Soc.2(4) :386-90; discussion 394-5, 2005).

Myeloid differentiation primary-response protein 88 (Myd88) : Myd88 is an adaptor protein involved in signaling in the primary pathways for TLR activation in DCs (Rudd, B. D., J. immunol, 178 (9) : 5820-7, 2007). Myd88 has shown to be downregulated in patients with Crohn 's Disease both before and after TLR-activation, indicating that Myd88 is involved in the deregulation of the innate immune response in Crohn 's Disease patients (Naito Y. et al., Aliment Pharmacol Ther. 24,4:256-65, 2006).

Mitogen activated protein kinase (MAPK) : The signaling pathway involving MAPK signalling is considered as crucial for the induction and maintenance of chronic inflammation and its components thus emerge as interesting end points for anti-inflammatory drug screening as well as molecular targets of small molecule inhibitors for controlling inflammation. (G. Shett et al., Ann Rheum Dis. Sep 7, 2007).

Extracelluar signal Regulated Kinase (ERKl, 2) : ERKl and 2 are activated by MEK1/2, and involved in TNFα mRNA transport and hence TNFα secretion. Inhibition of ERK1/2 thus provides an interesting tool for prevention of TNFα secretion, which is one of the most potent inflammatory cytokines (Usluoglu N. et al., Eur J Immunol, 37(8) :2317-25, 2007, Karin M., Proc Am Thorac Soc. 2(4) :386-90; discussion 394-5, 2005).

Interferon regulatory factor (IRF) : IRFs are a group of transcription factors activated by either TLR-agonists or cytokines. The function and expression of different types of IRFs has been analysed in various immune associated diseases, and several indications have shown that subtypes of IRFs are associated with development of e.g. psoriasis, Crohn 's disease and arthritis (Sigurdsson S. et al., Arthritis Rheum.56(7):2202-10, 2007, Clavell M. et al., J Pediatr Gastroenterol Nutr. 30(l) :43-7, 2000). C-Jun N-terminal Kinase (JNK) : CpG stimulation induces rapid Syk-dependent phosphorylation of the protein kinase JNK in DCs which leads to production of the proinflammatory cytokines IL6, TNFα and IL12p40. JNK is critical for the function of inflammatory cells in autoimmune diseases and has been suggested as a therapeutic target for treatment of inflammatory diseases. Thus, JNK downregulation by anti-inflammatory components in DCs is an interesting end-point for identification of such reagents (LeibundGut-Landmann, S. et al., nat immunol. 8(6) : 630-638, 2007, Liu G. et al., Curr Opin Investig Drugs. 6(10):979-87, 2005).

Fatty acid-binding proteins (FABPs) : FABPs act as intracellular receptors for hydrophobic compounds and regulates inflammatory pathways. DCs deficient for FABPs are poor producers of pro-inflammatory cytokines and FABP-deficient mice exhibit reduced clinical symptoms of experimental autoimmune encephalomyelitis (Reynolds, J. M. et al. Journal of immunol, 179:313-321, 2007).

Glycogen synthase kinase 3 (GSK-3) : GSK-3 is linked to neurologic diseases such as Alzheimer but also to non neurologic diseases as diabetes mellitus (Wagman, AS., et al.

Curr Pharm Des. 10: 1105-1137, 2004). GSK-3 is a multifunctional intracellular enzyme and a candidate regulator of DC function as it mediates differentiation and activation of proinflammatory DCs. GSK-3 enhances production of pro-inflammatory cytokines (Rodionova, E. et al. Blood, 109: 1584-1592, 2007).

COX-2 : COX-2 has for more than a decade been an attractive pharmaceutical target for treatment of cancer and inflammatory diseases, mainly through inhibition of the activity of COX-2, thereby preventing the generation of prostaglandins at the diseased site. Many different selective COX-2 inhibitors are currently on the market or in clinical trials for treatment of variety of inflammatory diseases (Patrignani P., et al., Brain Res Brain Res Rev. Apr;48(2):352-9, 2005, Tsatsanis C. et al., Int J Biochem Cell Biol.;38(10) : 1654-61, 2006).

Inducible nitric oxide synthase (iNOS) : iNOS is essential for the up-regulation of the inflammatory response through its production of nitric oxide (NO). NO promotes inflammation and likely participates in destructive mechanisms in the rheumatoid joint. NO is not only involved in inflammation but also in vaso- and bronchodilation as well as a large group of other physiologically relevant conditions. Therefore, iNOS inhibition might not be a suitable approach for systemic treatment of inflammation, but test components able to suppress iNOS activity and concomitant NO release might be useful for local alleviation of inflammation where the test compound is not administered systemically (Cuzzocrea S. Curr Pharm Des.l2(27) :3551-70, 2006, Mulrennan S. A, Treat Respir Med. 3(2) :79-88, 2004). Lipoxygenases: 15-lipoxygenase 1 and 5-lipoxygenase (5-LO) are involved in the synthesis of leukotrienes in leukocytes, which are strong chemoattractants for immune cells, (e.g. LTB4). Leukotrienes are also strong activators of DCs differentiated with GM-CSF and TNFα, and 5-LO expression has been identified in distinct DC phenotypes in vivo. (Spanbroek R, et al. Blood. Dec l;96(12) :3857-65, 2000, Sharma J. N., and Mohammed LA, Inflammopharmacology. Mar;14(l-2) : 10-6, 2006).

Indoleamine-pyrrole 2,3 dioxygenase (IDO) : IDO is a marker for DCs that are able to induce T-cell tolerance, and induction of IDO has been proposed as a therapeutic strategy in treatment in autoimmune disease. The expression of IDO in DCs is therefore a favourable marker for anti-inflammatory test components, and therefore an interesting end-point for the present DC screening model (Penberthy W. T. Curr Drug Metab. Apr;8(3) :245-66. 2007).

Prostaglandin synthases: Prostaglandin synthases are involved in the generation of PGE2, which is secreted from inflammatory cells upon their activation. Prostaglandin synthase activity correlates with the inflammatory state of immune cells like DCs (Teloni R., et al., Immunology. Jan;120(l) :83-9, 2007, Sheibanie AF., J Immunol. 178(12), 8138-47, 2007). In clinical inflammatory conditions such as chronic periodontitis and Type 1 diabetes mellitus, prostaglandin synthase is increased (Araya AV. et al., Eur Cytokine Netw. 14(3): 128-33, 2003).

Thromboxane synthase: Thromboxane synthases are responsible for generation of thromboxanes, which are produced in excess in diseases like inflammatory bowel disease. Thromboxane synthase inhibitors are being developed due to their potential antiinflammatory activity in patients with autoimmune disease, and thromboxane synthase expression in lamina propria cells occurs in active inflammatory bowel disease. The increased expression of thromboxane synthases in inflammatory DCs is a suitable end-point for assessing the inflammatory response in DCs (Carthy E. et al., J Clin Pathol. May;55(5) :367-70, 2002).

5-LO activating protein (FLAP) : FLAP is strongly induced in DCs differentiated with GM-CSF and TNFα (Spanbroek R., et al. Blood. Dec l;96(12) :3857-65, 2000), and is strongly upregulated in inflammation, atherosclerosis and metabolic diseases and could serve as an important marker of inflammatory status in dendritic cells (Back M. et al., Circ Res., 13; 100(7) :946-9, 2007).

In one embodiment of the invention the intracellular marker is selected from the group consisting of intracellular enzymes and proteins, intracellular nucleotides such as RNA or mRNA, intracellular organic or inorganic molecules such as nitric oxide (NO) and lipid derivatives. In another embodiment at least one intracellular marker, preferably a protein, is involved in signaling processes downstream of Toll like receptor activation. In another embodiment at least one intracellular marker, preferably a protein, is involved in signaling processes downstream of pattern recognition receptor activation. In another embodiment at least one intracellular marker, preferably protein, is involved in signaling processes downstream of cytokine or chemokine receptor activation. In another embodiment at least one intracellular marker, preferably protein, is involved in signaling processes responsible for synthesis and/or generation of eicosanoids. In another embodiment at least one intracellular marker and preferably protein is involved in signaling processes responsible for synthesis and/or generation of prostaglandins. In another embodiment at least one intracellular marker, preferably protein, is involved in signaling processes responsible for synthesis and/or generation of leukotrienes. In another embodimentat least one intracellular marker, preferably protein, is involved in signaling processes responsible for synthesis and/or generation of thromboxanes.

Functional endpoints:

Dendritic cells not only possess the ability to induce inflammation-associated proteins and secrete inflammatory mediators, but also to initiate the crucial first step in adaptive immunity through activation of naϊve T cells. In order for naϊve T cells to be activated it must recognize a foreign peptide bound to self MHC molecules and simultaneously receive a co-stimulatory signal delivered by the APC. The most potent activators of naϊve T cells are mature DCs, and these are thought to initiate most, if not all, T-cell responses in vivo. The activation of T cells leads to their proliferation and the differentiation of their progeny into effector T cells having a variety of functions.

Until recently, the known universe of adaptive CD4+ T cell responses has been encompassed by the Thl-Th2 paradigm. Development of T helperl (ThI) cells, which evolved to enhance clearance of certain intracellular pathogens, is coupled to the actions of IFNγ and IL12, whereas the Th2 cells, which evolved to enhance the clearance of parasites is coupled to IL4. Another newly described linage of CD4+ T cells is the Thl7 cells. Thl7 cells are characterized by production of IL17 and have likely evolved to enhance host clearance of a range of pathogens distinct from those targeted by ThI and Th2 cells.

The benefits of adaptive CD4+ T cell responses, however has a price. Inappropriate or poorly controlled effector T cells can cause autoimmunity or allergy and have deleterious effects when directed against self antigens or ubiquitous environmental or commensal flora antigens, which unlike most pathogens, cannot be effectively cleared. In this setting, persistent effector T cell responses drive chronic inflammatory disorders such as autoimmunity and allergy. In general it has been thought that ThI cells are responsible for organ-specific autoimmunity, i.e. diabetes mellitus and multiple sclerosis, whereas Th2 cells mediate eczema, allergy and asthma. However, also Thl7 cells with specificity for self- antigens are highly pathogenic and lead to the development of inflammation and severe autoimmunity (Afzali B. et al., Clin Exp Immunol. 148(l):32-46, 2007).

Although a key mechanism whereby dysregulated effector responses are avoided is through intrathymic deletion of self-reactive clones, postthymic mechanisms are also critical as many antigens are developmentally or physically sequestered from developing thymocytes .

Hence, evolutionary pressure to match the development of adaptive effector T cell responses with corresponding regulatory T cell programs has probably been critical to the successful emergence of adaptive immunity. Today, several subsets of regulatory T (Treg) cells have been described, albeit with incompletely defined lineage relationships and functions.

