EP1373906A1 - Method for diagnosing chronic intestinal inflammatory diseases - Google Patents

Abstract

The invention relates to a method for the in vitro diagnosis of chronic intestinal inflammatory diseases, such as Crohn's disease or haemorrhagic rectal colitis. The invention also relates to the use of compounds, such as an antibody, ligand or nucleic probe capable of specifically recognising PPARη gene expression products, for the preparation of the composition or kit for diagnosing said diseases.

Description

METHOD FOR DIAGNOSING CHRONIC INFLAMMATORY BOWEL DISEASES

The present invention relates to a method for the in vitro diagnosis of inflammatory bowel disease, such as Crohn's disease or ulcerative colitis, as well as the use of compounds, such as an antibody, ligand or nucleic probe capable of specifically recognize gene expression products

PPARγ for the preparation of a composition or a diagnostic kit for these diseases.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBD) that predominantly strike young adults, progressing in flares interspersed with periods of remission. They constitute one of the major problems of iepato-gastroenterology. Indeed, these diseases strike young people and are of chronic or prolonged course, potentially throughout life. These diseases can affect the entire digestive tract, from the mouth to the anus. These diseases also frequently affect personal and professional life through the frequency of flare-ups, complications and the sometimes necessary recourse to surgery. As their cause is not known, these diseases have no curative treatment. Even if it is effective immediately, medical treatment has only a suspensive effect. In addition, the cost of such suspensive medical treatment, already high, increases with new drugs. The prevalence of IBD increases due to an increasing incidence until the 1970s and has remained stable since. Based on the 1991 incidence figures and the estimated median survival, it was thus estimated that in the year 2005, among 232 million Caucasian Americans, 580,000 will have CD and 742,000 will UC.

In France, data from the EPIMAD register in the north-west of the country show a stable incidence since 1988 of IBD: 5.6 for CD and 3.5 for UC. Using the same calculation rules as in the United States, we arrive at an expected figure in France of 120,000 MC and 80,000 RCH in 2005.

The diagnosis of IBD is based on a set of clinical, morphological and histological criteria. Lesions during CD can reach all segments of the digestive tract while UC affects only the colon. The existence of isolated colonic lesions therefore represents a delicate situation for diagnosing CD or UC or to differentiate IBD from other pathologies associated with intestinal inflammation such as, for example, bacterial, viral or parasite infections, or even vascular pathologies, pathologies secondary to taking medication, diverticulitis, etc.

Faced with the difficulty of establishing the diagnosis of CD or UC, the development of new diagnostic markers is of fundamental interest which will improve patient care.

Thus, there is today a great need to be able to have an effective and reliable marker for chronic inflammatory diseases of the intestine, such as Crohn's disease or ulcerative colitis, such a marker should preferably be used easy and inexpensive.

This is precisely the object of the present invention.

By Western blot and immunohistochemistry techniques, the inventors have surprisingly demonstrated a deficit in the expression of the receptor activated by proliferators of the PPARγ peroxisomes in the colonic mucosa of patients suffering from UC.

The inventors were thus able to demonstrate in a surprising manner that the level of expression of the gene coding for the PPARγ receptor (PPARγ for “Peroxysome

Proliferator-Activated Receptor gamma) on the cell surface of cells of the epithelium and / or cells of the lamina propria of the intestinal tissue was correlated with the diagnosis or prognosis of chronic inflammatory diseases of the intestine.

The inventors have also, and just as surprisingly, demonstrated that this rate of expression of PPARγ added to its distribution according to the cell type, epithelium cells and / or cells of the lamina propria, not only makes it possible to '' refine this diagnosis or prognosis, but also to diagnose or predict with precision the type of chronic inflammatory diseases of the intestine of the patient tested.

The receptor activated by PPARγ peroxisome proliferators is a receptor which controls the expression of a large number of genes involved in metabolism (Schoonjans, et al., Curr. Opin. Lipidol., 8: 159-166, 1997 1997 ). In vitro studies have already shown that activators of PPARγ limit the production of inflammatory mediators such as inflammatory cytokines produced by human macrophages-monocytes (Ricote, et al., Nature, 391: 79-82, 1998; Jiang, and al. Nature, 391: 82-86, 1998) and by intestinal epithelial cells (Lefebvre, et al., Endocrinology, 162: 331-340, 1999). The in vivo utility of PPARγ agonists has also been described for the treatment of colitis induced in mice by the administration of sodium dextran sulfate (Su, et al., J. Clin. Invesi, 104: 383-9, 1999), or 2,4,6-trinitrobenzene sulfonic acid (Dubuquoy et al., Gastroenterol. Clin. Biol., 24: 719-24, 2000).

