WO2009030482A1

WO2009030482A1 - In vitro model for inflammatory diseases of the gut mucosa

Info

Publication number: WO2009030482A1
Application number: PCT/EP2008/007247
Authority: WO
Inventors: Sonja Schwarz; Jutta SCHRÖDER-BRAUNSTEIN; Felix Lasitschka; Stefan Meuer
Original assignee: Universitätsklinikum Heidelberg
Priority date: 2007-09-05
Filing date: 2008-09-04
Publication date: 2009-03-12

Abstract

The present invention relates to a method for screening compounds for anti-inflammatory activity in vitro by determining a modulation in the number of immune cells that are migrating out of the lamina propria of gut biopsies. The present invention further relates to a method for screening compounds for anti-inflammatory activity in vitro by determining a modulation of the expression of inflammatory markers of immune cells that are migrating out of the lamina propria of gut biopsies.

Description

In vitro model for inflammatory diseases of the gut mucosa

Background of the invention

The mucosa of the mammalian colon is the largest compartment of immune competent cells (T-lymphocytes, B-lymphocytes, myeloid cells, dendritic cells, mast cells) of the body. This compartment has not readily been available for ex-vivo examinations, which have been performed only in some cases using residual material from surgeries, whereby only a fraction of the relevant cells had to be isolated using laborious procedures. Thus, examinations of the immune competent cells of the gut were only possible in an isolated state. In order to functionally analyze these cells, an additional experimental simulation is required. Since immune competent cells are migrating in the body and functionally respond to their actual environment, each modification of said environment will also lead to a change in the functions of said cells. Thus, in-vitro examinations on isolated immune competent cells of the gut mucosa always include the risk of observing artefacts.

The two major types of inflammatory bowel diseases (IBD) are Crohn's disease and ulcerative colitis.

Crohn's disease occurs when the lining and wall of the intestines becomes inflamed and ulcers develop. Although Crohn's disease can occur in any part of the digestive system, it often occurs in the lower part of the small intestine where it joins the colon. The intestine becomes inflamed, meaning the lining of the intestinal wall reddens and swells. It can become irritated, causing it to bleed and preventing it from properly absorbing the nutrients from digested food. In ulcerative colitis, the large intestine becomes inflamed and ulcers may develop. Ulcerative colitis affects only the large intestine. The inflammation begins in the rectum (the last few inches of the large intestine where faeces are stored before they leave the body) and can affect only the rectum or the part of the large intestine that joins it. However, most kids and teens who have ulcerative colitis have the condition throughout their large intestines.

Drug discovery in the area of anti-inflammatory compounds has the potential to identify novel compounds that are more effective and/or better tolerated than existing anti-inflammatory compounds. Another important criterion for selection of a target is that suppression of such target does not induce immediate or long term deleterious effects or toxicity to the host.

It is an object of the present invention, to provide an improved screening method for identifying compounds that are of use in the treatment of inflammatory diseases of the gut. Another object of the present invention is, to provide improved methods for monitoring and treating inflammatory diseases of the gut.

The invention includes, in one aspect, an in vitro method for screening test compounds for anti-inflammatory activity, comprising the steps of (a) providing defined biopsy samples, preferably punch samples, from the at least upper wall of the gut of a mammalian subject (i.e. mucosa, lamina epithelialis, and lamina propria), preferably the complete wall of the gut, (b) adding a test compound to said samples, (c) removing and/or damaging the lumial epithelial cell layer of said samples of step (b), and (d) observing any modulation in the number of immune cells that are migrating out of the lamina propria. In the context of the present invention, a "modulation" can mean diminution or increase.

In another aspect, the invention provides a method for monitoring an inflammatory bowel disease (IBD) in a mammalian subject, comprising: (a) providing defined biopsy samples, preferably punch samples, from the at least upper wall of the gut of a mammalian subject receiving an anti-inflammatory treatment (i.e. mucosa, lamina epithelialis, and lamina propria), preferably the complete wall of the gut, (b) removing and/or damaging the lumial epithelial cell layer of said biopsy samples of step (a), and (c) observing the number of immune cells that are migrating out of the lamina propria, wherein a modulated, preferably reduced, number of immune cells that are migrating out of the lamina propria is indicative of an effective anti- inflammatory treatment. In the context of the present invention, a "modulation" can mean diminution or increase.

In yet another aspect, the invention provides a method for treating an inflammatory bowel disease (IBD) in a mammalian subject, comprising a method according to the present invention as above, wherein the treatment is based, at least in part, on the number of immune cells that are migrating out of the lamina propria as observed, and wherein a modulated, preferably reduced, number of immune cells that are migrating out of the lamina propria is indicative of an effective anti-inflammatory treatment. In the context of the present invention, a "modulation" can mean diminution or increase.

In yet another aspect, the invention provides kits for methods according to the present invention as above.

These and other objects and features of the invention will be more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings. For the purposes of the present invention, all references as cited are incorporated herein in their entireties by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic overview of the ,,walk-out" model as used according to the invention.

FIG. 2 shows a diagram regarding the effect of dexamethasone and AG490 on cell emigration in the ,,walk-out" model as used according to the invention.

FIG. 3 shows the inhibition of the activation by APDC. After addition of APDC (ammonium pyrrolidine dithiocarbamate, NFKB inhibitor) to the culture medium and culturing for 36h, a detection of the surface markers by means of FACS was performed.

FIG. 4 shows the inhibition of the activation by APDC. Activation markers on T-cells and macrophages are down-regulated by APDC. APDC has no influence on the number of cells as migrating (only concentrations of more than lOμmol inhibit the migration), but down- regulates activation markers.

FIG. 5 shows that CD33+ "walk-out"-macrophages (WO-LPMC) express higher levels of the LPS-binding receptors CD 14 and TLR4 compared to resting lamina propria macrophages (LPMO), and that the expression of other pattern recognition receptors (TLR2 and TLR6), the costimulatory molecules CD54, CD58, CD80/86, and CD70 as well as Fc receptors CD 16 and CD64 and the amino acid transporter CD98 were up-regulated on WO-LPMO in comparison to resting LPMO (Fig. 5a). Time course experiments reveal that expression of most surface molecules analysed on WO-LPMO increased during a period of 36 hours following tissue injury (Fig. 5b). CD3+ "walk-out" T lymphocytes also show an activated phenotype with expression of CD25, CD69, CD98 being up-regulated in comparison to resting LPT and PBT (Fig. 5c), and the expression of these surface molecules increased within the first 36 hours of culture (Fig. 5d).