Functional endpoints to assess DC phenotype and function may be defined by the outcome of T cell responses when T cells are added to the DCs. The following assays to measure T cell responses are relevant: Antigen specific or polyclonally stimulated T cell proliferation, mixed lymphocyte culture, Elispot, ⁵¹Cr-release assay, functional bioassays, FACS analysis, Immunofluorescence, ELISA and other similar assays for cytokine/chemokine measurement.

Naϊve T cells can be induced to commit to the particular lineages, ThI, Th2, Thl7 and Treg cells, based on mode of stimulation, antigen concentration, co-stimulation and cytokine milieu.

Suitable endpoints in ThI cells: ThI cells regulate antigen presentation and cellular immunity. Naϊve CD4+ T cells differentiate towards ThI in the presense of IL-12, which induces secretion of IFN-γ by Thl-cells. As an extracellular endpoint, IFN-γ is a pleotropic cytokine that plays an essential role in both innate and adaptive immunity. The essential role of IFN-γ is to activate macrophages in order to eliminate intracellular pathogens (Boehm, U. Annu rev immunol. 15:749-95, 1997). Other relevant extracellular endpoints for ThI cells may be as follows: GM-CSF, TNF-α, IL-2, IL-3, TNF-β, CXCL2, lymphotoxin-β, CD40 Ligand, Fas ligand, ICOS, OX40, LFA-I and isotype switching to IgG2a.

Intracellular endpoints in Thl-cells are associated with the process of ThI differentiation, which is regulated by the following key factores: T-bet, STAT4, H2.0-like homebox 1 (HLXl) and IRF-I and ERM. T-Box Transcription Factor Family Member, T-bet (T-bet) is the critical transcription factor, also known as Tbx21, which promotes ThI differentiation and inhibits Th2 differentiation (Dong, C. Nat Rev Immunol. 6:329-333, 2006). It has been shown that mice lacking T-bet fail to mount a ThI response, accompanied by an elevated Th2 profile leding to asthmatic conditions (Finotto, S., Science, 295:336-38, 2002). T-bet upregulates the inducible chain of the IL-12 receptor (II-12Rβ2) while suppressing Th2 associated factors.

H2.0-like homebox 1 (HLXl) is a Thl-specific homeobox gene, which is a target gene for T- bet. When expressed together, HLXl and T-bet have synergistic effects on IFN-γ production. HLXl expression is restricted to ThI but not Th2 clones (Mullen, AC, Nat Immunol, 10:652- 58, 2002)

STAT4 is an essential component of the IL12 and other signaling pathways and plays an important role in ThI differetiation. The IL-12/STAT4 pathway does not initiate IFN-γ expression but amplifies the amount of IFN-γ produced by individual cells (Mullen, AC, Science, 292:1907-10, 2004).

IRF-I and ERM are both induced in an IL12/STAT4- dependent manner in CD4 T cells. It has been suggested that they are directly involved in ThI differentiation or in IFN-γ gene transcription. ERM is ThI specific (Ouyang W., PNAS, 96:3888-93, 1999).

Other relevant intracellular endpoints in Thl-cells involve the protein level or activation state by e.g. phosphorylation of such proteins, which include: Jakl, Jak2, STATl, RIk, Itk, PLCγ, Rac2, MKK3/4/6, p38-MAPK, NFKB, JNK2, MyD88, IRAK, TRAF6, eomesodermin (EOMES), STATl, STAT3, NFAT, AP-I, NFKB, ATF-2, GADD45γ, GADD45β.

Suitable endpoints in Th2 cells: Th2 cells are important regulators in humoral immunity and allergic responses. Th2 cells differentiate from naive T cells in response to IL-4. Th2 cells mainly secrete IL-4, IL-5, IL-10, IL-13 and express CD40 Ligand, which are all suitable end- points to assess the functional phenotype of T-cells added to DCs in a MLC However, other relevant extracellular endpoints may be: IL-3, GM-CSF, TGF-β, CCLIl, CCL17 and isotype switching to IgGl and IgE. As a hallmark cytokine that directs Th2 development IL-4 is produced by the Th2 cells themselves and modulates many Th2 effector functions. IL-4 mediates MHC class II upregulation and promotes antibody isotype switching to IgE and IgGl (Nelms K., Annu rev Immunol, 17:701-738, 1999).

Relevant intracellular key endpoints which specifically regulate Th2 differentiation and cytokine expression, and which might be suitable as endpoints to assess DC-induced functional T-cell phenotypes comprise GATA-binding protein 3 (GATA3), STAT6 and MAF. GATA3 is a Th2 specific master transcription factor which promotes Th2 development and simultaneously suppresses ThI programs (Usui. T., Immunity, 18:415-428, 2003). Transgenic mice bearing a dominant negative form of GATA3 display reduction in Th2 cell- mediated allegic lung inflammation (Zhang, DH., Immunity, 11 :473-482, 1999).

STAT6: IL-4 promotes the development of Th2 through the action of STAT6 which is necessary for maximal Th2 development (Takeda, K. Nature, 380:627-630, 1996). In studies with a Th2-eliciting parasite infection, the primary IL-4 response remained intact in STAT6-/- mice, while secondary and Th2 memory responses were abrogated (Finkelman FD., J immune. 164:2303-2310, 2006).

MAF is a Th2 specific transcription factor that has an important role in IL-4 production once the Th2 programme has been established (Ho, I., Cell., 85:973-983, 1996). Overexpression of MAF in transgenic mice, disrupts the Thl/Th2 balance towards a Th2 skewed phenotype, and is sufficient to confer protection in autoimmune disease models (Pauza, M. E., Diabetes. 50: 39-46, 2001).

Other relevant intracellular endpoints in Th2-cells may be: Jakl, Jak2, STAT3, NFATcI,

NFATc2, CnA, ITK, PLCγ, Ras, Jnkl, p38, Erk, JUNB, Itch, p50, IRF4, NIP45, STAT5 (Mowen K. A., Immunol Rev. 202:203-222, 2004).

Suitable endpoints in Thl7 cells: Thl7 cells comprise a recently identified Th lineage that has a pro-inflammatory role in autoimmunity and tissue inflammation. Thl7 differentiation is initiated by TGF-β and IL-6, whereas in vivo IL-23 is additionally required for Thl7 cell differentiation. The extracellular key endpoints for Thl7 cells are IL-17A, IL-17F, TNF-α, IL- 21 and IL-22 (Infante-Duarte, C, J immunol, 165:6107-6115, 2000).

IL-17A is a pro-inflammatory mediator which stimulates the production of IL-6, IL-lβ, nitric oxide, PGE2, G-CSF and several chemokines: CXCLl, CCL2, CXCL2, CCL7, CCL20 as well as matrix metalloproteases (KoIIs, JK., immunity, 21 :467-476, 2004). IL-17 expression was found to be associated with many inflammatory diseases in humans such as rheumatoid arthritis and asthma (Dong, C, Nat rev Immunol. 6: 329-333, 2006)

The intracellular endpoints responsible for Th 17 differentiation are still poorly understood, however, STAT3 and the transcription factor retinoic acid related orphan receptor-γt (RORγt) mediate Thl7-cell lineage commitment. The orphan nuclear receptor RORγt is the key transcription factor that mediates differentiation of Thl7 cells (Ivanov, I., Cell. 126: 1121- 1133, 2006). RORγt is required for the expression of IL17A and IL-17F in response to IL-6 and TGF-β. Mice with RORγt deficient T cells have attenuated autoimmune disease and lack tissue-infiltrating Thl7 cells (Ivanov, I., Cell. 126: 1121-1133, 2006). Signaling via IL-6 activates STAT3 in Thl7 cells (Chen, Z., PNAS. 103: 8137-8142, 2006). It has been shown that there is an absolute requirement for STAT3 signaling in Thl7 differentiation and that STAT3 signaling also mediates a relative inhibition of ThI differentiation. Moreover STAT3 activation has been observed as an important transcription factor in a number of autoimmune diseases such as multiple sclerosis (Lock, C, Nat Med., 8:500-508, 2002) and systemic lupus erythematosus (Chabaud, M., Arthritis Rheum., 42: 963-970, 1999). Thus STAT3 is a candidate target for Thl7 dependent autoimmune disease immunotherapy that could selectively inhibit pathogenic immune pathways.

Othe relevant intracellular endpoints to assess the functional phenotype of T cell development into Thl7 cells in the proposed screening model could include Smads, Socs3, Actl, IkB, JNK, Erk, p38-MAPK, NF-kB, TAKl, TRAF3 and TRAF6.

Suitable endpoints in Regulatory T cells (Treqs) : Besides effector T-cel I subsets being ThI, Th2 and Thl7 cells, CD4 T-cells can differentiate into distinct regulatory subsets characterized by their ability to suppress adaptive T cell responses and prevent autoimmunity. While at least one class of Treg cells, naturally occurring (nTreg) cells develops intrathymically, other Tregs develop from naive CD4 T cell precursors in the periphery, socalled induced, or adaptive Tregs (aTregs). Suitable extracellular endpoints for Treg cells are the high-affinity component of the IL-2 receptor (CD25) IL-2, the glucocorticoid-induced tumor necrosis factor- receptor-related (GITR) protein, Folate receptor 4 (FR4), neuropilin-1, LAG-3, IL7R α-chain (CD127), CTLA4, OX-40, CD103, 41BB, TLR 4/5/7/8, membrane-bound TGF-β, TGF-β, IL-10.

The central intracellular endpoint of nTreg cells is the transcription factor FOXP3 (forkhead box P3). FOXP3 play an essential role in the development and function of nTreg cells. However, the underlying molecular mechanisms by which FOXP3 functions remains to be elucidated (Campbell, Dj. Nat rev immunol. 7: 305-310, 2007). The importance of FOXP3 is highlighted by experiments in which mutations in FOXP3 results in fatal autoimmune lymphoproliferative disease due to lack for nTreg cells. Similarly, humans with IPEX (immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome), suffering from multiorgan autoimmune disorders have mutations in FOXP3 and lack functional nTreg cells (Bennett, C. L., Nature Genet. 27:20-21, 2001).

Other relevant intracellular endpoints for Treg cells may be: ReI family transcription factors, NFAT, NF-KB, LAT-PLC-γl, STAT5, SMAD2/3/4 (Long, E. Transplantation. 84: 459-461, 2007). Murine Dendritic Cell Model

Dendritic cells from different mammals may be used in the present invention. Dendritic cells of murine origin are prepared in vitro from DC precursor cells by treating these with a differentiation-inducing composition for a predetermined period of time. The differentiation- inducing composition consists of one or more of the following cytokines and chemokines: IL4, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), Flt-3 ligand, stem cell factor (SCF), IL3, TGFβ or TNFα. This differentiation treatment results in immature dendritic cells after a period of 4-12 days, and typically 6-8 days. Our present screening model is based on murine bone marrow derived cells as dendritic precursor cells that develop into immature dendritic cells after 8 days of differentiation in the presence of GM-CSF. Hence, in one embodiment the dendritic cells used in the present invention are immature dendritic cells. Other useful sources of DC- precursor cells are monocytes, stem cells, stem cell lines, cancer cell lines, spleen, thymic and lymph node dendritic cells (which might constitute a mixture of dendritic cell precursors, immature dendritic cells and mature dendritic cells).