We can also cite:

- US patent document 5,861,274 published on January 19, 1999 which describes the nucleic and peptide sequence of the human PPARγ receptor; - the publication of Desreumaux et al., 1999 (Gastroenterology, 117, 1, 73-81, 1999) which describes an aberrant expression of PPARγ in the mesenteric adipose tissue of patients suffering from CD and a lack of regulation of PPARγ by TNFα at the adipocyte level;

- the publication of Lefebvre et al., 1999 (J. of Endocrinology, 162, 3, 331-340,1999) which describes the expression of PPARγ in the digestive tract in mice and humans;

- the publication of Su Chinyu et al., 1999 (J. of Clinical Investigation, 104, 4, 383-389, 1999) which describes the anti-inflammatory role of PPARγ agonists in vitro and in vivo in an experimental model of colitis in mice induced by dextran sodium sulfate; - Patent document 6,166,049 published on December 26, 2000, which describes the use of PPARγ agonists in the treatment or prevention of Syndrome X; and finally

- the two documents Hafraoui et al., 1999 (Gastroenterology, 116, 4, Part. 2, page A689, 1999 and Immunology Letters, 69, 1, 161-162, 1999) which describe a difference in expression of mRNA of PPARγ during UC vs CD and patients controlled by quantitative PCR.

The present invention therefore relates to an in vitro method for the diagnosis or for the prognosis of an inflammatory bowel disease in a patient, characterized in that it implements a step of comparison of the quantity of product d expression of the gene coding for PPARγ in a sample of intestinal tissue, said sample comprising at least cells of the epithelium and / or of the intestinal lamina propria, relative to a control sample. The present invention also relates to a method according to the invention, characterized in that said sample of intestinal tissue comprises at least cells of the epithelium and of the intestinal lamina propria and in that it further implements a step of comparing the distribution of the expression product of said PPARγ gene between said intestinal epithelium and lamina propria cells on the patient's sample.

The above diagnostic methods according to the invention are preferably characterized in that they comprise the steps of the methods defined below.

The present invention thus relates to an in vitro method for determining the quantity of expression product of the gene coding for PPARγ (PPARγ gene) on a sample of intestinal tissue, said sample comprising at least cells of the epithelium and / or intestinal lamina propria, characterized in that it comprises the following stages: a) the taking of a sample of intestinal tissue from a patient likely to have or progress to an inflammatory disease of the intestine, said sample comprising at least cells from the intestinal epithelium and / or lamina propria; b) bringing the expression product of the PPARγ gene into contact with a compound capable of binding specifically to said expression product of the PPARγ gene, under conditions allowing the formation of a complex between said compound and said expression product , said compound being labeled or capable of being labeled in order to obtain a signal representative of the quantity of said expression product present in the sample and, if necessary, making it possible to locate said expression product; and c) the quantification or display of the signal obtained in step b).

The present invention also comprises a method according to the invention, characterized in that said sample of intestinal tissue comprises at least cells of the intestinal epithelium and of the intestinal lamina propria, and in that it further implements a step for comparing the distribution of the expression product of said PPARγ gene between said intestinal epithelium and lamina propria cells on the patient's sample. Preferably, said sample is a biopsy of intestinal tissue removed from the patient's colon or small intestine, especially intestinal tissue removed from the patient's colon.

In the methods according to the present invention, any conventional procedure or test can be used to result in step b) of said methods in obtaining a detectable and / or quantifiable signal representative of the quantity of said expression product. present in the sample, depending on the expression product sought, preferably the polypeptide corresponding to the translation product of said PPARγ gene, in particular by the so-called “Western blot” method or by immunohistochemistry well known to man of the art, or its transcription product (mRNA) encoding said PPARγ receptor, in particular by the so-called in situ hybridization method also well known to those skilled in the art, methods which will not be developed in the present description. However, reference may be made to the examples below for the so-called “Western blot” method or by immunohistochemistry. In a particularly preferred embodiment, the method according to the invention is characterized in that said expression product of said PPARγ gene is the translation product of said PPARγ gene, and in that the compound capable of specifically binding to said product d expression of the PPARγ gene in step b) is chosen from the following compounds: - a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor, or one of its fragments; and

- a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor.

By “translation product of said PPARγ gene” is meant in particular the polypeptide corresponding to the PPARγ receptor expressed by the intestinal mucosa, in particular on the surface of the cells of the intestinal epithelium and lamina propria.

The amino acid sequence of the human PPARγ receptor is in particular described in the document US Pat. No. 5,861,274 (SEQ LD No. 2). In general, for the preparation of monoclonal antibodies or their fragments directed against the PPARγ receptor, reference may be made to the techniques which are in particular described in the “Antibodies” manual (Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Publications pp. 726, 1988) or to the technique of preparation from hybridomas described by Kôhler and Milstein, Nature, 256: 495-497, 1975.

The monoclonal or polyclonal antibodies which can be used in the methods according to the invention can be obtained for example from the cell of an animal immunized against the PPARγ protein, or one of its fragments comprising the specific epitope (determinant of the protein responsible for the specific interaction with the antibody).

Said PPARγ receptor protein, or a fragment thereof, may in particular be produced, according to the usual procedures, by genetic recombination from a nucleic acid sequence contained in the cDNA sequence coding for the PPARγ receptor protein, or by peptide synthesis from an amino acid sequence included in the peptide sequence of the OZF protein.

The monoclonal or polyclonal antibody fragments according to the invention comprise any fragment of said monoclonal antibody capable of binding to the epitope of the PPARγ protein on which the monoclonal or polyclonal antibody from which said fragment is derived is attached. Examples of such fragments include in particular monoclonal or polyclonal single chain antibodies or monovalent fragments Fab or Fab 'and divalent fragments such as F (ab') 2, which have the same binding specificity as the monoclonal antibody of which they are from. A fragment according to the invention may also be a single chain Fv fragment produced by methods known to those skilled in the art and as described for example by Skerra, et al., Science, 240: 1038-1041, 1988 and King et al., Biochemical J., 290: 723-729, 1991.