FIG. 6 shows that the up-regulation of surface receptor protein expression in WO-LPMC was accompanied by an increased transcription of the corresponding genes in these cells -at least for the markers/molecules analysed (Fig. 6a). Kinetics of gene expression were similar to that of protein expression for the surface receptors analysed in WO-LPMC (Fig. 6b). The expression of TLR4 stayed at constant levels during the preparation steps followed by a decrease after 12h of culture (Fig. 6c)

FIG. 7 shows cytokine antibody arrays that were used to screen "walk-out" organ culture supernatant for the presence of cytokines and chemokines as markers (Fig. 7).

FIG. 8 shows that increased gene expression of IL-I beta, IL-6 and MIP-I beta was observed in WO-LPMC when compared to LPMC and PBMC (Fig 8a). Gene transcription of all three parameters peaked at 12h (or potentially earlier) of organ culture (Fig. 8b and Fig. 8c).

FIG. 9 shows that the expression of numerous parameters up-regulated in WO-LPMC (e.g. IL-Ib, IL-6, CD54) depends on activation of the transcription factor NF-kB. In the presence of APDC, surface expression of CD16, (CD31), CD54, CD58, CD86 and CD98 was down- regulated in WO-LPMO, while CD69 expression was decreased in WO-LPT (Fig. 9a and Fig. 9b). Furthermore, gene transcription of CD54, CD86, IL-lbeta, IL-6, IL8 and MIP-lbeta was reduced in WO-LPMC in the presence of this compound (Fig. 9c).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., 1989, and Ausubel FM et al., 1993, for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process typically includes both transcription and translation, but in some cases may refer to transcription in the absence of translation. Screening typically includes functional assays for biological activity, protein assays and assays for marker mRNA, as further described herein. The terms "treating", "treatment" and "therapy" as used herein relative to a mammalian, particularly human subject or patient refer to curative therapy, prophylactic therapy, and preventative therapy. As used herein, the term "about" refers to a deviation of +/- 10% from the value as given.

The invention is directed, in one aspect, to an in vitro method for screening test compounds for anti-inflammatory activity, comprising the steps of (a) providing defined biopsy samples, preferably punch samples, from the at least upper wall of the gut of a mammalian subject (i.e. mucosa, lamina epithelialis, and lamina propria), preferably the complete wall of the gut, (b) adding a test compound to said samples, (c) removing and/or damaging the lumial epithelial cell layer of said samples of step (b), and (d) observing any modulation in the number of immune cells that are migrating out of the lamina propria. In the context of the present invention, a "modulation" can mean diminution or increase.

In the present model, pharmaceutical candidate compounds can be examined for their effect in the mammalian or human system, whereby a situation is maintained that reflects the actual situation in the gut. Systems that have been used until today are based on cell culture experiments such as examinations on Caco-monolayers, animal experiments with DSS-induced Colitis in the mouse, or colorectal ex-plant cultures.

These cell culture systems have the disadvantage that based on their homogeneity and their origin from tumour cells no physiological system can be represented. Isolated immune competent cells of the blood, likewise, do not offer the possibility of examining inflammatory modulators in the intestinal micro-environment. In animal models, always the question of the transferability on the human is questionable. Until today, ex-plant-cultures pursued a similar approach, nevertheless without making use of the specific effects of the generation of an inflammatory reaction through the loss/damage of the epithelial layer.

After the surgical harvest of colon tissue, punches of the upper wall (i.e. mucosa, lamina epi- thelialis, and lamina propria), preferably the whole wall, of the mammalian gut are obtained. These samples are preferably incubated under defined in-vitro conditions. If the luminal epithelial cell layer is damaged/removed, a spontaneous activation of immune competent mucosa cells and their migration from the tissue into the culture supernatant takes place. These so-called "walk-out-cells" have a phenotype that corresponds to the cells of the inflammatory colon mucosa. This system thus allows to exactly analyze the "natural" or "pathologic" start of the inflammation of the colonic mucosa by measuring a selection of parameters (change of expression of cell-surface molecules/receptors, production of cytokines and chemokines), as well as the number of cells that are migrating out of the lamina. At the same time, the effect of pharmaceutical substances on these initial inflammatory mechanisms of the colonic mucosa can be determined.

For the examination of the function, no external stimuli are required, since a "natural" inflammatory reaction is started that is very similar to the in-vivo situation for the generation of chronic inflammatory bowl diseases. The system can easily be standardized, avoids artefacts through preparative steps of the isolation of relevant cells, and reflects the interaction of bowl cell populations.

Martinsson et al (in Martinsson T. Ropivacaine inhibits serum-induced proliferation of colon adenocarcinoma cells in vitro. J Pharmacol Exp Ther. 1999 Feb;288(2):660-4) describe ropivacaine as being investigated for the treatment of ulcerative colitis. A study was per- formed to evaluate the effect of ropivacaine on the proliferation of human colon adenocarcinoma cells (HT-29 and Caco-2) in vitro. Ropivacaine inhibited the growth of HT-29 and Caco-2 cells in a dose-dependent manner. Lidocaine, hydrocortisone, and 5 -aminosalicylic acid were found to be less potent than ropivacaine in inhibiting proliferation. Ropivacaine caused a dose-dependent membrane depolarization that appeared to correlate with the inhibited cell proliferation, whereas the effect was not related to inhibition of leukotriene B4 or prostaglandin E2.

Axelsson et al. (in Axelsson LG, Landstrδm E, Bylund-Fellenius AC. Experimental colitis induced by dextran sulphate sodium in mice: beneficial effects of sulphasalazine and olsa- lazine. Aliment Pharmacol Ther. 1998 Sep;12(9):925-34) describe animal models of inflammatory bowel disease are artificial and more or less representative of human disease. However, the dextran sulphate sodium (DSS) induced intestinal inflammation model has recently been shown to fulfill some pathological criteria for an adequate experimental model. DSS was used to induce intestinal inflammation in conventional Balb/c mice and athymic nu/nu CD- l(BR) mice, and the well-documented 5-aminosalicylic acid (5-ASA) based anticolitis drugs sulphasalazine (SASP) and olsalazine (OLZ) were used to study therapeutic effects. Parameters which have been shown to reflect DSS-induced intestinal inflammation (body weight, colon length, spleen weight, diarrhoea, and rectal bleeding) were measured in the Balb/c mice.