The immature dendritic cells are treated with the test component (small molecules, macromolecules, natural extracts, microorganisms etc) prior to, simultaneously with or after addition of test component. The pro-inflammatory cocktail comprises cytokines, chemokines, prostaglandins and ligands (including nucleotide based macromolecules such as poly I:C and GA-polymers) binding to receptors such as pattern recognition receptors (PRR) including but not limited to toll like receptors (TLR-ligands). A pro-inflammatory cocktail is developed by analyzing pro-inflammatory components alone and in various combinations and selecting the combination that is the most effective and/or efficient at (a) inducing secretion of pro-inflammatory cytokines and chemokines, in particular high level of IL12 secretion, from the dendritic cells (b) minimizing induction of ILlO secretion and/or (c) inducing higher expression of maturation markers like CD40, CD80, CD86, CD83, CCR7, MHCII compared to immature dendritic cells. Once mature, the dendritic cells have decreased endocytic and phagocytic activity. After a predetermined period of time between 12 and 96 hours, and typically between 16 and 24 hours, the effect of the test component on the effect of the pro-inflammatory cocktail is determined by analyses of the growth media from the treated dendritic cell culture for cytokine and chemokine profile, including but not limited to ILlO, IL12, RANTES, MCPl, TNFα and IL6. The analyses further comprise measuring the expression of maturation markers of the treated dendritic cells.

The suppressive ratio is dependent upon the time interval between addition of test component and the pro-inflammatory cocktail. In one embodiment, the test component is added 1-8 hours prior to the pro-inflammatory cocktail. The suppressive ratio obtained increases with the length of time between addition of the test component and the proinflammatory cocktail for an anti-inflammatory test component. Based upon IL12 concentrations and using a time interval of 2 hours, a high suppressive ratio (>1.5) corresponds to a test component with potential anti-inflammatory activity, whereas a low suppressive ratio (<1) corresponds to a test component with poor anti-inflammatory activity, or possibly even pro-inflammatory activity.

The method for in-vitro determination of the in vivo anti-inflammatory effect of a test component according to the present invention provides a number of applications.

In one aspect the present invention provides the use of the method for predicting the ability of a test component to prevent or alleviate an inflammatory condition in a human, comprising subjecting the potential test component to said method for in-vitro determination of the anti-inflammatory effect. In one embodiment the inflammatory condition is selected from an inflammatory bowel disease such as Crohn 's disease or ulcerative colitis, rheumatoid arthritis, psoriasis, eczema, allergy, systemic lupus erythromatosis, or other autoimmune or allergic diseases.

In another aspect the present invention provides the use of the method for quantification of the in-vitro effect of a test component on the pro-inflammatory phenotype of said mammalian dendritic cells resulting from contacting said mammalian dendritic cells with said pro-inflammatory cocktail. In one embodiment said quantification is carried out for at least two different concentrations of said test component to provide a dose-response curve.

Human Dendritic Cell Model

A dendritic cell based human model for screening and selection of anti-inflammatory compounds is made by developing blood monocytes, CD34+ progenitor cells, stem cells, stem cell lines, or cancer cell lines of haematopoetic origin into immature dendritic cells by incubating the cells with a differentiation inducing composition comprising IL4, GM-CSF, M- CSF, Flt-3 ligand, SCF, IL3, TGFβ or TNFα without serum, or with serum. Typically, immature dendritic cells are developed from CD14+ and/or CDlIc+ monocytes over a period of 6-10 days, in the presence of GM-CSF and IL4.

The immature dendritic cells are treated with a range of different cocktails of pro- inflammatory components to select for a specific pro-inflammatory cocktail that provides high levels of IL12 expression (p40 or p70 subunit) and secretion, and increased expression of maturation markers like CD40, CD80, CD86, CD83, CCR7, MHCII. The cocktail components used for human dendritic cells have the same physiological effects as the cocktail components used for the mouse dendritic cells, but are of human rather than of murine origin where relevant. A typical pro-inflammatory cocktail for maturation of human dendritic cells consists of one or (usually) more of the following components: poly I:C (range 1-50 ug/ml), IFNα/γ (range 100-5000 units/ml), ILlβ(range 1-50 ng/ml), IL6 (range 1-50 ng/ml), IL12 (range 1-50 ng/ml), TNFα (range 5-250 ng/ml), TGFβ(range 1-50 ng/mf)

The test component for screening of anti-inflammatory activity is added prior to, simultaneously with or after addition of the pro-inflammatory cocktail, and most likely the test component is added to the immature dendritic cells 2-4 hours prior to addition of the pro-inflammatory cocktail.

After 4-96 hours of incubation, and more specifically between 12-48 hours, the conditioned media is analysed for the presence and levels of IL12 (subunit p40 or p70), IL6 or TNFα or other extracellular or intracellular markers of inflammation. A second possibility is to detect dendritic cell maturation markers such as CD40, CD80, CD86, CD83, CCR7, MHCII. A third possibility is to assess functional end-points by applying the MLC-reaction to the treated DCs. If the test component is able to suppress the induction of IL12 (subunit p40 or p70), IL6 or TNFα and/or for some test component suppress expression of maturation markers like CD40, CD80, CD86, CD83, CCR7, MHCII or other of the aforementioned end-points it would be considered to be anti-inflammatory and therefore to have potential clinical applications such as in the treatment of human inflammatory conditions like gastrointestinal inflammations (which include but are not limited to Crohn 's disease and ulcerative colitis), rheumatoid arthritis, psoriasis, eczema, allergy, systemic lupus erythromatosis or other autoimmune or allergic diseases.

Discussion of experiments.

In our present exemplification the immature dendritic cells derived from murine bone marrow cells were treated for 16 to 18 hours with various combinations and concentrations of pro-inflammatory compounds to optimize for a cocktail-induced dendritic cell secretion of high levels of IL12 and low levels of ILlO. The optimal cocktail constitutes the proinflammatory cocktail which in this example comprises CpG thioate nucleotide sequence and IFNγ. The test component being tested was added to the immature dendritic cells 1-8 hours prior to the addition of the pro-inflammatory cocktail and typically 2 hours after addition of the test compound.

A compound exhibiting in vitro anti-inflammatory activity is capable of suppressing the production and secretion of pro-inflammatory cytokines and chemokines like e.g. IL12, RANTES, MCPl, TNFα and IL6 either intracellular^ or extracellularly in the growth media, and/or suppression of expression of the aforementioned maturation markers or other membrane bound, intracellular or extracellular markers of inflammation or functional end- points.

The method described above was validated by demonstrating the expected suppressive activity of two commonly known anti-inflammatory compounds, the glucocorticoid dexamethasone and the prostaglandin D2 (PGD2) in the model. Both compounds were able to reduce the pro-inflammatory phenotype comprising synthesis of IL12, IL6 and TNFα in a dose dependent manner in the dendritic cells.

The ability of the in vitro method described above to predict the in vivo anti-inflammatory effect of a test component was assessed by determining the anti-inflammatory activity of Lactobacillus strains and comparing said activity to the ability of these strains to reduce the development of colitis in a trinitrobenzene sulfonate (TNBS) induced mouse model which is a model for human Crohn 's disease. It was found that a high suppressive ratio as defined below correlated with the ability to reduce the development of colitis.

Dendritic cell based in vitro models have previously been examined for the effects of biological and disease associated molecules to influence DC maturation. A. Takahashi et al., (Cancer Immunol Immunother. 53, 543-550, 2004), have analysed the effect of the human protein VEGF and its influence on DC maturation. The effect of VEGF was analysed in the context of exploring the potential negative impact of VEGF on DC mediated immune responses in relation to cancer development. The authors treated in vitro differentiated immature DCs with VEGF together with either a DC-maturing cocktail consisting of TNFα, PGE2, IL6 and ILlβ, or LPS, where all components were added simultaneously. After 7 days of incubation the IL12p70 level was analysed in conditioned media, and potential of the DCs to stimulate allogenic T-cell proliferation. VEGF was shown to slightly reduce the ability of LPS to induce IL12p70 secretion and T-cell proliferation. Our present invention differs from this publication on several points: first, A. Takahashi et al., did not consider timeing between addition of VEGF and maturation stimuli (LPS or cocktail), second, the cocktail and LPS used as maturation stimuli were not optimized to induce a predetermined proinflammatory component like IL12p70, third, the matured DC phenotypes were not compared to an inflammatory disease phenotype in terms of e.g. cytokine pattern, and fourth, the authors do not mention the use of DCs in screening of anti-inflammatory compounds, but in the context of exploring the effect of normally occurring and biologically relevant proteins for the development of mature DCs in tumors of cancer patients, and its impact for the immune system in fighting the tumor. A number of groups have studied the effect of naturally occurring immunoregulating cytokines and chemokines in relation to studies of DC function under normal physiological and pathofysiological conditions. The nature of these studies focus on unraveling the complexity of DC biology, and in particular to study the effect of different cytokines in relation to DC maturation, development and function. To some extent these studies use in vitro models of DCs and different maturation stimuli including defined cocktails. Examples of such studies are:

Grauer O et al.,(J Neurooncol. 82(2), 151-61, 2007), have studied the involvement of tumor produced TGFβ2 and its involvement in escape from immune surveillance in gliomas. They showed that TGFβ2 was involved in inhibition of DC maturation, believed to be important for immune mediated T-cell activation against tumor antigens. When DCs were matured with cocktails comprising 1) TNFα, PGE2, IL6 and ILlβ, or 2) Poly I:C, or 3) R848, TNFα, IFNγ and ILlβ, TGFβ2 was no longer able to inhibit DC maturation. The authors suggest that DCs matured by these cocktails could play a role in efficient immunotherapy for cancer patients. The authors do not discuss issues relating to anti-inflammatory screening models, discovery of anti-inflammatory molecules or the similarity of the matured DCs to DCs from inflammatory diseases.

In another example, Teloni et al., (Immunology, 120, 83-89, 2006), have analysed the intracellular marker of inflammation COX-2, and its expression dependent on the presence of IL4. Human DCs were made from monocytes in the presence of IL4 and GM-CSF under the differentiation process to immature DCs. The continued presence of IL4 blocked the LPS induced expression of COX-2 in the DCs, whereas depletion of IL4 allowed COX-2 expression. This work differs from our present invention in the fact that IL4 is used for the differentiation process from monocytes to immature DCs, and is considered a requirement for DC development. IL4 can in this respect not be considered as an anti-inflammatory drug candidate, but rather a component of the immune system that is believed to influence the normal DC development and function. The authors do not mention screening models or antiinflammatory components.