The monoclonal or polyclonal antibody fragments of the invention can be obtained from the monoclonal or polyclonal antibodies as described above by methods such as digestion with enzymes, such as pepsin or papain and / or by cleavage of the bridges disulfides by chemical reduction. In another way, the monoclonal or polyclonal antibody fragments can be synthesized by automatic peptide synthesizers such as those supplied by the company Applied Biosystems, etc., or can be prepared manually using techniques known to man. of art and as described for example by Geysen, et al., J. rmmunol. Methods, 102: 259-274, 1978. The monoclonal or polyclonal antibodies, or their fragments, as well as the specific ligands capable of binding specifically to said translation product can also, according to the invention, be in marked form in order to obtain a detectable and / or quantifiable signal. The antibodies, or their fragments, or said labeled ligands according to the invention include for example antibodies or ligands called immunoconjugates which can be conjugated for example with enzymes such as peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetyl cholinesterase, lysozyme, malate dehydrogenase or glucose-6 phosphate dehydrogenase or by a molecule such as biotin, digoxigenin or 5-bromo-deoxyuridine. Fluorescent markers can also be conjugated to antibodies, or their fragments, or to said ligands, and include in particular fluorescein and its derivatives, fluorochrome, rhodamine and its derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl , umbelliferone, etc. In such conjugates, the antibodies, or their fragments, or the said ligands can be prepared by methods known to those skilled in the art. They can be coupled to enzymes or fluorescent markers directly or through a spacer group or a linking group such as a polyaldehyde, such as glutaraldehydeque, ethylenediaminetetraacetic acid (EDTA), diethylene acid -triaminepentaacetic (DPTA), or in the presence of coupling agents such as periodate, etc. The conjugates comprising markers of fluorescein type can be prepared by reaction with an isothiocyanate.

Other conjugates can also include chemiluminescent markers such as luminol and dioxetanes or bioluminescent markers such as luciferase and luciferin.

Among the markers which can be attached to the monoclonal or polyclonal antibody, or one of their fragments, or to said ligands, mention may also be made of radioactive markers which can be detected by known means such as the gamma counter or scintillation counter, by autoradiography, etc. In the methods according to the present invention, any conventional procedure or test can be implemented to result in step b) of said methods in obtaining a detectable and / or quantifiable signal representative of the quantity of said product of expression present in the sample, in particular by “Western blot” or by immunohistochemistry.

In these so-called “Western blot” or immunohistochemistry techniques, the specific complex formed between the translation product of the PPARγ gene with a compound capable of binding specifically to said translation product may result from the contacting of an antibody, or one of its fragments, or of a ligand specific for the PPARγ receptor with said translation product.

In such detection and / or quantification, said test can be a competition or sandwich test, or any test known to those skilled in the art dependent on the formation of an antibody-antigen or ligand-receptor immune complex.

The antibody, or one of its fragments, or the ligand can also be immobilized. This immobilization can be carried out on numerous supports known to those skilled in the art. These supports may in particular include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, or natural or modified celluloses. These supports can be either soluble or insoluble. Mention may in particular be made of solid supports such as polystyrene beads, microtiter plates, which are well known in the field of immunoassays and which will not be developed in the present description.

For example, a preferred method involves immunoenzymatic processes according to the ELISA technique, by immunofluorescence or immunoluminescence, or radioimmunological (RIA), gold labeling or equivalent.

The specific techniques and reagents enabling the detection, identification, localization and / or assay of the antigen-antibody or ligand-receptor complexes which can be used in the methods of the invention, may of course be associated, in the case where the antibodies or ligands participating in the complex are not already immunoconjugated or labeled, with the appropriate reagents. These suitable reagents will be, for example, immunoconjugated or labeled antibodies, as described above, capable of specifically recognizing the antibodies or ligands participating in said complex. The chromogenic substrates specific for the conjugated enzymes and the control reagents with positive, negative and quantitative control will also be associated with these techniques or reagents. By "natural or synthetic agonist or antagonist ligand" of the PPARγ receptor is meant herein any compound other than an anti-PPARγ antibody capable of binding specifically to the PPARγ receptor.

Among these agonist or antagonist ligands, those chosen from natural agonists of the prostaglandin J2 type, polyunsaturated fatty acids and synthetic agonists of the thiazolinedione type are preferred.

In a preferred embodiment, the method according to the invention is of the “Western blot” type and is characterized in that, before step b):

- the total proteins are extracted from the sample taken; then - separated and transferred to membrane (Western blot); and in that in step c), the signal obtained in step b) is quantified.