Zhao et al (in Zhao Z, Satsu H, Fujisawa M, Hori M, Ishimoto Y, Totsuka M, Nambu A, Ka- kuta S, Ozaki H, Shimizu M. Attenuation by dietary taurine of dextran sulfate sodium- induced colitis in mice and of THP-I -induced damage to intestinal Caco-2 cell monolayers. Amino Acids. 2007 JuI 6) describes the effects of dietary taurine on the experimental colitis induced by dextran sulfate sodium (DSS) in mice. Taurine supplementation significantly attenuated the weight decrease, diarrhea severity, colon shortening, and the increase in the colonic tissue myeloperoxidase activity induced by DSS. Taurine also significantly inhibited the increase in the expression of a pro-inflammatory chemokine, macrophage inflammatory protein 2 (MIP-2), but not of interleukin (IL)-I beta or tumor necrosis factor (TNF)-alpha mRNA. Furthermore, taurine significantly protected the intestinal Caco-2 cell monolayers from the damage by macrophage-like THP-I cells in an in vitro coculture system.

Fletcher et al. (in Fletcher PS, Elliott J, Grivel JC, Margolis L, Anton P, McGowan I, Shat- tock RJ. Ex vivo culture of human colorectal tissue for the evaluation of candidate microbi- cides. AIDS. 2006 Jun 12;20(9): 1237-45) describe the establishment of an in vitro model to evaluate rectal safety and the efficacy of microbicide candidates. Human colorectal explants were cultured at the liquid-air interface on gelfoam rafts. Phenotypic characterization of HIV- 1 target cells was performed by fluorescence-activated cell sorter analysis. HIV-I infection was determined by the measurement of p24 antigen release, viral RNA, and proviral DNA accumulation.

In accordance with the first aspect of the invention, the samples as described above are used to screen for compounds having anti-inflammatory activity, as evidenced by a test compound's ability to modulate, preferably reduce or diminish, the number of immune cells that are migrating out of the lamina propria.

Typically, and increasing amounts of the test compound, e.g., 0, 10, 100, 1000, 10,000 mM are added to the samples. After a suitable incubation time, e.g., 12-36 hours, the number of immune cells that are migrating out of the lamina propria and/or level of the detectable marker (preferably selected from cell surface markers, expression markers, cytokines, chemokines) in the medium is measured, to determine if the compound, at any concentration, has resulted in a reduction of the expression diminution of said detectable-marker protein.

Compounds to be tested typically will include known anti-inflammatory compounds, such as glucocorticoids, salicylates, cyclosporin, and rapmycin, such as analogs generated as part of a chemical library including known anti-inflammatory compounds, prostaglandin inhibitor or COX-2 inhibitors . Other candidates are small polypeptides of nucleic acid aptamer compounds, e.g., members of a polypeptide or DNA aptamer library. Compounds identified as anti-inflammatory compound candidates may be further tested in defined screening systems, such as animal model systems, to further assess the potential of the compound as an antiinflammatory agent.

It will be appreciated that the screening format is readily adaptable to high-throughput screening (HTS), for example, by simultaneously screening a large number of sample in the microliter wells of a multiwell plate, such as one having 96, 384, 720 or larger numbers of wells. The wells are readily assayed for compound effect. Test compounds which show evidence of producing an effect can then be retested for more precise dose response, to further determine the potential value of the compound as an anti-inflammatory compound. When a test compound that (i) reduces the number of immune cells that are migrating out of the lamina propria and/or level of the detectable marker in the medium, and (ii) is active at a pharmaceutically practical level has been identified, the compound may be further assayed to develop its pharmacological profile. Such tests may include in vitro cell-culture studies to determine the effect of the identified compound, the ability of the compound to inhibit inflammation in suitable animal model systems, and the toxicology profile of the compound in animals.

In addition, when test compounds are identified, the compound may be further developed by standard drug-design or combinatorial-structure approaches to seek more active analogs, and/or compounds with reduced toxicity. Modification can be effected by a variety of methods known in the art, which include without limitation the introduction of novel side chains or the exchange of functional groups like, for example, introduction of halogens, in particular F, Cl or Br, the introduction of lower alkyl groups, preferably having one to five carbon atoms like, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl or iso-pentyl groups, lower alkenyl groups, preferably having two to five carbon atoms, lower alkynyl groups, preferably having two to five carbon atoms or through the introduction of, for example, a group selected from the group consisting of NH₂, NO₂, OH, SH, NH, CN, aryl, heteroaryl, COH or COOH group.

In a further embodiment of the method of the present invention the test compound identified as outlined above, which may or may not have gone through additional rounds of modification and selection, is admixed with suitable auxiliary substances and/or additives. Such substances comprise pharmacological acceptable substances, which increase the stability, solubility, biocompatibility, or biological half-life of the anti-inflammatory test compound or comprise substances or materials, which have to be included for certain routs of application like, for example, intravenous solution, sprays, Band-Aids or pills.

Preferred is a screening method according to the invention, wherein said immune cells are selected from T-lymphocytes, B-lymphocytes, myeloid cells, dendritic cells, and mast cells.

In an alternate embodiment of the screening method according to the invention, steps (b) and (c) are inverted, i.e. the biopsy sample can be contacted with a test compound before or after removing and/or damaging the lumial epithelial cell layer of said biopsy samples. The test compound can also be included in the medium to be added.

Preferred is a screening method according to the invention, wherein the biopsy samples are incubated in a suitable growth medium following step (c).

Further preferred is a screening method according to the invention, wherein said samples are punches of the upper wall (i.e. mucosa, lamina epithelialis, and lamina propria), preferably the complete wall of the gut. Preferably, said punches have a surface area of between about 50 mm^" and 100 mm^". Most preferably, ail punches as examined have an essentially identical surface area.

Further preferred is a screening method according to the invention, wherein about 3.5 ml of growth medium are added per surface area of about 100 mm².

Preferred is a screening method according to the invention, wherein the growth medium is RPMI/PSGGABC+10%FCS. Of course, other suitable media can be used as well.

In an alternate embodiment of the screening method according to the invention, the number of immune cells that are migrating out of the lamina propria is compared with a biopsy sample to which no test compound has been added in step (b). Preferably, said comparing comprises the generation of a calibration curve for said biopsy sample to which no test compound has been added in step (b). Most preferably, said calibration curve is based on the parameters surface area, growth medium, and number of immune cells that are migrating out of the lamina propria.

Further preferred is a screening method according to the invention, wherein all biopsy samples as examined are derived from the same mammalian subject.