A follicular dendritic cell line (HK) model was published by Lee et al., (MoI Immunol, 44(12), 3168-72, 2007). The follicular dendritic cells were treated with TNFα as the proinflammatory stimulus, and the effect on COX-2 and prostacyclin analysed as inflammatory end-points. When indomethacin or a selective COX-2 inhibitor was added, COX-2 activity was reduced. This follicular DC model differs from the present invention at several points. First, follicular dendritic cells are very different from the dendritic cells suggested used in this present invention, comprising dendritic cells of myeloid (mDC) or plasmacytoid (pDC) origin. Follicular DCs are mainly present in the lymphoid follicles and not believed to be present in the tissue or circulation as gatekeepers of immune survelliance as mDCs and pDCs. Second, they possess no ability to phagocytose foreign particles like mDCs, which is relevant for the present invention in relation to several screening aspects, e.g. for usefulness of screening of anti-inflammatory microorganisms where phagocytosis of the microorganisms and intracellular antigen processing are important for the DC-function. Third, follicular DCs process foreign antigen on a much longer timescale compared to mDC and pDCs, which makes them less suitable in a screening model. Finally, the follicular dendritic cell line model shown by Lee et al., use an immortalized cell line, which most likely does not posess the same repertoire of TLR and PRR expression, as well as intracellular signaling pathways, which are required for the screening method mentioned in this present application.

A final example is mentioned, describing a study where the effect of pathogens are analysed in respect to DC function, and in particular in relation to the inhibition of a proper immune response from the host towards the infectious organisms. Sa-Nunes et al., (J Immunol, 179, 3, 1497-1505, 2007), have shown that the saliva from Ixodes scapularis (blood sucking arthropod) have the ability to inhibit dendritic cell maturation when added simultaneously with LPS. The work by Sa-Nunes et al., does not involve the optimization and use of a proinflammatory cocktail to induce a range of disease associated and pre-determined cyto- or chemokines as in the screening model according to the present invention. Secondly, Sa- Nunes et al., does not disclose the use of DCs in relation to screening of anti-inflammatory components.

Probiotics

Based on the predictive abilities of the method, cf. above, a collection of Bifidobacterium and Lactobacillus strains were analysed for anti-inflammatory activity. Two strains, BI98 and BI504 with high suppressive ratio were selected. Such strains are likely to possess antiinflammatory activity in vivo and are tested for their ability to reduce level of inflammation in relevant mouse models. An anti-inflammatory effect in mouse models will indicate a likely clinical effect in humans.

BI504 is a human fecal isolate. It was isolated from a healthy 38 years old male person in the year 2002. It belongs to the genus Bifidobacterium and based on 16S rDNA sequencing it has been shown to belong to the B. bifidum species (see figure 13). Pulsed field gel electrophoresis (PFGE) has been conducted to verify that it constitutes a unique strain (see figure 11 B). The strain exhibits good adhesion properties as judged by determining the number of cells capable of adhering to a confluent layer of differentiated Caco-2 cells. BI504 shows a reproducible and potent suppression of the inflammatory response in the in vitro dendritic cell model with an average suppressive ratio of 7.3±3.0 (see figure 10 B). BI504 has been deposited at DSMZ under the accession number DSM 19158.

BI98 is a human fecal isolate. It was isolated from a healthy 30 years old male person in the year 2001. It belongs to the genus Bifidobacterium and based on 16S rDNA sequencing it has been shown to belong to the B. bifidum species (see figure 12). PFGE have been conducted to verify that it constitutes a unique strain (see figure 11 B). The strain exhibits excellent adhesion properties as judged by determining the number of cells capable of adhering to a confluent layer of differentiated Caco-2 cells. BI98 shows a reproducible and potent suppression of the inflammatory response in the in vitro dendritic cell model with an average suppressive ratio of 5.3±2.8 (see figure 10 A). BI98 has been deposited at DSMZ under the accession number DSM 19157.

In another aspect the present invention provides at least one Bifidobacterium bifidum selected from strains having a suppressive ratio indicative of anti-inflammatory effect, such as BI98, BI504, a homolog, descendant or mutant thereof.

In one embodiment the present invention relates to the use of at least one Bifidobacterium bifidum selected from strains having a suppressive ratio indicative of anti-inflammatory effect, such as BI98, BI504, a homolog, descendant or mutant thereof, as a probiotic.

Probiotics, as defined by the Food and Agricultural Organization of the United Nations (FAO), are "live microorganisms administered in adequate amounts which confer a beneficial health effect on the host." The microorganisms referred to in this definition are most often associated with dietary supplements and constitutes potentially beneficial bacteria or yeast, with lactic acid bacteria (LAB) as the most common microbes used. LAB have been used in the food industry for many years, because they are able to convert sugars (including lactose) and other carbohydrates into lactic acid. LAB are generally regarded as safe organisms, i.e. they have GRAS (Generally Recognized As Safe) status with most national regulatory agencies, and have historically provided the characteristic sour taste of fermented dairy foods such as yoghurt. They are used in dairy products as a preservative, since their metabolism lowers the pH and create fewer opportunities for contaminating organisms to grow.

Probiotic bacterial cultures are intended to assist the body's naturally occurring gut flora to reestablish itself. They are sometimes recommended by doctors and, more frequently, by nutritionists, after treatment with antibiotics. Probiotics may have antimicrobial, immunomodulatory, anticarcinogenic, antidiarrheal, antiallergenic and antioxidant activities. Claims have been made that probiotics can strengthen the immune system. Bifidobacteria are LAB belonging to the genus Bifidobacterium which are Gram-positive, non-motile, often branched anaerobic bacteria. Bifidobacteria are one of the major genera of bacteria that make up the gut flora, the bacteria that reside in the colon. Bifidobacteria aid in digestion, and have been reported to be associated with a lower incidence of allergies and also prevention of some forms of tumor growth. Some bifidobacteria are being used as probiotics.

The gastrointestinal tract represents a complex ecosystem in which a delicate balance exists between the intestinal microflora and the host. The microflora is principally comprised of facultative anaerobes and obligate anaerobes. Approximately 95% of the intestinal bacterial population in humans is comprised of obligate anaerobes, including Bifidobacterium, Clostridium, Eubacterium, Fusobacterium, Peptococcus, Peptostreptococcus and Bacteroides. Approximately 1% to 10% of the intestinal population is comprised of facultative anaerobes, including Lactobacillus, Escherichia coli, Klebsiella, Streptococcus, Staphylococcus and Bacillus. Aerobic organisms are not present in the intestinal tract of healthy individuals with the exception of Pseudomonas, which is present in very small amounts. Most of the bacteria are present in the colon where the bacterial concentration ranges between 10¹¹ to 10¹² colony-forming units (CPU) per milliliter.

The intestinal microflora is important for maturation of the immune system, the development of normal intestinal morphology and in order to maintain a chronic and immunologically balanced inflammatory response. The microflora reinforce the barrier function of the intestinal mucosa, helping in the prevention of the attachment of pathogenic microorganisms and the entry of allergens. Some members of the microflora may contribute to the body's requirements for certain vitamins, including biotin, pantothenic acid and vitamin B₁₂. Alteration of the microbial flora of the intestine, such as may occur with antibiotic use, disease and aging, can negatively affect its beneficial role.

The probiotics that are marketed as nutritional supplements and in functional foods, such as yogurts, are principally the Bifidobacterium species and the Lactobacillus species. Probiotics are sometimes called colonic foods. Most of the presently available probiotics are bacteria. Saccharomyces boulardii is an example of a probiotic yeast.

The following describe the various bacteria and yeasts which are used as probiotics. These bacteria and yeasts are examples of test components which antiinflamatory effect can be determined suing the present invention.

Bifidobacteria are normal inhabitants of the human and animal colon. Newborns, especially those that are breast-fed, are colonized with bifidobacteria within days after birth. The population of these bacteria in the colon appears to be relatively stable until advanced age when it appears to decline. The bifidobacteria population is influenced by a number of factors, including diet, antibiotics and stress. Bifidobacteria are gram-positive anaerobes. They are non-motile, non-spore forming and catalase-negative. They have various shapes, including short, curved rods, club-shaped rods and bifurcated Y-shaped rods. Their name is derived from the observation that they often exist in a Y-shaped or bifid form. The guanine and cytosine content of their DNA is between 54 mol% and 67mol%. They are saccharolytic organisms that produce acetic and lactic acids without generation of CO₂, except during degradation of gluconate. They are also classified as lactic acid bacteria (LAB). Bifidobacteria used as probiotics include Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium thermophilum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium lactis.

Lactobacilli are normal inhabitants of the human intestine and vagina. Lactobacilli are gram- positive facultative anaerobes. They are non-spore forming and non-flagellated rod or coccobacilli. The guanine and cytosine content of their DNA is between 32 mol% and 51 mol%. They are either aerotolerant or anaerobic and strictly fermentative. In the homofermentative case, glucose is fermented predominantly to lactic acid. Lactobacilli are also classified as lactic acid bacteria (LAB). Lactobacilli used as probiotics include Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus GG (Lactobacillus rhamnosus or Lactobacillus casei subspecies rhamnosus), Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum and Lactobacillus salivarus. Lactobacillus plantarum 299v strain originates from sour dough. Lactobacillus plantarum itself is of human origin.

Lactococci are gram-positive facultative anaerobes. They are also classified as lactic acid bacteria (LAB). Lactococcus lactis (formerly known as Streptococcus lactis) is found in dairy products and is commonly responsible for the souring of milk. Lactococci that are used or are being developed as probiotics include Lactococcus lactis, Lactococcus lactis subspecies cremoris (Streptococcus cremoris), Lactococcus lactis subspecies lactis NCDO 712, Lactococcus lactis subspecies lactis NIAI 527, Lactococcus lactis subspecies lactis NIAI 1061, Lactococcus lactis subspecies lactis biovar diacetylactis NIAI 8 W and Lactococcus lactis subspecies lactis biovar diacetylactis ATCC 13675.

Saccharomyces belongs to the yeast family. The principal probiotic yeast is Saccharomyces boulardii. Saccharomyces boulardii is also known as Saccharomyces cerevisiae Hansen CBS 5296 and S. boulardii. S. boulardii is normally a nonpathogenic yeast. S. boulardii has been used to treat diarrhea associated with antibiotic use. Streptococcus thermophilus is a gram-positive facultative anaerobe. It is a cytochrome-, oxidase- and catalase-negative organism that is nonmotile, non-spore forming and homofermentative. Streptococcus thermophilus is an alpha-hemolytic species of the viridans group. It is also classified as a lactic acid bacteria (LAB). Steptococcus thermophilus is found in milk and milk products. It is a probiotic and used in the production of yogurt.