The separation will be carried out in particular by electrophoresis in acrylamide gel (PAGE), the transfer being carried out on membranes well known to those skilled in the art such as, in particular, nitrocellulose or PNDF membranes. In a particularly preferred embodiment, the method according to the invention is of the “immunohistochemistry” type and is characterized in that prior to step b):

- a histological section is made of the sample taken, said sample having been, if necessary, fixed in a solution of paraformaldehyde or formaldehyde (at about 4%) and included in paraffin; and in that in step c), the signal obtained in step b) is displayed. The samples taken having been previously fixed in a solution of paraformaldehyde or formaldehyde and included in paraffin, may have been stored for several days or less at 4 ° C or in frozen form. In another aspect, the method according to the invention is characterized in that said expression product of said PPARγ gene is the ARΝm transcription product of said PPARγ gene and in that the compound capable of binding specifically to said expression product of PPARγ gene in step b) is chosen from nucleic acid sequences, where appropriate labeled, capable of hybridizing specifically (under conditions of high stringency) with a fragment of said ARΝm. Among the methods making it possible to detect (or locate) and / or quantify the mRNA of the PPARγ gene, the so-called in situ hybridization method well known to those skilled in the art is preferred.

The nucleic acid sequences, where appropriate labeled, capable of hybridizing specifically (under conditions of high stringency) with a fragment of said mRNA will be probes, preferably labeled, or oligonucleotide primers.

Said probes or primers can for example be drawn from the sequence SEQ ID No. 1 as described in application US 5,861,274 (cDNA coding for the PPARγ receptor). Specific hybridization means that the hybridization is carried out under conditions of high stringency, in particular under conditions of temperature and ionic strength such that they allow hybridization to be maintained between two globally complementary DNA fragments.

By way of illustration, high stringency conditions of the hybridization step for the purpose of defining the nucleotide sequences described above are advantageously as follows.

Hybridization is carried out at a preferred temperature of 65 ° C., in the presence of a 6 × SSC buffer, 5 × Denhardt's solution, 0.5% SDS and 100 μg / ml of DNA from salmon sperm. 1 x SSC corresponds to 0.15 M NaCl and 0.05 M sodium citrate and a solution of 1 x Denhardt corresponds to 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin.

The washing steps can, for example, be carried out for 5 to 30 min. at 65 ° C, in 2 x SSC or 1 x SSC buffer and 0.1% SDS. The high stringency hybridization conditions described above for a polynucleotide of defined size will be adapted by those skilled in the art for oligonucleotides of larger or smaller size, according to the teaching of Sambrook, J., et al., Molecular cloning, A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. The oligonucleotide probes or primers, and in general the nucleic acid sequences according to the invention, will have a minimum size of 15 bases and fragments of at least minus 20, 25, 30 or 50 bases. The techniques making it possible to quantify and / or localize a nucleic sequence and which can be used in the methods of the invention are well known to those skilled in the art and will not be developed exhaustively here. Mention may be made, without being limited to the techniques described below. In general, the detection, localization (distribution) and / or specific assay of the mRNA of the PPARγ gene in the biological sample will include the following steps for in situ hybridization (steps b) and c) of the methods according to the invention. 'invention):

- bringing an oligonucleotide probe, if appropriate labeled, into contact with the biological sample, the mRNA contained in the biological sample having, where appropriate, previously been made accessible for hybridization, under conditions allowing hybridization of the probe to the mRNA contained in the biological sample;

- detection, localization and / or assay of the hybrid formed between the oligonucleotide probe and the mRNA of the biological sample.

In the case where the PPARγ gene mRNA is quantified from an extract of total RNA from the sample taken, optionally enriched in mRNA, in general, the quantification of the mRNA of the PPARγ gene in the prepared extract comprises the following steps of the so-called quantitative RT-PCR method (steps b) and c) of the methods according to the invention):

- obtaining a cDNA from the mRNA of the PPARγ gene using specific primers and a reverse transcriptase;

- the specific amplification of the PPARγ cDNA obtained by a method called PCR (polymerase chain reaction) or related PCR using a specific pair of primers;

- quantitative analysis of amplification products. Another method can also be cited for carrying out the quantification of the mRNA of the PPARγ gene from a total RNA extract from the sample taken, optionally enriched in mRNA, said method comprising the following steps (steps b ) and c) methods according to the invention):

- bringing a first oligonucleotide probe specific for the mRNA of the PPARγ gene or, where appropriate, its amplification products, immobilized on a support with an extract of the total RNA from the sample, into contact, or , if necessary, with the PCR products of the mRNA of the PPARγ gene obtained using primers specific, under conditions allowing hybridization of the first probe to the RNA of said sample or, where appropriate, to said PCR products;

contacting the hybrid formed between said first probe immobilized on a support and said RNAs or, where appropriate, said PCR products, where appropriate after elimination of the nucleic acids which have not hybridized with the first probe, with a second oligonucleotide probe, in particular labeled capable of hybridizing to the mRNA of the PPARγ gene or, where appropriate, to its PCR products, then elimination of the second probes which have not hybridized; and

- quantitative analysis of the triplexes thus formed. The term “related PCR” will be understood to denote all the methods using direct or indirect reproductions of the nucleic acid sequences, or else in which the labeling systems have been amplified. These techniques are of course known. In general, it is the amplification of DNA by a polymerase; when the original sample is an RNA, reverse transcription should be carried out beforehand. There are currently many methods for this amplification, for example the so-called NASBA “Nucleic Acid Sequence Based Amplification”, TAS “Transcription based Amplification System”, LCR “Ligase Chain Reaction”, “Endo Run Amplification” (ERA), “ Cycling Probe Reaction ”(CPR), and SDA“ Strand Displacement Amplification ”well known to those skilled in the art. Also known to those skilled in the art, the nucleic acid quantification techniques in which, for example, the nucleic acid to be quantified is amplified by a PCR type method and in the presence of a standard nucleic acid of the same size and quantity. known and capable of hybridizing to the same primers as the target nucleic acid. The invention also comprises a method according to the invention, characterized in that the quantification or visualization (the image) of the signal obtained in step b) is compared, and where appropriate, the distribution of the product. expression of said PPARγ gene between said intestinal epithelium and lamina propria cells on the patient sample with those obtained for a control (or control) sample. In a preferred embodiment, said control sample is a sample of intestinal tissue taken from a healthy patient or suffering from an inflammatory disease known bowel disease, preferably chosen from Crohn's disease (CD) or ulcerative colitis (UC).