Further preferred is a screening method according to the invention, wherein said test compound is selected from antibiotics, glucocorticoids, antibodies, cytokines, chemokines, and immune-suppressive agents. An alternate embodiment of the screening method according to the invention, furthermore comprises the step of detecting at least one surface marker and/or cytokine marker and/or chemokine marker in the immune cells that are migrating out of the lamina propria. Preferably, said surface marker and/or cytokine marker and/or chemokine marker is selected from CDl Ib, CD14, CD16, CD25, CD31, CD38, CD40, CD54, CD58, CD64, CD69, CD70, CD80, CD86, CD98, CD180, TLRl, TLR2, TLR3, TLR4, TLR6, TLR8, HLA-DR, FGF-7, IGFBP-2, IL-I beta, IL-5, IL-6, IL-7, LIGHT, MCP-I, Acrp30, GRO, GRO-alpha, HGF, ICAM-I, IL-8, sTNF-RI, TIMP-2, uPAR, ALCAM, Cardiotrophin-1, CD 14, ICAM-2, IGF-II, IL-5Ra, IL-IRII, M-CSF R, MMP-I, MMP-9, MMP-13, Angiogenin, PDGF Ra, VE- TXΓC_™

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A iv/ifΛ_Λj-i o« jji wi^iαuij' , ~ n,iα«-iijv,v«_i is selected from G-CSF, and IL-6. Further preferred is a screening method according to the invention, wherein said detecting comprises detecting the protein and/or mRNA expression, preferably by FACS or rtPCR.

Further preferred is a screening method according to the invention, wherein said mammal is a human.

Another aspect of the present invention is then directed at a kit for screening, comprising materials for performing a method according to the invention as above, optionally together with instructions for use.

In another aspect, the invention provides a method for monitoring an inflammatory bowel disease (IBD) in a mammalian subject, comprising: (a) providing defined biopsy samples, preferably punch samples, from the at least upper wall of the gut of a mammalian subject receiving an anti-inflammatory treatment (i.e. mucosa, lamina epithelialis, and lamina propria), preferably the complete wall of the gut, (b) removing and/or damaging the lumial epithelial cell layer of said biopsy samples of step (a), and (c) observing the number of immune cells that are migrating out of the lamina propria, wherein a modulated, preferably reduced, number of immune cells that are migrating out of the lamina propria is indicative of an effective antiinflammatory treatment. In the context of the present invention, a "modulation" can mean diminution or increase.

A further aspect of the present invention is then directed at an improved method for treating an inflammatory bowel disease (IBD) in a mammalian subject, comprising a method accord- ing to the present invention as above, wherein the treatment is based, at least in part, on the number of immune cells that are migrating out of the lamina propria as observed, wherein a modulated, preferably reduced, number of immune cells that are migrating out of the lamina propria is indicative of an effective anti-inflammatory treatment. In the context of the present invention, a "modulation" can mean diminution or increase. Preferably, said inflammatory bowel disease (IBD) is selected from Crohn's disease and ulcerative colitis.

Preferred is a method according to the invention, wherein said mammal is a human.

Further preferred is a method according to the invention, wherein said immune cells arc selected from T-lymphocytes, B-lymphocytes, myeloid cells, dendritic cells, mast cells.

Preferred is a method according to the invention, wherein said biopsy samples are punches of the complete wall of the gut. Said punches preferably can have a surface area of between about 50 mm² and 100 mm². More preferably, all punches as examined have an essentially identical surface area.

Preferred is a method according to the invention, wherein about 3.5 ml of growth medium are added per surface area of about 100 mm².

Further preferred is a method according to the invention, wherein said growth medium is RPMI/PSGGABC+10%FCS. Of course, other suitable media can be used as well.

An alternate embodiment of the method according to the invention, the number of immune cells that are migrating out of the lamina propria is compared with a biopsy sample of a subject that has not been treated. Preferably, said comparing comprises the generation of a calibration curve for said biopsy sample. More preferably, said calibration curve is based on the parameters surface area, growth medium, and number of immune cells that are migrating out of the lamina propria.

Further preferred is a method according to the invention, wherein all biopsy samples are derived from the same mammalian subject. Even further preferred is a method according to the invention, wherein said anti-inflammatory treatment is selected from agents selected from antibiotics, glucocorticoids, and immune- suppressive agents. Further preferred is an anti-inflammatory treatment comprising the administration of the pharmaceutical composition to the subject, comprising an antiinflammatory test compound as identified with a screening method according to the invention as described herein.

A further aspect of the present invention is then directed at an improved method for treating and/or monitoring an inflammatory bowel disease (IBD) in a mammalian subject as above, furthermore comprising the step of detecting at least one surface marker and/or cytokine marker and/or chemokine marker in the immune cells that are migrating out of the lamina propria. Preferably said surface marker and/or cytokine marker and/or chemokine marker is selected from CDl Ib, CD14, CD16, CD25, CD31, CD38, CD40, CD54, CD58, CD64, CD69, CD70, CD80, CD86, CD98, CDl 80, TLRl, TLR2, TLR3, TLR4, TLR6, TLR8, HLA-DR, FGF-7, IGFBP-2, IL-lbeta, IL-5, IL-6, IL-7, LIGHT, MCP-I, Acrp30, GRO, GRO-alpha, HGF, ICAM-I, IL-8, sTNF-RI, TIMP-2, uPAR, ALCAM, Cardiotrophin-1, CD 14, ICAM-2, IGF-II, IL-5Ra, IL-IRII, M-CSF R, MMP-I, MMP-9, MMP-13, Angiogenic PDGF Ra, VE- Cadherin, MiP-I β, MCP-I, TNFα, S0CS3, GADD45β, and G-CSF. More preferably, marker is selected from G-CSF, and IL-6. Preferred is a method of the present invention, wherein said detecting comprises detecting the protein and/or mRNA expression, preferably by FACS or rtPCR.

A further aspect of the present invention is then directed at a kit for monitoring the treatment of an IBD, comprising materials for performing a method according to the present invention, optionally together with instructions for use.

A yet further aspect of the present invention is the use of a pharmaceutical composition of the invention for the production of a medicament for the treatment of IBD as described herein.

Finally, a further aspect of the present invention is a method for identifying new targets in the treatment of IBD, comprising comparison of different markers as described herein in an untreated sample (normal gut) and in a sample from the gut of an IBD-patient, selective modulation of those parameters that are identified as modulated between normal gut and the gut of an IBD-patient, and identifying the disease-state of the inflammation, in order to identify new targets in the treatment of IBD, which can be used in the methods for screening as described herein.

Specific examples of the methods and compositions as described above are set forth in the following examples. However, it will be apparent to one of ordinary skill in the art that many modifications are possible and that the examples are provided for purposes of illustration only and are not limiting of the invention unless so specified.