Enterococci are gram-positive, facultative anaerobic cocci of the Streptococcaceae family. They are spherical to ovoid and occur in pairs or short chains. Enterococci are catalase- negative, non-spore forming and usually nonmotile. Enterococci are part of the intestinal microflora of humans and animals. Enterococcus faecium SF68 is a probiotic strain that has been used in the management of diarrheal illnesses.

In another aspect the present invention provides a composition comprising at least one Bifidobacterium bifidum according to the present invention.

Compositions of the invention may be prepared by admixture, suitably at ambient temperature and atmospheric pressure, usually adapted for oral administration. Such compositions may be in the form of tablets, capsules, oral liquid preparations, conventional food products, powders, granules, lozenges, reconstitutable powders or suspensions. In one embodiment the composition comprising at least one Bifidobacterium bifidum according to the present invention is an edible composition such as yogurt, cheese, nutritional supplement, tablet etc.

In another embodiment the composition comprising Bifidobacterium additionally comprises one or more acceptable excipients. It will be appreciated that an acceptable excipient will be well known to the person skilled in the art of probiotic composition preparation. Examples of such acceptable excipients include: sugars such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose and xylose; sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, palatinol, reduced glutinous starch syrup and reduced glutinous maltose syrup; emulsifiers such as sucrose esters of fatty acid, glycerin esters of fatty acid and lecithin; thickeners (stabilizers) such as carrageenan, xanthan gum, guar gum, pectin and locust bean gum; acidifiers such as citric acid, lactic acid and malic acid; fruit juices such as lemon juice, orange juice and berry juice; vitamins such as vitamin A, vitamin B, vitamin C, vitamin D and vitamin E; and minerals such as calcium, iron, manganese and zinc.

In another embodiment the composition comprising a Bifidobacterium according to the present invention additionally comprises one or more socalled bifidogenic factors, e.g. a compound stimulating the growth of Bifidobacteria and which are resistant to digestion in the upper gastrointestinal tract. In one embodiment the bifidogenic factor is selected from the group consisting of fructo-oligosaccharides (e.g. short-chain oligosaccharides comprised of D-fructose and D-glucose, containing from three to five monosaccharide units), inulins (e.g having an average degreee of polymerization of 10 to 12), isomalto-oligosaccharides (such as isomaltose, panose, isomaltotetraose, isomaltopentaose, nigerose, kojibiose, isopanose and higher branched oligo-saccharides), lactilol (4-0-(beta-D-galactopyranosyl)- D-glucitol being a disaccharide analogue of lactulose), lactosucrose, lactulose, pyrodextrins, soy oligosaccharides, transgalacto-oligosaccharides (TOS) and xylo-oligosaccharides.

Tablets and capsules for oral administration may be in unit dose form, and may contain one or more conventional excipients, such as binding agents, fillers, tabletting lubricants, disintegrants, and acceptable wetting agents. The tablets may be coated according to methods well known in pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be in the form of a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), preservatives, and if desired, conventional flavourings or colourants.

In one embodiment, the composition of the invention is formulated as a conventional food product, more preferably, a dairy based product (e.g. fermented milk, vegetable milk, soybean milk, butter, cheese or yoghurt) or fruit juice. The composition may be formulated as a food or drink for adult and infant humans and animals. In an alternative embodiment, the composition is formulated as a lyophilised or spray-dried powder.

In an embodiment, bifidobacteria according to the present invention is combined with other bifidobacteria or other probiotic bacteria such as: bacteria belonging to the genus Lactobacillus such as Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus gallinarum, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus kefir, Lactobacillus paracasei, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. bulgaricul, Lactobacillus helveticus, Lactobacillus zeae and Lactobacillus salivalius; bacteria belonging to the genus Streptococcus such as Streptococcus thermophilus; bacteria belonging to genus Lactococcus such as Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis; bacteria belonging to the genus Bacillus such as Bacillus subtilis; and yeast belonging to the genus Saccharomyces, Torulaspora and Candida such as Saccharomyces cerevisiae, Torulaspora delbrueckii and Candida kefyr. In another embodiment the edible composition comprises Bifidobacteria in a concentration to provide a dose in the range from about 10⁵ to about 10¹⁴ colony forming units (CFU), such as from about 10⁸ to about 10¹² CFU, or about 10¹⁰ to 10¹² CFU.

In another embodiment the composition is for external application on humans such as a creme, e.g. a skin creme or a vaginal creme. In another embodiment of the invention the composition is a pharmaceutical composition.

In one embodiment said at least one Bifidobacterium bifidum is used to prevent or alleviate an inflammatory condition. In another embodiment said inflammatory condition is at least in part due to an inflammatory bowel disease. In antoher embodiment said inflammatory bowel disease is Crohn 's disease or ulcerative colitis. In another embodiment said inflammatory condition is at least in part due to rheumatoid arthritis, psoriasis, systemic lupus erythromatosis, eczema or allergy.

The invention further provides a method of treatment and/or prophylaxis of the above disorders, in a human or animal subject, which comprises administering to the subject a therapeutically effective amount of at least one Bifidobacterium bifidum selected from strains having a suppressive ratio indicative of anti-inflammatory effect, such as BI98, BI504 or a homolog, descendant or mutant thereof.

Bifidobacterium bifidum selected from strains having a suppressive ratio indicative of anti- inflammatory effect, such as BI98, BI504 or strains derived therefrom, may be used in combination with other therapeutic agents, for example, other medicaments known to be useful in the treatment and/or prophylaxis of gastrointestinal diseases, cancer, cholesterol excesses, allergies and infection.

Thus, as a further aspect of the invention, there is provided a combination comprising Bifidobacterium bifidum selected from strains having a suppressive ratio indicative of an anti-inflammatory effect, such as BI98, BI504, a homolog, descendant or mutant thereof, together with a further therapeutic agent or agents.

The combinations referred to above may conveniently be presented for use in the form of a probiotic composition and thus probiotic compositions comprising a combination as defined above together with one or more excipients comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined probiotic compositions.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having," "including," or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subject matter recited in the claims and/or aspects appended hereto.

The present invention is further illustrated by the following examples which, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately or in any combination thereof, be material for realising the invention in diverse forms thereof.

EXAMPLES

Example 1.

How to select a pro-inflammatory cocktail to stimulate dendritic cells into an inflammatory phenotype, and the subsequent validation of the inflammatory dendritic cell functional phenotype.

The present screening model is based on the initial optimization of a pro-inflammatory cocktail that is capable of maturing immature dendritic cells into an inflammatory phenotype, characterized by secretion of high levels of IL12 and/or other inflammatory related cytokines and chemokines like IL6, IL12, RANTES (CCL5), TNFα, MCPl (CCL2), MlPlα (CCL3) and KC-chemokine (CXCLl) and a low level of ILlO. Based on the ability to screen anti-inflammatory compounds and microorganisms as depicted on Figure 1 and 2, a strong inflammatory response must be identified. A matrix combining multiple dendritic cell activators, receptor ligands or other components defined as the pro-inflammatory cocktail, must be tested in order to select the most potent cocktail combination (see figure 3, 17 and 20 as examples). Once a combination of the above mentioned compounds with high expression of IL12 and TNFα and low expression of ILlO is determined, the ability of the selected cocktail to induce a maturation phenotype can be analyzed. Such analyses comprise typically expression of maturation markers, exemplified on figure 4, 24, 25 and 29, with cocktail induced elevation of expression of CD40, CD80, CD83, CD86 and MHC-II. The cocktail initially identified might be optimized in relation to concentration relevant for the induction of the dendritic cell phenotype (see figure 5 A), where a dose-dependent IL12 stimulation is seen. The concentration of each cocktail component, in this case CpG and IFNγ for mouse DCs and cocktails shown in Fig. 17C for human DCs, can be varied in order to optimize for the most potent ratio and concentration of cocktail components. This is exemplified in figure 5 B, showing that the concentrations at 20 ng/ml IFNγ and 1 μM CpG are not crucial for a strong IL12 response, but also that no other combination of the two is capable of inducing a stronger response. To ensure that the selected cocktail induces an inflammatory response by analysis in other immune related aspects, a cytokine array was used for identification of other cytokines and chemokines induced by the selected cocktail. Figure 6 A, shows that this present cocktail (20 ng/ml IFNγ and 1 uM CpG), also causes induction and secretion of IL6, RANTES (CCL5), TNFα, MCPl (CCL2), MlPlα (CCL3) and KC- chemokine (CXCLl), all inflammatory related proteins involved in recruiting of inflammatory cells and sustainment of an inflammatory condition. Thus, each of these aforementioned cytokines and chemokines could serve as detection markers in the present model system for assessment of anti-inflammatory compounds. Quantitative analysis of some key inflammation markers are shown by ELISA analyses of dendritic cells treated with this present pro-inflammatory cocktail (figure 6B). Both IL6, IL12 (p40/p70) and TNFα are shown to be dramatically induced by the cocktail. The functional phenotype of the proinflammatory cocktail was determined by assaying the response of purified allogeneic CD4+T-cells from mouse spleen or in a mixed leukocyte reaction (MLR). In figure 6 C it is seen that cocktail treated dendritic cells incubated with cocktail for 16 h, washed and then exposed to purified CD4+T-cells, were able to induce secretion of IFNγ after a 4 day incubation period. Non-treated dendritic cells (column 1), and non-APC activated CD4+ T- cells (column 3) were not able to induce secretion of IFNγ. A similar result was seen when total mouse spleen cells were applied to dendritic cells treated the same way, although the MLR response showed some IFNγ secretion when added to the immature dendritic cells

(column 4). Cocktails can further be selected based on their ability to induce NO-production as an example of a secreted inflammatory marker which is not a cytokine or chemokine (figure 14), or based on changes in intracellular markers of inflammation like iNOS or COX- 2, exemplified in figure 15 and 16. Finally, cocktails can be selected based on their ability to induce a DC-phenotype that can stimulate T-cell proliferation in a MLC reaction (figure 26 and 30). Altogether, this example describes the selection of pro-inflammatory cocktails, and the subsequent characterization to assure that the pro-inflammatory cocktail treated dendritic cells have matured into an inflammatory phenotype.