The present invention very particularly and very preferably relates to an in vitro method of diagnosis by immunohistochemistry of ulcerative colitis (UC) in a patient likely to have or progress to an inflammatory bowel disease from a histological section of a sample of intestinal tissue previously taken from this patient, said sample comprising at least cells of the intestinal epithelium, characterized in that it comprises the following step: a) contact of said histological section with a compound capable of specifically binding to the translation product of the PPARγ gene, under conditions allowing the formation of a complex between said compound and said translation product, said compound being labeled or capable of being labeled in order to obtain a marking or a signal representative of the quantity of said translation product present in the sample and, l e if necessary, making it possible to locate said translation product of the PPARγ gene; and b) quantifying or visualizing the intensity of the marking or of the signal obtained in step a) at the level of the cells of the intestinal epithelium.

The inventors have indeed demonstrated in a completely surprising manner, that it was possible to diagnose with very high sensitivity and a specificity close to 100% a ulcerative colitis (UC) in a patient by immunohistochemistry carried out from a histological section of a sample of intestinal tissue taken from this patient, this sample comprising at least cells of the intestinal epithelium, this in particular with respect to the sensitivity and / or the specificity obtained by a diagnostic test carried out by quantitative PCR on the transcription product of the PPARγ gene or by a diagnostic test carried out by Western Blot on the translation product of the PPARγ gene from a sample where the cells of the intestinal epithelium have not been correctly isolated. Indeed, the inventors have been able to demonstrate for the first time that only the observation of the expression deficit of the translation product of the PPARγ gene in the cells of the intestinal epithelium made it possible to carry out such a specific and also sensitive diagnosis. Such a diagnostic test carried out by immunohistochemistry can be easily exploited commercially, little expensive, this compared to a test carried out by quantitative PCR or Western Blot where it will be necessary to isolate the cells of the intestinal epithelium from the sample taken to avoid any contamination with neighboring non-epithelial cells in the sample. Indeed, any presence of non-epithelial cells during the total extraction of the mRNA coding for PPARγ (for the PCR test) or during the extraction of the total proteins (for the Western Blot test) will likely increase the quantity of a transcripton or translation product of the PPARγ gene. Such prior isolation of the cells of the intestinal epithelium from the sample taken in an attempt to carry out a test by quantitative PCR or by Western blot making it possible to diagnose ulcerative colitis (UC) in a patient with the sensitivity and specificity that l 'can be obtained by immunohistochemistry is difficult to perform and can be considered commercially.

In an even more preferred embodiment, the invention relates to an in vitro method of diagnosis by immunohistochemistry of ulcerative colitis (UC) according to the present invention, characterized in that the quantification or the visualization of the signal intensity obtained at l step a) is compared to that obtained for a histological section obtained from a sample of a healthy patient.

In a more preferred embodiment, the invention relates to an in vitro method of diagnosis by immunohistochemistry of ulcerative colitis (UC) according to the present invention, characterized in that in step b), the case is observed if necessary, a reduction in the intensity of the marking or of the signal obtained in step a) in the cells of the intestinal epithelium originating from the sample of the patient likely to have one or to evolve towards a inflammatory bowel disease compared to those from a healthy patient sample. In a more preferred embodiment, the invention relates to an in vitro method of diagnosis by immunohistochemistry of ulcerative colitis (UC) according to the present invention, characterized in that said sample is a biopsy of intestinal tissue taken from the colon or the patient's small intestine.

In a more preferred embodiment, the invention relates to an in vitro method of diagnosis by immunohistochemistry of ulcerative colitis (UC) according to the present invention, characterized in that said sample is a biopsy of intestinal tissue taken from the colon of the patient. In a still more preferred embodiment, the invention relates to an in vitro method of diagnosis by immunohistochemistry of ulcerative colitis (UC according to the present invention, characterized in that said sample of the patient likely to have one or more '' progressing to inflammatory bowel disease is a sample of intestinal tissue taken from the patient's healthy or injured mucosa.

In a still more preferred embodiment, the invention relates to an in vitro method of diagnosis by immunohistochemistry of ulcerative colitis

(RCH) according to the present invention, characterized in that said compound capable of binding specifically to said translation product of the PPARγ gene in step a) is chosen from the following compounds:

- a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor; and

- a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor.

In another aspect, the invention relates to a diagnostic composition, characterized in that it comprises a monoclonal or polyclonal antibody, optionally labeled, directed against the PPARγ receptor.

The invention further relates to a diagnostic composition, characterized in that it comprises a natural or synthetic agonist or antagonist ligand, optionally labeled, of the PPARγ receptor, said ligands preferably being chosen from those mentioned above.