EXAMPLES

The human intestinal mucosa is colonized by a commensal bacterial flora consisting of an estimated number of 10¹⁴ micro-organisms. Intrusion of these potentially infectious organisms is mainly prevented by a tight layer of mucus and epithelial cells. Furthermore, a large number of immune cells are located in the intestinal lamina propria which form an important defense line against bacterial invasion in case the surface barrier is broken. Under normal conditions these cells display a specialized differentiation state: while lamina propria T lymphocytes (LPT) are hyperresponsive to co-stimulatory signals, resident macrophages (LPMO) exist in an "anergic" state lacking expression (or expressing only low levels) of PRR (CD 14, TLR2/4), complement receptors (CDl lb/c, CD 18), Fc receptors (CD 16/32/64/89) and costimulatory molecules (CD54, 58, 80, 86).

In this example, the effect of tissue damage on lamina propria macrophages and T lymphocytes as well as total mucosa was explored using a human in-vitro organ culture model ("walk-out" model) originally described by Mahida et al. (Mahida, Y. R., A. M. Galvin, T. Gray, S. Makh, M. E. McAlindon, H. F. Sewell, D. K. Podolsky. 1997. Migration of human intestinal lamina propria lymphocytes, macrophages and eosinophils following the loss of surface epithelial cells. Clin. Exp. Immunol. 109:377). Briefly, mucosal damage was induced by detachment of the epithelial layer of healthy mucosa pieces obtained from colon resections. Loss of epithelial cells caused directed emigration of LPMC through pores onto the denuded basal lamina / basement membrane. Emigrated resident macrophages (WO-LPMO) and lamina propria T lymphocytes (WO-LPT) as well as organ culture tissue were screened with regard to expression of pattern recognition /complement /Fc receptors as well as costimulatory molecules and T cell activation markers. Furthermore, cytokine and chemokine expression was determined in "walk-out" cells and in total mucosa. Finally, an attempt was made to identify signalling events involved in the activation of lamina propria macrophages and T lymphocytes following tissue damage.

EXAMPLE 1

Large bowel specimen are obtained from patients undergoing resection for colon cancer or ulcerative colitis or Morbus Crohn. Normal or inflamed mucosa (5* 5cm) is dissected from the submucosa near the resection margin. The fresh tissue is washed extensively in RPMI/PSGABC at 4⁰C. The Mucus is removed by incubation in HBSS/PSGABC containing ImM Dithiotreitol (DTT) for 15 min at room temperature. After washing the tissue is incubated in a shaking water bath in HBSS/PSGABC containing 0,7mM EDTA at 37⁰C for 30 minutes. This incubation is repeated twice with fresh medium until the supernatant is free of epithelial cells. Between each incubation step the tissue is washed once in HBSS/PSGABC without EDTA for 10 minutes at 37°C. After the last incubation step the tissue is washed four times for 10 minutes in HBSS/PSGABC, until the supernatant becomes clear.

Finally, the tissues is transferred to a Petri dish and punched with the appropriate punches. The punches are then incubated for 12-36h with or without the substances to be investigated in the appropriate amount of RPMI/PSGGABC +10%FCS in the appropriate microtiter plates. After this timeperiod emigrated cells, supernatant and remaining tissue can be further assessed via cell count, PCR, FACS and Cytokinearrays.

RNA-Lysates of "walked out cells" are prepared by centrifuging 500.000 cells at 8000 rpm for 20 sec at 4°C, discarding the supernatant and adding 400μl of MagnaPur Lysis buffer complemented with lOmg/ml DTT. Tubes are then mixed well and stored at -80⁰C for further studies.

RNA-Lysates of tissue samples are prepared by punching a 6mm in diameter sample from the desired tissue sample, transferring it into a Ribolyzer tube, which has been earlier filled with Tissue Lysis buffer II complemented with 10mg/ml DTT, and ribolyzing the tissue at speed 5.0 for 20 sec in a commercial Ribolyzer. Until further assessment tubes are stored at -20⁰C.

Up-regulation of surface receptors on lamina propria macrophages and T lymphocytes in response to tissue damage Following detachment of epithelial cells human intestinal mucosa was taken into culture. Emigrated ("Walk-out") lamina propria mononuclear cells (WO-LPMC) were harvested after 12, 24, 36 and 60 hours and compared to resting LPMC (obtained by collagenase digestion) as well as autologous peripheral blood mononuclear cells (PBMC) with regard to surface marker expression and cytokine/chemokine production.

CD33+ "walk-out"-macrophages (WO-LPMC) expressed higher levels of the LPS-binding receptors CD 14 and TLR4 compared to resting lamina propria macrophages (LPMO) (Fig. 5a). Furthermore, expression of other pattern recognition receptors (TLR2 and TLR6), the costimulatory molecules CD54, CDS 8, CD80/86, and CD70 as well as Fc receptors CD 16 and CD64 and the amino acid transporter CD98 were up-regulated on WO-LPMO in comparison to resting LPMO (Fig. 5a). Importantly, WO-LPMC expressed significantly higher levels of HLA-DR than resting LPMO. Time course experiments revealed that expression of most surface molecules analysed on WO-LPMO (see above) increased during a period of 36 hours following tissue injury (Fig. 5b). When compared to CD33+ PBMO, W0-LPM0 expressed lower levels of CD14, CD16, CD98, CD58, TLR2 and TLR6, whereas CD64 and CD70 were similarly expressed. Expression levels of CD54, CD80, TLR4, and HLA-DR on W0-LPM0 even exceeded those on PBMO.

CD3+ "walk-out" T lymphocytes also showed an activated phenotype with expression of CD25, CD69, CD98 being up-regulated in comparison to resting LPT and PBT (Fig. 5c). Expression of these surface molecules increased within the first 36 hours of culture (Fig. 5d).

Up-regulation of surface receptor protein expression in WO-LPMC was accompanied by an increased transcription of the corresponding genes in these cells -at least for the markers/molecules analysed (CD 14, TLR4, TLR2, CD54, CD86, and CD25; Fig. 6a). However, the weak up-regulation of TLR4 transcripts in W0-LPM0 does not correlate with the high surface expression of TLR4 on these cells, which even exceeds that of PBMO. This may indicate that post-transcriptional mechanisms are involved in the regulation. Kinetics of gene expression were similar to that of protein expression for the surface receptors analysed in WO- LPMC (Fig. 6b): while CD14 and CD25 transcript levels markedly increased between 12h and 36h of culture, maximum transcript levels of CD54 were observed at 12h. CD86 gene expression also reached high levels within 12h of culture, which were only marginally increased after 36h of culture. In tissue culture samples gene expression of CD 14 stayed at a constant level during the preparation steps until 36h of culture. TLR2, CD86 and CD25 expression increased up to 12h of culture, followed by a decrease after 36h of culture. Expression of TLR4 stayed at constant levels during the preparation steps followed by a decrease after 12h of culture (Fig. 6c).