Example 2:

Validation of the suitability of a pro-inflammatory dendritic cell based screening model for selection of anti-inflammatory candidates using known antiinflammatory drugs and/or compounds

A screening model for identification of anti-inflammatory compounds and probiotics, must be able to show that currently known anti-inflammatory compounds can be identified using this model. We therefore examined the ability of dexamethasone and prostaglandin D2 to suppress the pro-inflammatory cocktail induced secretion of IL12. Dex and prostaglandin D2 were added to the immature murine dendritic cells in 10-2000 nM (dexamethasone) and 1- 50 μM (prostaglandin D2) (see figure 7 A and B respectively). After 4 hours, the proinflammatory cocktail was added for another 18 hours, and the conditioned growth media analysed for levels of IL12. A dose-dependent suppression of IL12 secretion was seen with both dex and prostaglandin D2, in both cases without significant cell death (not shown). The demonstration that other inflammatory cytokines can be useful as read-out parameters in this anti-inflammatory screening model is shown by the ability of prostaglandin D2 to suppress the secretion of IL6 and TNFα also in a dose dependent manner, as seen in figure 8 A and B. Validation of the human DC based screening model is seen using several known anti-inflammatory reagents. Figure 16C shows that dex is able to suppress the ability of LPS, cocktail F2 and 7 to induce COX-2 expression as an intracellular target of inflammation in human DCs. COX-2 is involved in prostaglandin synthesis, and addition of COX-inhibitors to DCs prior to addition of cocktails show the ability of the non-specific COX-inhibitor indomethacin and the specific COX-2 inhibitor NS398 to suppress cocktail F and 10 (see fig. 21) induced protstaglandin E secretion in two different donors. Both cocktails were able to induce high levels of PGE2, shown in a screening assay for identification of the most potent cocktail (fig. 20). Dex was also able to suppress cocktail induced IL12 and TNFα secretion in human DCs (fig. 23). Dex was added 6 or 24 h prior to addition of cocktail 5, 6, 8, 9 and 10, and conditioned media analysed for IL12 and TNFα. For almost all cocktails, there was a clear dose-dependent anti-inflammatory effect of dex, seen by suppression of IL12 and TNFα secretion, most pronounced after 24 h preincubation. These data show that known and clinically used anti-inflammatory drugs, do suppress the secretion of pro-inflammatory markers in both the murine and human DC based screening models, using these novel cocktails. Dex was also able to suppress cocktail induced membrane bound maturation markers on DCs. Figure 24 A and B shows that all cocktails except cocktail 8 are able to induce expression of maturation markers HLA-DR, CD40, 80, 83 and 86 on human DCs after 24 h incubation, measured by FACS analyses, compared to immature DCs. Dex was able to suppress the expression of these different membrane bound markes of inflammation to different levels, most pronounced was the inhibitory effect for cocktail 10, where e.g. CD86 was reduced to more than 50 % of cells treated with cocktail 10 alone.

Validation of the model using T-cell proliferation as end-point as stated in claim 4, was shown by the ability of the known anti-inflammatory compounds dex and lα,25 dihydroxy vitamin D3, to inhibit the cocktail mediated proliferation (vitamin D3) (figure 26 and 30B). Dex and vitamin D3 treatment of human DCs prior to addition of cocktail 6 and 10 (fig. 26), or addition to murine DCs (fig. 30B) suppressed the ability of the DCs to induce T-cell proliferation, compared to DCs treated with the cocktails alone. Dex alone, vitamin D3 alone, or both compounds combined, were able to suppress the cocktail treated DCs ability to stimulate T-cell proliferation (figure 26 and 30B). Using an intracellular end-point in T- cells, FoxP3, it was shown that dex was able to induce the level of FoxP3, known to be involved in induction of a tolerogenic phenotype, which is believed to be beneficial in an autoinmmune or inflammatory disease (figure 26C). Using an end-point for the present screening model related to T-cell secreted substances, T-cell secreted cytokines like IL5 and IFNγ were determined in conditioned media from MLR-assays using purified CD4+ T-cells (figure 27). IL5 and IFNγ secretion from the T cells were suppressed when the DCs used for the MLR was pretreated with dex and/or vitamin D3 prior to addition of cocktail 6. When cocktail 10 was used, IFNγ was markedly reduced using dex or vitamin D3 either alone or in combinations. Example 3:

Correlation of the dendritic cell based screening model with the effect of an antiinflammatory probiotic strain in an animal model of inflammation

A hall-mark of in vitro screening models is their predictive value for the effect of antiinflammatory compounds in animal models and eventually their predictive value in human clinical trials. One great obstacle for selecting anti-inflammatory compounds using in vitro screening models is exactly the lack of predictability to animal and human studies. Hence, we have correlated the anti-inflammatory activity data from our current screening model to the effect of probiotic strains on the inflammation score as seen in the TNBS induced mouse model of colitis, a model of human Crohn 's disease. Three Lactobacillus strains 1) Lactobacillus salivarius Ls 33, 2) Lactobacillus plantarum Lpll5 and 3) Lactobacillus acidophilus NCFM were tested in a preventive model of TNBS induced colitis. The strains were administered in a concentration of 10⁹ CFU to the mice orally five days prior to rectal administration of TNBS, and the colon of the mice scored for sign of inflammation two days later. Based on these inflammation scores, each strain was rated for its ability to reduce the inflammatory scores as seen in figure 9. Lactobacillus salivarius Ls 33 showed a significant and strong ability to reduce signs of inflammation. Our current screening model provided this strain with a suppressive ratio of 1.62, when the timing between addition of probiotic strain and the pro-inflammatory cocktail was fixed to 2 hours. A suppressive ratio of 1,62 means that this strain was able to suppress the pro-inflammatory cocktail induced secretion of IL12 to 60 % of the secretion seen with cocktail treated dendritic cells alone. The Lactobacillus plantarum Lpll5 strain showed only weak ability to suppress the inflammation in mice, and scored lower suppressive ratio in our screening model, with largely no change of IL12 response compared to cocktail only treated dendritic cells. Finally, Lactobacillus acidophilus NCFM was not able to suppress the TNBS induced colitis in mice and, in fact, caused an even more severe colitis in mice. Our in vitro screening model interestingly showed that this strain induced increased levels of IL12 compared to dendritic cells treated solely with the pro-inflammatory cocktail, and therefore reached a very low suppressive ratio of 0.83 (see figure 9). The timing between addition of test component and the proinflammatory cocktail is important for the relative level of the suppressive ratio. In the present example, this interval was fixed to 2 hours, however, if this time interval is increased, the IL12 secretion is further reduced, thereby increasing the suppressive ratio. This information is important in designing a specific model. The most useful time intervals have in our hands been identified between 2-4 hours. Example 4:

Selection and determination of potency of the two anti-inflammatory candidates BI98 and BI504.

In order to use the present screening model for selection of potent probiotic strains with anti-inflammatory activity, potentially useful for alleviation of inflammatory conditions in humans suffering from gastrointestinal inflammatory conditions, arthritis or other autoimmune diseases, we screened a large panel of probiotic strains for their ability to suppress our pro-inflammatory cocktail induced IL12 secretion. A large proportion of the strains showed no suppression, corresponding to suppressive ratios below 1. One example is shown in figure 10 C, represented with the response seen when the Lactobacillus acidophilus LX37 is added to the dendritic cells at concentrations of 10 and 100 μg/ml two hours prior to addition of the pro-inflammatory cocktail. The strain showed an average suppressive ratio of 0.74±0,27, correlating with a significant immune stimulatory activity. In contrast, two bifidobacteria, BI98 and BI504 were able to suppress the pro-inflammatory cocktail to a large extent, with average suppressive ratios of 5.3±2.8 and 7.3±3.0 respectively (figure 10 A and B).

Generally, strains of Lactobacillus showed a window of suppressive ratios from 0,5 to approximately 5, with an average suppressive ratio of 35 different strains at 1,2, when the time length between addition of probiotic strain and cocktail was fixed to 2 hours. Strains of Bifidobacterium showed a window of suppressive ratios from 0,7 to approximately 10, with an average suppressive ratio of 25 different strains at 2,6, when the time length between addition of probiotic strain and cocktail was fixed to 2 hours. Among a group of 25 different Bifidobacterium strains, BI98 and BI504 showed highest suppressive ratios, in contrast to non-suppressing strains BI435 and BI46 (Table, figure 10, D). BI98 and BI504 which represent potent suppressive strains in vitro with a 2 hours time interval and BI46 which represents a non-suppressive strain in vitro are compared in an animal model of inflammation to determine the level of suppression necessary to classify a strain of Bifidobacterium as an anti-inflammatory in vivo. Hence, the BI98 and BI504 strains are very potent and interesting candidates for testing in the mouse model of TNBS induced colitis as described in Example 3, and potentially later in human clinical trials for their antiinflammatory effect. The BI98 strain was further analyzed for its suppressive activity in a dose-dependent manner in murine DCs from 1 to 1000 μg/ml (see figure 11 A). This concentration range corresponds approximately to a probiotic celhdendritic cell ratio at: 1 μg/ml corresponds to a ratio at 0.03-0.05, 10 μg/ml corresponds to a ratio at 0.3-0.5, 100 μg/ml corresponds to a ratio at 3-5, 1000 μg/ml corresponds to a ratio at 30-50. The BI98 and BI504 were both isolated from human feces from two different individuals, and to exclude that they were identical the two strains were fingerprinted using pulsed field gel electrophoresis (PFGE) of Spel restriction enzyme digests of genomic DNA. Figure 11 B shows that the strains have different PFGE profiles and therefore are different. Hence, BI98 and BI504 represents two very potent anti-inflammatory probiotic strains.. BI98 and BI504 were further tested for their ability to suppress cocktail induced IL12 secretion in the human DC model (figure 22). The probiotic strains were added to immature human DCs derived from human donor blood in a dose-dependent manner, in doses from 1-200 ug/ml. Both strains were able to suppress cocktail 6 and 10 induced IL12 secretion more than 10 fold, with IC50 concentrations below 1,0 ug/ml for cocktail 10 and below 10 ug/ml for cocktail 6 (figure 22).

Example 5:

Determination of suppressive ratio in relation to timing between addition of test component and pro-inflammatory cocktail

The timing between addition of test components like probiotic strains, and the pro- inflammtory cocktail affects the absolute value of the suppressive ratio. This interval must be the same for comparison of suppressive ratios in order to assess the anti-inflammatory potential of a test component including a probiotic strain. When testing microorganisms like probiotic strains, the immature dendritic cells become mature by the probiotic treatment alone, mainly in a non-inflammatory maturation process. The implication is that less effect of the addition of the pro-inflammatory cocktail is seen if the cocktail is added a long time after a probiotic strain is added. An example of this phenomena is seen in Fig 11 C and D. The strongly suppressing strain BI98 blocked efficiently the response from the proinflammatory cocktail, when added 4 hours or more before the cocktail. The lactobacillus acidophilus LX37 was not able to suppress the pro-inflammatory cocktail added 2 hours after LX37 while the addition of the pro-inflammatory cocktail after 4 or 6 hours reduced the IL12 secretion to levels close to control value (pro-inflammtory cocktail alone). The timeing between addition of the anti-inflammatory component and the cocktail was also seen to be important for the human model, where dex worked more efficiently in suppressing IL12 and TNFα when added 24 h prior to addition of cocktail compared to 6 h before (figure 23). In designing the specific screening model, the timing between addition of the test component and the pro-inflammatory cocktail is important as is the timing of the assay for cytokine response (IL12, ILlO) relative to addition of test component and pro-inflammatory cocktail. Example 6:

Identification of nitric oxide secreted from pro-inflammatory DCs and suppression of NO secretion by anti-inflammatory probiotics

Immature murine dendritic cells were exposed to the two anti-inflammatory Bifidobacterium strains, BI98 and BI504, and after 4 h a pro-inflammatory cocktail was added to the DCs. In parallel, cocktail, LPS and the two strains BI98 and BI504 were added to immature DCs. After 24 h incubation the conditioned DC-media was collected and the NO-content was determined using Griess reagent as described (Promega, Madison, WI). The pro- inflammatory cocktail, LPS and IFNγ + LPS all induced NO-secretion to the media, as a marker of pro-inflammatory activity according to claim 2. The two Bifidobacterium strains, BI98 and BI504, induced minimal NO production by themselves, whereas a Lactobacillus strain acidophilus X37, which induce high levels of IL12p70 stimulated NO secretion much more strongly, and was more potent than cocktail and LPS by themselves. When the two Bifidobacterium strains, BI98 and BI504 were added prior to addition of cocktail (fig. 14, last two columns) they were able to suppress the cocktail induced NO-secretion at different levels.