The invention further relates to a diagnostic composition, characterized in that it comprises a nucleic sequence, optionally labeled, capable of hybridizing specifically with a fragment of the mRNA or of the cDNA coding for the PPARγ receptor, preferably comprising at least 15, 20, 25, 30, 50 or 75 bases.

In another aspect, the subject of the present invention is the use of a compound chosen from:

- a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor;

- a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor; or a nucleic sequence, if appropriate labeled, capable of hybridizing specifically with a fragment of the mRNA or of the cDNA coding for the PPARγ receptor, for the preparation of a composition intended for the diagnosis or prognosis of inflammatory disease of the gut in a patient.

Preferably, the use according to the invention is characterized in that said inflammatory bowel disease is a chronic disease, in particular Crohn's disease (CD) or ulcerative colitis (UC).

Also preferably, the use according to the invention is characterized in that said inflammatory bowel disease is an acute disease of the intestine in a patient.

In another even more preferred aspect, the present invention relates to the use of a compound chosen from:

- a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor; or

a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor, for the preparation of a composition intended for the diagnosis or prognosis of ulcerative colitis (UC). The kits or kits for the diagnosis of inflammatory bowel disease, especially chronic disease such as Crohn's disease (CD) or ulcerative colitis (UC) are advantageously part of the invention.

The present invention thus relates in a last aspect to a kit or necessary for the diagnosis or prognosis of inflammatory bowel disease in a patient, characterized in that it comprises: a) at least one of the compounds chosen from following compounds:

- a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor;

- a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor; or

- a nucleic sequence, where appropriate labeled, capable of hybridizing specifically with a fragment of the ΑRNm coding for the PPARγ receptor; b) where appropriate, the reagents for constituting the medium conducive to the formation of a complex between said compound as defined in a) and an expression product of the PPARγ gene; c) where appropriate, the reagents for the quantification or visualization of complexes possibly formed between said compound as defined in a) and an expression product of the PPARγ gene; d) if appropriate, a control sample, preferably an extract of total proteins from a sample of intestinal tissue or a section of intestinal tissue taken from a healthy patient and / or from a patient suffering from an inflammatory disease of the intestine of known type.

Finally, the present invention relates preferably to a kit or kit necessary for the diagnosis by immunohistochemistry of ulcerative colitis (UC) in a patient likely to have or progress to an inflammatory bowel disease, characterized in that it comprises: a) at least one of the compounds chosen from the following compounds:

- a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor; or

- a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor; and b) where appropriate, the reagents for constituting the medium suitable for the formation of a complex between said compound as defined in a) and the translation product of the PPARγ gene; c) where appropriate, the reagents allowing the quantification or visualization of complexes possibly formed between said compound as defined in a) and the translation product of the PPARγ gene; d) where appropriate, a control histological section of intestinal tissue removed from a healthy patient and / or from a patient suffering from an inflammatory bowel disease of known type, on which which histological control sections, if any, the immunohistochemistry labeling of the translation product of the PPARγ gene has been carried out beforehand; e) if applicable, an explanatory note showing images of histological sections obtained after immunohistochemical labeling of the gene translation product PPARγ for histological sections of intestinal tissue from samples of healthy patients, patients with ulcerative colitis and / or patients with Crohn's disease.

The kits or kits necessary for the diagnosis of inflammatory bowel disease according to the present invention may also include an explanatory notice indicating to the user the levels and / or distribution of expression products of the PPARγ gene expected for samples of samples from a healthy patient suffering from inflammatory bowel disease, in particular chronic disease such as Crohn's disease (CD) or ulcerative colitis (UC), as indicated in FIGS. 1A, 1B, 2A and 2B below.

Other characteristics and advantages of the invention appear in the following description with the examples and the figures, the legends of which are shown below.

Caption of Figures Figures 1 A and 1B

FIG. 1A represents a Western blot produced for a protein extract obtained from a biopsy of a healthy patient (control, three representative patients), of a patient suffering from Crohn's disease (noted CD or MC, three representative patients) or suffering from ulcerative colitis hemorrhagic (noted UC or RHC, three representative patients). This Western blot highlights a decrease in the intensity of the band corresponding to the PPARγ protein (by approximately 53 kDa) for patients with Crohn's disease compared to the healthy control, a decrease which is even more accentuated for the patients. with ulcerative colitis.

FIG. 1B represents for each sample tested (18 for the samples of healthy patient (control), 18 for the samples of patient suffering from CD and 15 for the samples of patient suffering from UC), the quantity of protein PPARγ expressed, quantity expressed in optical density for 50 μg of total protein. Figures 2A. 2B and 2C FIGS. 2A to 2C represent a histological section resulting from an intestinal biopsy performed at the level of the colon in which the PPARγ receptors have been immunolabeled.

FIG. 2A represents the histological section of a healthy patient (control), FIG. 2B, that of a patient suffering from CD and FIG. 2C, that of a patient suffering from UC. In these figures, we can observe in particular:

- Figure 2A: a high density of labeling on epithelial cells for the control sample with a low density of labeling for cells of the lamina propria; - Figure 2B: a high density of labeling on epithelial cells, with a decrease in the intensity of labeling, as well as a high density of labeling on cells of the lamina propria; and

- Figure 2C: an absence of labeling (or very weak) on the epithelial cells and on the cells of the lamina propria.