"Walk-out" organ culture supernatant contains potent pro-inflammatory cytokines and chemokines

Cytokine antibody arrays were used to screen "walk-out" organ culture supernatant for the presence of cytokines and chemokines as markers (Fig. 7). Markers that were up-rεgulatεd at least 10-fold in "walk-out" organ culture supernatant when compared to medium control/ organ culture supernatant of intact mucosa, are listed in Table 1 , below. Functionally, these markers are involved in immune regulation, tissue repair or chemotaxis. Time course experiments reveal that most of these cytokines/chemokines were detected in the supernatant after 12h of tissue culture. IL-8 and MMP- 13 were already detectable in the 2h tissue culture supernatant whereas angiogenin appeared first in the 36h tissue culture supernatant.

Table 1: positive cytokines and chemokines in cytokine antibody arrays

Angiogenin** IGFBP-2**

CD14** IL-I beta**

FGF-7** IL-6**

GRO** IL-8**

GRO-alpha** MCP-I**

HGF** M-CSF R

ICAM-I ** MMP-13**

ICAM-2** uPAR

** These cytokines and chemokines are known to be up-regulated in inflammatory bowel diseases.

Up-regulation of selected cytokines and chemokines was also confirmed on the transcriptional level in "walk-out" LPMC and mucosal tissue. As shown in Fig. 8a, increased gene expression of IL-I beta, IL-6 and MIP-I beta was observed in WO-LPMC when compared to LPMC and PBMC. Gene transcription of all three parameters peaked at 12h (or potentially earlier) of organ culture (Fig. 8b). In mucosal tissue, significant up-regulation of IL-I beta, IL-6, MIP- lbeta as well as MCP-I and G-CSF occurred very rapidly, i.e. within 2h, after starting to in- flict tissue damage. Peak gene expression levels were reached at 4h (IL-I beta, MIP-I beta, MCP-I), 12h (IL-6), and 36h (G-CSF) (Fig. 8c).

The exclusive or predominant induction of G-CSF, MCP-I, and IL-6 in mucosal tissue when compared to WO-LPMC suggests that these factors are mainly produced by non-immune cells, e.g. fibroblast or myo-fibroblasts. No or only very low protein and gene expression of IL-2, IFN-g, TNF-a, and IL-IO was observed in WO-LPMC and mucosal tissue.

Activation of "walk-out" LPMC is largely suppressed in the presence of the NfkappaB inhibitor APDC

The expression of numerous parameters up-reguiated in WO-LPMC (e.g. IL-Ib, IL-6, CD54) depends on activation of the transcription factor NF-kB. The inventors therefore determined the effect of APDC, an NFkB inhibitor, on activation of WO-LPMC. In the presence of APDC, surface expression of CD 16, (CD31), CD54, CD58, CD86 and CD98 was down- regulated in WO-LPMO, while CD69 expression was decreased in WO-LPT (Fig. 9a and Fig. 9b). Furthermore, gene transcription of CD54, CD86, IL-lbeta, IL-6, IL8 and MIP-lbeta was reduced in WO-LPMC in the presence of this compound (Fig. 9c). Interestingly, emigration of LPMC was not affected by treatment with APDC.

Table 2: Increase of surface markers on CD3 positive "walk-out" T-cells

Table 3: Increase of surface markers on CD33 positive ,,walk-out" macrophages

Table 4: Increase of the transcripts in walk-out LPL compared with LPL from collagenase- digest

In the PCR an increased expression of several inflammatory markers could be shown. Parameters that were also detected on the protein level by means of FACS are indicated in bold.

φ = 1-15 times, φφ = 16-130 times, φφφ = 131-1500 times the base level expression

MATERIALS AND METHODS

Tissues/samples

All human studies were approved by the ethics committee of the University of Heidelberg and were performed in accordance with the principles laid down in the Declaration of Helsinki. Informed consent was obtained from the patients. Gut specimens were derived from individuals undergoing resection for localized colon cancer or benign colonic diseases. Colonic mucosa being microscopically normal was dissected from the surgical specimen near the resection margin and immediately processed for isolation of lamina propria cells.

Antibodies Following antibodies were used for flow cytometry: CD3 FITC, CD 3PE, CD3 PerCP clone SK7 (Becton, Dickinson and Company (BD)), CDl Ib PE clone ICRF44 (BD Pharmingen), CD 14 FITC clone MφP9 (BD), CD25 PE clone M-A251 (BD Pharmingen), CD31 FITC clone WM59 (BD Pharmingen), CD33 FITC , CD33 PE, CD33 PerCP-Cy5.5 clone 67.6 (BD), CD38 FITC clone HB-7 (BD), CD54 FITC clone MCA532F (serotec), CD58 FITC clone 1C3 (BD Pharmingen), CD64 FITC clone 22 (immunotech), CD69 PE clone L78 (BD), CD70 PE clone Ki-24 (BD), CD80 PE clone L307.4 (BD), CD86 PE clone 2331 (FUN-I) (BD Pharmingen), CD98 FITC clone UM7F8 (BD Pharmingen), HLA-DR PerCP clone L243 (BD), TLR2 PE clone TL2.1 (imgenex), TLR4 FITC clone HTAl 25 (imgenex). For proliferation assays CD2 monoclonal antibodies Ml and M2 were produced in our own laboratory. Mouse monoclonal antibody 3PT was kindly provided by Drs S. F. Schlossman and E. L. Reinherz, Dana-Farber Cancer Institute, Boston, USA. Ly294002. Coating of plates for CD3 proliferation assays was performd with AffiniPure Goat Anti-Mouse IgG and IgM (dianova).