Example 7:

Identification of intracellular targets cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) induced in pro-inflammatory DCs and potential suppression of these targets by anti-inflammatory components

Inducible nitric oxide synthase (iNOS or NOS2) is the intracellular marker for an inflammatory cell that produce and secrete the inflammatory mediator nitric oxide, as measured in DC media in fig. 14. iNOS was analysed using Western blot of DC protein lysates in fig. 15. Immature murine (A) and human (B) DCs were treated with the indicated reagents, either untreated (lane 1), or different cocktails, LPS or probiotic strains with different ability to influence DC maturation and cytokine secretion. In untreated cells iNOS was undetectable, but in cocktail and LPS treated DCs iNOS was strongly induced as sign of treatment with a pro-inflammatory reagent or cocktail. The 9 probiotics showed various iNOS induction, and remarkably weak induction was seen with Bifidobacterium strains, BI98 and BI504 (fig. 15A, lane 8 and 9). The same phenomena was seen with these probiotics when human DCs were treated (fig. 15B, lane 8 and 9). Several cocktails optimized for this model also increased iNOS expression, including cocktail F, F2, 6 and 7. Cocktail 10 did not induce iNOS to the same extent. In the same protein lysates, COX-2 was analysed as an intracellular marker of inflammation and end-point according to claim 3, also using Western blot of DC protein lysates, as shown in fig. 16. In murine DCs (A), COX-2 was present a low levels in untreated DCs, but induced in cocktail and LPS treated DCs. COX-2 was strongly induced as sign of treatment with a pro-inflammatory reagent or cocktail. All 9 probiotic strains induced COX-2 at similar levels. In human DCs, COX-2 was induced by both LPS, and cocktail F, F2, 6, 7 and 10 (fig. 16B). As validation of the present screening model for identification of anti-inflammatory reagents using intracellular markers of inflammation as endpoints according to claim 3, dex was added prior to addition of selected cocktails (fig. 16C). Dex was able to reduce expression of COX-2 stimulated by LPS and cocktail F2 and 7, compared to cells which were not treated with dex (compare lane 2 vs 3, 4 vs 5, and 8 vs 9, in fig. 16C).

Example 8:

A functional endpoint to assess DC phenotype and function by T cell proliferation in a mixed lymphocyte culture (MLC).

The mixed lymphocyte culture (MLC) reaction is used to assess the in vitro lymphocyte recognition and proliferation in response to DCs treated with cocktails or anti-inflammatory compounds. DCs and T cells from two individuals were mixed together in tissue culture for several days. DCs from incompatible individuals will stimulate T cells to proliferate significantly, whereas DCs from compatible individuals will not. In a one-way primary MLC reaction, the DCs from one individual are inactivated by treatment with mitomycin c, thereby allowing only the untreated remaining population of T cells to proliferate in response to the treated DCs. T cell proliferation is measured by incorporation of radioactive thymidine (in the DNA of proliferating T-cells) in T-cells present in the MLC after contact with the treated DCs. In figure 26 A and B, human DCs were treated with either cocktail 6 or 10, dex alone, vitamin D3 alone, dex and vitamin D3 in combination, or all three test reagents 6 or 24 h prior to addition of cocktails. DCs were mixed with CD4-purified T-cells in different ratios from 1 : 10 to 1 :640, since one DC can activate multiple T-cells. In figure 26A, cocktail 10 induced a stronger T-cell response than immature DCs, in donor 1, which was seen for all DC:T-cell ratios, and in donor 2 from a ratio of 1 :40. When the DCs were pretreated with dex, the ability of the DCs to stimulate T-cell proliferation was reduced, most significantly at ratios from 1 :80. DCs pretreated with dex alone were very poor T-cell stimulators, whereas immature DCs showed some stimulating ability (fig. 26A). Pretreatment for 24 h with dex and/or vitamin D3 could reduce the ability to stimulate a T-cell response by both cocktail 6 and 10 at all DC:T-cell ratios tested (fig. 26B). This shows, that known reagents and drugs with anti-inflammatory properties can be identified also using T-cell proliferation as an end- point according to claim 4.

Example 9:

A functional endpoint to assess the ability of an anti-inflammatory component to induce tolerance using allogeneic dendritic cell and MLCs

In order to assess the development of tolerogenic DCs by treatment with test components, a secondary MLC is performed. DCs derived from the same donor as in the primary MLC, will instead of the test compound be stimulated/activated maximally and used as stimulator cells in a secondary MLC. The responding T cells in the secondary MLC will be the once from the primary MLC reaction. If the test compound treated DCs induce T cell hyporesponsiveness in the primary MLC and DCs from the same donor after full activation still induce T cell hyporesponsiveness in the secondary MLC we will truly know that the test compound mediates tolerogenic DCs. As control in murine studies a third part of incompatible fully activated DCs will be used to stimulate an alloreaction in the same T cell population.

Example 10:

Identification of intracellular markers in T-cells contacted with treated DCs

In order to assess the development of tolerogenic DCs by treatment with test components, thereby counterbalancing the proinflammatory stimuli, the intracellular T-cell endpoint FoxP3 was analysed in MLCs where the DCs were treated with the known tolerance inducing drug dex (fig. 26C). CD4+ T cells were added to the DCs after 24 h treatment, and after 4 days the content of intracellular FoxP3 as an intracellular T-cell end-point was determined. In both donors, the dex treated DCs stimulated FoxP3 intracellular^ in the T-cells with approximately 50 % (10,7 to 15,3 % and 5,0 to 7,5%) (fig 26C). Dex was also able to induce FoxP3 content in T-cells when DCs were pretreated with dex for 24 h and later with cocktail 10. In this case dex increased FoxP3 content from 32,3 to 35,9 % and 15,3 to 18,6 % in the two donors. This indicates, that dex by its ability to suppress the DC maturation into a proinflammatory phenotype, can programme the DCs to increase the amount of T- cells with a tolerogenic phenotype, believed to counterbalance a pro-inflammatory condition. This example thus relates to claim 4 of the present screening model, where the intracellular marker FoxP3 changes its expression level by an increase in protein content in T-cells, which correlates with induction of a tolerogenic or potentially anti-inflammatory response.

Example 11:

Identification of linage specific cytokines in the MLC reaction

The functional phenotype of T cell responses is classified according to their cytokine secreting capability. In this setting the MLC reaction is used to address the type of T cell linage, ThI, Th2, Thl7 or Treg, induced in the MLC using DCs treated with a proinflammatory cocktail or a test component for assessment of anti-inflammatory activity. Human (Fig 27) and murine (Fig. 31) DCs were used in a MLC as stimulator cells and allogenic naive T cells as responders. After 5 days of culture the cytokine profile in the supernatant was analysed by ELISA for IFNγ (Thl-response), IL4 and IL5 (Th2-response). Cocktail 6 treated DCs induced IL5 strongly in T-cells being contacted with the DCs, whereas IFNγwas slightly induced. Both IL5 and IFNγ secretion were reduced by pre-treatment of the DCs with dex, vitamin D3 or a combination of these. The secretion of IFNγ was also reduced by treatment with dex, vitamin D3 or a combination of these when the DCs were subsequently treated with cocktail 10. In cocktail treated murine DCs, T-cells showed a reduction in IL4 secretion compared to immature DCs. Pretreatment of the DCs with dex reduced IL4 secretion to very low levels. This example thus describes the use of T-cell secreted extracellular end-points in the present screening model, according to claim 4.

Example 12:

Use of DC endpoints by detecting the activation state of intracellular markers

DCs treated with pro-inflammatory reagents initiate intracellular signalling cascades, involving activation of intracellular proteins by phosphorylation. DCs treated with TNFα or LPS for 10 and 30 min were analysed by Western blot using antibodies against Iκβa recognizing either the phosphorylated or activated form of the protein (fig.28 upper pane, P- Iκβa), or total amounts of Iκβa (fig. 28, lower panel, Iκβa). After 10 min, Iκβa was not activated by phosphorylation (upper panel), but after 30 min, there was a clear increase in phosphorylated (activated) Iκβa for both TNFα or LPS treated DCs, where the total amount of the protein was not changed (lower panel). Using the activation state by phosphorylation as an intracellular marker as end-point in DCs, the cells can be pretreated with a test reagent, which potentially can block the ability of the cells to activate Iκβa by phsophorylation. Thus, this example shows and describes the use of an intracellular marker of inflammation in DCs, whose activation state measured by phosphorylation can be used as an end-point for the present screening model.

Claims

1. Method for in-vitro determination of the in vivo anti-inflammatory effect of a test component such as a molecule, a mixture of molecules, a microorganism, a fraction of cells or combinations thereof, comprising the steps of a) contacting said test component, mammalian dendritic cells and a pro-inflammatory cocktail, b) quantifying the secretion of at least one pro-inflammatory cytokine and/or chemokine from said mammalian dendritic cells, c) comparing said secretion of at least one pro-inflammatory cytokine and/or chemokine with that of a reference test wherein said mammalian dendritic cells have not been contacted with said test component but have been contacted with said pro-inflammatory cocktail, d) to establish the extent to which said test component reduces secretion of said at least one pro-inflammatory cytokine and/or chemokine.

2. Method for in-vitro determination of the in vivo anti-inflammatory effect of a test component such as a molecule, a mixture of molecules, a microorganism, a fraction of cells or combinations thereof, comprising the steps of a) contacting said test component, mammalian dendritic cells and a pro-inflammatory cocktail, b) quantifying the secretion of at least one pro-inflammatory compound or marker which is not a cytokine and/or chemokine from said mammalian dendritic cells, c) comparing said secretion of at least one pro-inflammatory compound or marker which is not a cytokine and/or chemokine with that of a reference test wherein said mammalian dendritic cells have not been contacted with said test component but have been contacted with said pro-inflammatory cocktail, d) to establish the extent to which said test component reduces secretion of said at least one pro-inflammatory compound or marker which is not a cytokine and/or chemokine.