EXAMPLES Patients

All the patients analyzed gave their consent for this study, the conditions of which were approved by the local ethics committees. The diagnosis of Crohn's disease and ulcerative colitis was established according to the usual criteria (Rutgeerts, P., et al., Gastroenterology, 99: 956-963, 1990). Example 1 Quantification of PPARγ in the Colon by Western Blot

Total protein extracts are obtained by homogenization of colon biopsies in a lysis buffer consisting of PBS (phosphate buffer in saline solution) with 1% NP-40 (™ Nonidet P40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and a classic cocktail of protease inhibitors (Fajast, L., et al., J. Biol. Chem., 272: 18779-18789, 1997).

Separation of total proteins (50 μg), transfer to PNDF membrane

(polyvinylidene difluoride) (cf. FIG. 1A) and the immunodetection of PPARγ by incubation of the membrane with a polyclonal rabbit antiserum for 12 hours

(dilution 1/500, TEBU, Le Perray en Yvelines, France) were carried out as previously described (Lefebvre, et al., 1999). The complex is revealed by chemiluminescence (ECL, Amersham, UK), as indicated in the supplier's protocol. The results are expressed in optical density unit (OD) for 50 μg of total proteins (cf. FIG. 1B). Example 2: Immunolabelling of PPARγ in the colon The colon biopsies are fixed in 4% paraformaldehyde, included in the paraffin and cut to 4 micrometers for immunohistochemistry and immunofluorescence.

The sections are preincubated for 30 minutes at room temperature in a blocking solution containing Pavidine D and biotin (™ Blocking Kit, SP2001, Nector Laboratories, Burlingame, CA, USA). They are then exposed to a polyclonal rabbit antiserum directed against PPARγ (dilution 1/50, WAK-CHEMIE, Bad Soden, Germany) for two hours at room temperature. The sections are washed in PBS containing 0.05% of Triton X-100 ™ and incubated with a secondary goat anti-rabbit antibody biotinylated (dilution 1 / 500th for 30 minutes, Dako, Trappes, France). The immune complex is detected by avidin-biotin coupled with peroxidase

(™ ABCOMPLEX / HRP, Dako, Trappes, France) and revealed using 3,3'-diaminobenzidine (DAB, Dako, Trappes, France). As a negative control, the primary antibody is replaced by an irrelevant rabbit serum. Statistical methods Comparisons of the mean ± ESM of the amounts of mRNA and of the PPARγ receptor protein between patients with UC, CD and control patients were analyzed by the non-parametric test ANONA by Kruskal-Wallis. The differences are judged statistically different for a p <0.05. Example 3: Results By Western blotting techniques (cf. FIG. 1A) and immunohistochemistry (cf. FIGS. 2A to 2C), it was demonstrated, in the intestinal epithelial cells, for the first time a deficit of expression of the receptor activated by PPARγ peroxisome proliferators in healthy or injured colonic mucosa, especially in patients with UC, and to a lesser extent in patients with CD (visible in the intensity of labeling in immunohistochemistry ). This expression deficit is thus easily demonstrated in patients with UC (cf. FIGS. 1B and 2C). This deficit for CD also appears significant and can be put also in evidence by observing the intensity of immunostaining in immunohistochemistry (cf. FIG. 2B).

Concerning CD, the immunohistochemistry technique highlights a gain in expression of the PPARγ receptor at the level of the lamina propria cells, while the expression at the level of these cells does not appear or only very weakly for the CHR. or for healthy patients.

Concerning the cells of the lamina propria, one can also note a deficit of expression for the patients suffering from UC compared to the healthy controls.

The detection of PPARγ in the colonic mucosa is a new marker enabling rapid diagnosis of UC and / or CD. This detection is possible by performing biopsies made in healthy or injured mucosa during an endoscopic examination which can be limited to the exploration of the first 20 cm of the colon at the level of the rectum and sigmoid.

The tests carried out on all of the samples analyzed show that the sensitivity and specificity is 100% in MC and RCH.

The techniques used are simple and can be performed routinely by all pathology laboratories.

Among the other advantages of the method according to the invention, there may be mentioned in particular the fact that: - biopsies can be performed both in healthy mucosa and injured mucosa, during periods of quiescence or disease outbreak; and

- rectal biopsies only require a short endoscopy, without general anesthesia, without colic preparation.

The invention also relates to a diagnostic device comprising means suitable for implementing the various steps of the method described above.