Preparation of lymphocytes and tissue culture supernatant

Lamina propria mononuclear cells were isolated according to a modified method of Bull and Bookman [Bull, 1977 #33]. Briefly, the mucosal layer was dissected from the fresh tissue and washed extensively in RPMI 1640 (Gibco) and antibiotics. Mucus was removed by shaking tissue samples in wash media with DTT (lμM, from Sigma). Subsequently, the mucosa was incubated in a shaking water bath at 37°C with 0.7mM EDTA (Sigma) in HBSS without

Ca 2+ and Mg 2+ for 30 minutes to remove epithelial cells. After each inkubation step the tissue was washed with HBSS. This incubation was repeated three times. After extensive washing, the tissue was separated in two parts. One part was cut into 2-4 mm pieces and digested in a shaking waterbath at 37⁰C for 10-12 hours by 45 U/ml collagenase (Sigma) and 27 U/ml deoxyribonuclease I (Sigma) in RPMI 1640 containing 2% fetal calf serum (Sigma), 2% L- glutamine (Gibco) and antibiotics. The resulting cell suspension was separated from undigested tissue by filtration through a 70μm nylon mesh (Becton Dickinson). For further isolation of lamina propria T lymphocytes, the cell suspension was subjected to Percoll (GE Healthcare) density gradient centrifugation followed by Ficoll-Hypaque (GE Healthcare) density gradient centrifugation to remove dead cells. Cells of the second mucosal part were isolated according to a method of Mahida et al. (Ma- hida et al. 1997). In brief, the denuded mucosal sample was placed in tissue culture dishes (greiner) and cultivated in RPMI 1640 containing 10% fetal calf serum, 2% L-glutamine and antibiotics at 37°C for 12 to 6Oh. After detaching the adhearend cell at 4°C, lymphcytes were harvested and washed. The supernatant was used directly or was aliquoted and frozen at - 80°C.

Peripheral blood was taken during the operation. Peripheral blood lymphocytes were obtained by Ficoll-Hypaque (GE Healthcare) density gradient centrifugation.

Tissue culture in addition of APDC

Fresh tissue samples were washed in RPMI 1640 (Gibco) with antibiotics in absence and addition of APDC in 4 different concentrations (10OnM, ImM, 10 mM, 10OmM). After that the tissue was punched in pieces of lcm². These pieces were treated as descrided above in addition of APDC to all media. Following the tissue samples were cultured in 6-well plates (nunc) for 36h. At last LPL were harvested as described above.

Gene expression analysis

5x10⁵ PBL and LPL were collected in 400μl lysis buffer from the MagnaPure mRNA Isolation Kit I (Roche Diagnostics) and mRNA was isolated with the MagnaPure-LC device using the mRNA-I standard protocol. The elution volume was set to 50μl. An aliquot of 8.2 μl RNA was reverse transcribed using AMV-RT and oligo- (dT) as primer (First Strand cDNA synthesis kit, Roche) according to the manufactures protocol in a thermocycler. After termination of the cDNA synthesis, the reaction mix was diluted to a final volume of 500 μl and stored at - 20°C until PCR analysis.

Primer sets optimized for the LightCycler (RAS, Mannheim, Germany) were developed and provided by SEARCH-LC GmbH, Heidelberg. The PCR was performed with the LightCycler FastStart DNA Syber Green I kit (RAS) according to the protocol provided in the parameter specific kits. To control for specificity of the amplification products, a melting curve analysis was performed. No amplification of unspecific products was observed. The number of transcripts was calculated from a standard curve, obtained by plotting known input concentrations of four different plasmids at log dilutions to the PCR-cycle number (CP) at which the detected fluorescence intensity reaches a fixed value. This approach dramatically reduced variations due to handling errors over several logarithmic dilution steps.

To correct for differences in the content of mRNA, the calculated transcript numbers were normalized according to the expression of the housekeeping gene cyclophilin B. Values were thus given as transcripts per 1000 transcripts of CPB.

Flow Cytometry

Flow Cytometry was performed with a FACSCalibur (BD Biosciences) and data was analyzed with the CellQuestPro software.

For FACS staining 1 x 10⁵ to 1 x 10⁶ cells were labelled with a cocktail of CD3, CD33 and a third antibody in three different colours. After measurement cells were gated CD33 positive, CD3 negative for monocytes and CD3 positive, CD33 negative for T-cells.

Proliferation assays

5 x 10⁵ cells per well were planted on 96 well roundbottom plates (nunc) for CD2 proliferation assays or 96 well flatbottom plates (nunc) for CD3 proliferation assays. After 3 days of culture 3H-Thymidin was added and samples were cultured futher 18 houres. Proliferation was measured by counting the increasse of 3H-Thymidin uptake.

Stimulation with supernatant

The supernatant was fractionised by centrifugal filtration in Centriprep filter units (Millipore) with a cut off at 3kD. The retentate was diluted with RPMI 1640 (Gibco) with antibiotics to the initial volume, 10% FCS was added to the filtrate.

LPL and PBL were then cultured with filtrate, retentate, supernatant and culture medium after 30 min, Ih, 2h, 4h, 8h, 24h, 36h rtPCR was performed. PBL and LPL were analysed by flow cytometry after 24h of culture. Tranwell Experiments

For transwell experiments 6-well hanging culture inserts (Millipore) were used according to the protocoll. 5 x 10⁶ PBL or LPL were planted in each well of a 6-well plate (Falcon), the denuded mucosa was punched in pieces of lcm² and placed in the insert. For negative con- troll only culture media was appended in the inserts. APDC 0 was added in concentrations of 100 nM, lμM, lOμM and lOOμM. After 24 hour of culture cells were detached by shaking Ih at 4⁰C. LPL and PBL were analysed with rtPCR and flow cytometry.

Cytokine antibody arrays

For the antibody arrays frozen tissue culture supernatant from 12, 24, 36 and 60 hour culture was defrosted under running water. RayBio Human Cytokine Antibody Arrays VI, VII, VIII C Series 2000 (Raybiotech) that detect 174 different proteins were used according to the protocoll.

Claims

1. An in vitro method for screening test compounds for anti-inflammatory activity, comprising:

(a) providing defined biopsy samples from the at least upper wall of the gut of a mammalian subject,

(b) adding a test compound to said biopsy samples,

(c) removing and/or damaging the lumial epithelial cell layer of said biopsy samples of step (b), and

(d) observing any modulation in the number of immune cells that are migrating out of the lamina propria.

2. The method of claim 1, wherein said immune cells are selected from T-lymphocytes, B-lymphocytes, myeloid cells, dendritic cells, mast cells.

3. The method of claim 1 or 2, wherein steps (b) and (c) are inverted.

4. The method of any of claims 1 to 3, wherein the biopsy samples are incubated in a suitable growth medium following step (c).

5. The method of any of claims 1 to 4, wherein said biopsy samples are punches of the complete wall of the gut.

6. The method of any of claims 1 to 5, wherein said punches have a surface area of between about 50 mm² and 100 mm².

7. The method of any of claims 1 to 6, wherein all punches as examined have an essentially identical surface area.

8. The method of any of claims 1 to 7, wherein about 3.5 ml of growth medium are added per surface area of about 100 mm².