3. Method for in-vitro determination of the in vivo anti-inflammatory effect of a test component such as a molecule, a mixture of molecules, a microorganism, a fraction of cells or combinations thereof, comprising the steps of a) contacting said test component, mammalian dendritic cells and a pro-inflammatory cocktail, b) quantifying the expression level or activation state of at least one intracellular or membrane bound marker of inflammation from said mammalian dendritic cells, c) comparing said expression level or activation state of at least one intracellular or membrane bound marker of inflammation with that of a reference test wherein said mammalian dendritic cells have not been contacted with said test component but have been contacted with said pro-inflammatory cocktail, d) to establish the extent to which said test component reduces the expression level or activation state of said at least one intracellular marker or membrane bound marker of inflammation.

4. Method for in-vitro determination of the in vivo anti-inflammatory effect of a test component such as a molecule, a mixture of molecules, a microorganism, a fraction of cells or combinations thereof, comprising the steps of a) contacting said test component, mammalian dendritic cells and a pro-inflammatory cocktail, b) determining the functional phenotype of said dendritic cells by applying T-cell cultures and quantifying at least one intracellular or extracellular endpoint or the proliferation of the T-cells that have been contacted with said dendritic cells, c) comparing said at least one intracellular or extracellular endpoint or the proliferation in said T-cell cultures that have been contacted with said dendritic cells with that of a reference test wherein said mammalian dendritic cells have not been contacted with said test component but have been contacted with said pro-inflammatory cocktail, d) to establish the extent to which said test component changes the level of said at least one intracellular or extracellular endpoint or the proliferation in T-cell cultures as a marker of dendritic cell functional phenotype.

5. The method according to claim 1, wherein said at least one pro-inflammatory cytokine and/or chemokine is selected from the group consisting of IL12 (p40/p70), TNFα, IFNy, IFNα, IL6, RANTES, IPlO, MCPl/2, MIPIa and mixtures thereof.

6. The method according to any of claims 1-2, wherein said pro-inflammatory cocktail has been designed to increase secretion of at least one factor selected from IL12 (p40/p70), TNFα, IL6, RANTES, MCPl/2 and MIPIa, while concomitantly decreasing ILlO secretion.

7. The method according to any of claims 1-2, wherein said pro-inflammatory cocktail has been designed to increase secretion of at least one factor from the dendritic cells selected from IL12 (p40/p70), TNFα, IL6, RANTES, MCPl/2 and MIPIa, IL2, IL4, IL5, IL13, IL17, IL23, IL15, IL18, TGFβ, TARC, MDC, prostaglandins, leukotrienes, thromboxanes, nitric oxide (NO) and fatty acids.

8. The method according to claim 2, wherein said pro-inflammatory cocktail has been designed to increase the expression level of at least one extracellular endpoint as said at least one pro-inflammatory compound.

9. The method according to claim 2, wherein said at least one pro-inflammatory compound is selected from the group consisting of lipid derivatives such as eicosanoids, sphingolipids, prostaglandins, phospholipids, bioactive lipids, fatty acids (poly or monounsaturated and even saturated fatty acids), leukotrienes, oxylipins, thromboxanes, and inorganic molecules such as nitric oxide (NO).

10. The method according to any of claims 3-4, wherein said pro-inflammatory cocktail has been designed to increase the expression level of at least one intracellular, extracellular or membrane bound endpoint.

11. The method according to any of claims 3 or 5-6, wherein said pro-inflammatory cocktail has been designed to increase the expression level or activation state of at least one intracellular or membrane bound endpoint.

12. The method according to any of claims 4 or 10, wherein said pro-inflammatory cocktail has been designed to increase said intracellular or said extracellular endpoint or said proliferation of in T-cell cultures that have been contacted with said dendritic cells.

13. The method according to claim 10, wherein said pro-inflammatory cocktail comprises at least one compound selected from the group consisting of ILlβ, IL6, TNFα, IFNy, IFNα, RANTES, IPlO, PGE2, TSLP, IL4, IL5, ILlO, IL13, TGFβ, MCPl/2, MIPIa, Poly I:C, CpG-or

ODN-oligonucleotides, LPS, R848, peptidoglycan, LTA, Pam3Cys (and its derivatives), MALP- 2, paclitaxel, flagellin, lipomannan, FSL-I, gardiquimod, R837, loxoribine, profilin and zymosan.

14. The method according to claim 13, wherein said pro-inflammatory cocktail comprises at least one compound selected from the group consisting of ILlβ, IL6, IFNy, IFNα, RANTES,

IPlO, PGE2, TSLP, IL4, IL5, ILlO, IL13, TGFβ, MCPl/2, MIPIa, Poly I:C, R848, LTA, Pam3Cys (and its derivatives), MALP-2, paclitaxel, flagellin, lipomannan, FSL-I, gardiquimod, R837, loxoribine, profilin and zymosan.

15. The method according to claim 14, wherein said pro-inflammatory cocktail does not contain any of the compounds LPS, TNFα, peptidoglycan and CpG-oligonucleotides.

16. The method according to claim 14, which comprises at least two compounds selected from the group consisting of ILlβ, IL6, TNFα, IFNγ, IFNα, RANTES, IPlO, PGE2, TSLP, IL4, IL5, ILlO, IL13, TGFβ, MCPl/2, MIPIa, Poly I:C, CpG-or ODN-oligonucleotides, LPS, R848, peptidoglycan, LTA, Pam3Cys (and its derivatives), MALP-2, paclitaxel, flagellin, gardiquimod, R837, loxoribine, lipomannan, FSL-I, profilin and zymosan.

17. The method according to any of claims 3 or 5, wherein said pro-inflammatory cocktail has been designed to change said activation state of said at least one intracellular endpoint.

18. The method according to claim 6, wherein the ratio between secreted IL12 (p40/p70) and ILlO is at least 3 mg IL12 (p40/p70) per mg ILlO, such as at least 6 mg IL12 (p40/p70) per mg ILlO, or such as at least 9 mg IL12 (p40/p70) per mg ILlO.

19. The method according to any of the preceding claims, wherein said pro-inflammatory cocktail comprises IFN-γ.

20. The method according to claim 19, wherein said pro-inflammatory cocktail comprises IFN-γ in a concentration to contact the dendritic cells with a IFN-γ concentration in the range from about 1 ng/ml to about 200 ng/ml, such as in the range from about 5 ng/ml to about 50 ng/ml.

21. The method according to any of the preceding claims, wherein said pro-inflammatory cocktail comprises CpG oligonucleotide.

22. The method according to any of the preceding claims, wherein said pro-inflammatory cocktail comprises at least one member of the group consisting of IL6, TNFα, poly I:C and

ILlβ.

23. The method according to any of the preceding claims, wherein in step a) said proinflammatory cocktail comprises IFN-γ in a concentration to contact the dendritic cells with about 20 ng/ml IFN-γ and CpG-oligonucleotide in a concentration to contact the dendritic cells with about 1 μM CpG-oligonucleotide.

24. The method according to any of the preceding claims, wherein said test component is selected from the group consisting of a compound, a small chemical entity, a macromolecule such as a peptide, a protein, a polysaccharide, an oligonucleotide, a nucleic acid, a lipid, a sugar, a natural extract, a cell fragment, a microorganism, a bacterium and mixtures and combinations thereof.

25. The method according to claim 4, wherein said extracellular or intracellular endpoint is selected from the group consisting of IL2, IL4, IFNγ, ILlO, TGFβ, IL5 and IL13, IL17, IL6, lipid derivatives of prostaglandins, leukotrienes, eicosanoids, bioactive lipids and fatty acid derivatives.

26. The method according to any of claims 1-4, wherein in step a) said mammalian dendritic cells are contacted with said test component prior to said mammalian dendritic cells being contacted with said pro-inflammatory cocktail.

27. The method according to claim 26, wherein in step a) said mammalian dendritic cells are contacted with said test component at least one hour, such as at least 2-3 hours after said mammalian dendritic cells having been contacted with said pro-inflammatory cocktail.

28. The method according to any of the preceding claims, wherein said dendritic cells are immature dendritic cells.

29. The method according to any of the preceding claims, wherein said mammalian dendritic cells are human dendritic cells.

30. The method according to any of the preceding claims, wherein said mammalian dendritic cells are non-human dendritic cells, such as murine dendritic cells.

31. The method according to claim 30, wherein said mammalian dendritic cells are murine dendritic cells originating from murine bone marrow.

32. Use of the method according to any of the preceding claims for predicting the ability of a test component to prevent or alleviate an inflammatory condition in a human, comprising subjecting the potential test component to said method for in-vitro determination of the anti-inflammatory effect.

33. The use according to claim 32, wherein said inflammatory condition is selected from an inflammatory bowel disease such as Crohn 's disease or ulcerative colitis, rheumatoid arthritis, psoriasis, systemic lupus erythromatosis, eczema and allergy.

34. Use of the method according to any of claims 1-31 for quantification of the in-vitro effect of a test component on the pro-inflammatory phenotype of said mammalian dendritic cells resulting from contacting said mammalian dendritic cells with said pro-inflammatory cocktail.

35. Use according to claim 34, wherein said quantification is carried out for at least two different concentrations of said test component to provide a dose-response curve.

36. Bifidobacterium bifidum BI98 (DSM 19157) or a homolog, descendant or mutant thereof which exhibits probiotic activity.

37. Bifidobacterium bifidum BI504 (DSM 19158) or a homolog, descendant or mutant thereof which exhibits probiotic activity.

38. Composition comprising at least one Bifidobacterium bifidum selected from the strains according to any of claims 36-37.

39. Composition according to claim 38, which is an edible composition such as yoghurt, cheese, nutritional supplement, tablet, etc.

40. Composition according to claim 38, which is for external application on humans such as a creme, e.g. a skin creme or a vaginal creme.

41. The composition according to claim 38, which is a pharmaceutical composition.

42. Use of at least one Bifidobacterium bifidum selected from the strains according to any of claims 36-37 as a probiotic.

43. The use according to claim 42, wherein said at least one Bifidobacterium bifidum is used to prevent or alleviate an inflammatory condition.

44. The use according to claim 43, wherein said inflammatory condition is at least in part due to an inflammatory bowel disease.

45. The use according to claim 44, wherein said inflammatory bowel disease is Crohn 's disease or ulcerative colitis.

46. The use according to claim 43, wherein said inflammatory condition is at least in part due to rheumatoid arthritis, psoriasis, systemic lupus erythromatosis, eczema or allergy.