9. The method of claim 4 or 8, wherein said growth medium is RPMI/PSGGABC+10% FCS.

10. The method of any of claims 1 to 9, wherein the number of immune cells that are migrating out of the lamina propria is compared with a biopsy sample to which no test compound has been added in step (b).

11. The method of claim 10, wherein said comparing comprises the generation of a calibration curve for said biopsy sample to which no test compound has been added in step (b).

12. The method of claim 11, wherein said calibration curve is based on the parameters surface area, growth medium, and number of immune cells that are migrating out of the lamina propria.

13. The method of any of claims 1 to 12, wherein all biopsy samples are derived from the same mammalian subject.

14. The method of any of claims 1 to 13, wherein the modulation is a reduction or increase.

15. The method of any of claims 1 to 14, wherein said test compound is selected from antibiotics, glucocorticoids, antibodies, cytokines, chemokines, and immune-suppressive agents.

16. The method of any of claims 1 to 15, furthermore comprising the step of detecting at least one surface marker and/or cytokine marker and/or chemokine marker in the immune cells that are migrating out of the lamina propria.

17. The method of claim 16, wherein said surface marker and/or cytokine marker and/or chemokine marker is selected from CDl Ib, CD14, CD16, CD25, CD31, CD38, CD40, CD54, CD58, CD64, CD69, CD70, CD80, CD86, CD98, CDl 80, TLRl, TLR2, TLR3, TLR4, TLR6, TLR8, HLA-DR, FGF-7, IGFBP-2, IL-I beta, IL-5, IL-6, IL-7, LIGHT, MCP-I, Acrp30, GRO, GRO-alpha, HGF, ICAM-I, IL-8, sTNF-RI, TIMP-2, uPAR, ALCAM, Cardiotrophin-1, CD 14, ICAM-2, IGF-II, IL-5Ra, IL-IRII, M-CSF R, MMP-I, MMP-9, MMP- 13, Angiogenin, PDGF Ra, VE-Cadherin, MiP-lβ, MCP- 1, TNFα, S0CS3, GADD45β, and G-CSF.

18. The method of claim 16 or 17, wherein said marker is selected from G-CSF, and IL-6.

19. The method of any of claim 16 to 18, wherein said detecting comprises detecting the protein and/or mRNA expression, preferably by FACS or rtPCR.

20. The method of any of claims 1 to 19, wherein said mammal is a human.

21. A kit for screening, comprising materials for performing a method of any of claims 1 to 20, optionally together with instructions for use.

22. A method for monitoring an inflammatory bowel disease (IBD) in a mammalian subject, comprising:

(a) providing defined biopsy samples from the at least upper wall of the gut of a mammalian subject receiving an anti-inflammatory treatment,

(b) removing and/or damaging the lumial epithelial cell layer of said biopsy samples of step (a), and

(c) observing the number of immune cells that are migrating out of the lamina propria, wherein a modulated, preferably reduced, number of immune cells that are migrating out of the lamina propria is indicative of an effective anti-inflammatory treatment.

23. A method for treating an inflammatory bowel disease (IBD) in a mammalian subject, comprising a method according to claim 22, wherein the treatment is based, at least in part, on the number of immune cells that are migrating out of the lamina propria as observed, and wherein a modulated, preferably reduced, number of immune cells that are migrating out of the lamina propria is indicative of an effective anti-inflammatory treatment.

24. The method two according to claim 22 or 23, wherein said inflammatory bowel disease (IBD) is selected from Crohn's disease and ulcerative colitis.

25. The method of any of claims 22 to 24, wherein said mammal is a human.

26. The method of any of claims 22 to 25, wherein said immune cells are selected from T- lymphocytes, B-lymphocytes, myeloid cells, dendritic cells, mast cells.

27. The method of any of claims 22 to 26, wherein said biopsy samples are punches of the complete wall of the gut.

28. The method of any of claims 22 to 27, wherein said punches have a surface area of between about 50 mm² and 100 mm².

29. The method of any of claims 22 to 28, wherein all punches as examined have an essentially identical surface area.

30. The method of any of claims 22 to 29, wherein about 3.5 ml of growth medium are added per surface area of about 100 mm².

31. The method of claim 30, wherein said growth medium is RPMI/PSGGABC+10% FCS.

32. The method of any of claims 22 to 31, wherein the number of immune cells that are migrating out of the lamina propria is compared with a biopsy sample of a subject that has not been treated.

33. The method of claim 32, wherein said comparing comprises the generation of a calibration curve for said biopsy sample.

34. The method of claim 33, wherein said calibration curve is based on the parameters surface area, growth medium, and number of immune cells that are migrating out of the lamina propria.

35. The method of any of claims 22 to 34, wherein all biopsy samples are derived from the same mammalian subject.

36. The method of any of claims 22 to 35, wherein said anti-inflammatory treatment is selected from agents selected from antibiotics, glucocorticoids, antibodies, cytokines, chemokines, and immune-suppressive agents.

37. The method of any of claims 22 to 36, furthermore comprising the step of detecting at least one surface marker and/or cytokine marker and/or chemokine marker in the immune cells that are migrating out of the lamina propria.

38. The method of claim 37, wherein said surface marker and/or cytokine marker and/or chemokine marker is selected from CDi Ib, CD14, CDlό, CD25, CD31, CD38, CD40, CD54, CD58, CD64, CD69, CD70, CD80, CD86, CD98, CD 180, TLRl, TLR2, TLR3, TLR4, TLR6, TLR8, HLA-DR, FGF-7, IGFBP-2, IL-lbeta, IL-5, IL-6, IL-7, LIGHT, MCP-I, Acrp30, GRO, GRO-alpha, HGF, ICAM-I, IL-8, sTNF-RI, TIMP-2, uPAR, ALCAM, Cardiotrophin-1, CD 14, ICAM-2, IGF-II, IL-5Ra, IL-IRII, M-CSF R, MMP-I, MMP-9, MMP- 13, Angiogenin, PDGF Ra, VE-Cadherin, MiP- lβ, MCP- 1, TNFα, SOCS3, GADD45β, and G-CSF.

39. The method of claim 37 or 38, wherein said marker is selected from G-CSF, and IL-6.

40. The method of any of claim 37 to 39, wherein said detecting comprises detecting the protein and/or mRNA expression, preferably by FACS or rtPCR.

41. A kit for monitoring the treatment of an IBD, comprising materials for performing a method of any of claims 22 to 40, optionally together with instructions for use.

42. A pharmaceutical composition, comprising an anti-inflammatory test compound as identified with a screening method according to any of claims 1 to 21.