Claims

1. In vitro method for determining the quantity of expression product of the gene coding for PPARγ (PPARγ gene) on a sample of intestinal tissue, said sample comprising at least cells of the intestinal epithelium and or lamina propria , characterized in that it comprises the following stages: a) the taking of a sample of intestinal tissue from a patient likely to have one or progressing to an inflammatory bowel disease, said taking comprising at least intestinal epithelium and / or lamina propria cells; b) bringing the expression product of the PPARγ gene into contact with a compound capable of binding specifically to said expression product of the PPARγ gene, under conditions allowing the formation of a complex between said compound and said expression product , said compound being labeled or capable of being labeled in order to obtain a signal representative of the quantity of said expression product present in the sample and, if necessary, making it possible to locate said expression product; and c) the quantification or display of the signal obtained in step b). 2. Method according to claim 1, characterized in that said sample of intestinal tissue comprises at least cells of the intestinal epithelium and lamina propria, and in that it further implements a step of comparing the distribution of the expression product of said PPARγ gene between said intestinal epithelium and lamina propria cells on the patient's sample. 3. Method according to claim 1, characterized in that said sample is a biopsy of intestinal tissue taken from the patient's colon or small intestine. 4. Method according to claim 1, characterized in that said sample is a biopsy of intestinal tissue taken from the patient's colon. 5. Method according to claim 1, characterized in that said expression product of said PPARγ gene is the translation product of said PPARγ gene, and in that the compound capable of binding specifically to said expression product of PPARγ gene at l step b) is chosen from the following compounds: - a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor; and - a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor. 6. Method according to claim 5, characterized in that prior to step b): - the total proteins are extracted from the sample taken; then - separated and transferred to membrane (Western blot); and in that in step c), the signal obtained in step b) is quantified. 7. Method according to claim 5, characterized in that before step b): - A histological section is made of the sample taken, said sample having been, if necessary, fixed in a solution of paraformaldehyde or formaldehyde and included in parafinne; and in that in step c), the signal obtained in step b) is displayed. 8. Method according to claim 1, characterized in that said expression product of said PPARγ gene is the mRNA transcription product of said PPARγ gene, and in that the compound capable of binding specifically to said expression product of the PPARγ gene to step b) is chosen from nucleic acid sequences, where appropriate labeled, capable of specifically hybridizing with a fragment of said mRNA. 9. Method according to one of claims 1 to 8, characterized in that the quantification or visualization of the signal obtained in step b) is compared, and where appropriate, the distribution of the expression product of said gene. PPARγ between said intestinal epithelium and lamina propria cells on the patient's sample with those obtained for a control sample. 10. The method of claim 9, characterized in that said control sample is a sample of intestinal tissue taken from a healthy patient or suffering from a chronic inflammatory bowel disease, preferably chosen from Crohn's disease (CD) or ulcerative colitis (UC). 11. In vitro method of diagnosis by immunohistochemistry of ulcerative colitis (UC) in a patient likely to have or progress to inflammatory bowel disease from a histological section of a sample of intestinal tissue previously taken from this patient, said sample comprising at least cells of the intestinal epithelium, characterized in that it comprises the step following: a) bringing said histological section into contact with a compound capable of specifically binding to the translation product of the PPARγ gene, under conditions allowing the formation of a complex between said compound and said translation product, said compound being marked or capable of being marked in order to obtain a marking or a signal representative of the quantity of said translation product present in the sample and, where appropriate, making it possible to locate said translation product of the PPARγ gene; and b) quantifying or visualizing the intensity of the marking or of the signal obtained in step a) at the level of the cells of the intestinal epithelium. 12. In vitro diagnostic method by immunohistochemistry of ulcerative colitis (UC) according to claim 11, characterized in that the quantification or visualization of the signal intensity obtained in step a) is compared to that obtained for a histological section. from a healthy patient sample. 13. In vitro method of diagnosis by immunohistochemistry of ulcerative colitis (UC) according to claim 11 or 12, characterized in that in step b), a reduction in the intensity of the labeling is observed, where appropriate. of the signal obtained in step a) at the level of the cells of the intestinal epithelium originating from the sample of the patient likely to have one or to evolve towards an inflammatory disease of the intestine compared with those originating from 'a healthy patient sample. 14. Method according to one of claims 11 to 13, characterized in that said sample is a biopsy of intestinal tissue taken from the colon or small intestine of the patient. 15. The method of claim 14, characterized in that said sample is a biopsy of intestinal tissue taken from the patient's colon. 16. Method according to one of claims 11 to 15, characterized in that said sample of the patient likely to have one or progress to an inflammatory bowel disease is a sample of intestinal tissue taken from healthy or injured mucosa of the patient. 17. Method according to one of claims 11 to 16, characterized in that said compound capable of specifically binding to said translation product of the PPARγ gene in step a) is chosen from the following compounds: - a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor; and - a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor. 18. Diagnostic composition, characterized in that it comprises a monoclonal or polyclonal antibody, where appropriate labeled, directed against the PPARγ receptor. 19. Diagnostic composition, characterized in that it comprises an agonist or antagonist ligand, natural or synthetic, if appropriate labeled, of the PPARγ receptor. 20. Diagnostic composition, characterized in that it comprises a nucleic sequence, where appropriate labeled, capable of hybridizing specifically with a fragment of the mRNA coding for the PPARγ receptor. 21. Use of a compound chosen from: - a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor; - a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor; or a nucleic sequence, if appropriate labeled, capable of hybridizing specifically with a fragment of the mRNA coding for the PPARγ receptor, for the preparation of a composition intended for the diagnosis or prognosis of inflammatory bowel disease in a patient. 22. Use of a compound according to claim 21, characterized in that said inflammatory bowel disease is a chronic disease. 23. Use of a compound according to claim 22, characterized in that said inflammatory bowel disease is Crohn's disease (CD) or ulcerative colitis (UC). 24. Use of a compound according to claim 21, characterized in that said inflammatory bowel disease is an acute disease of the intestine in a patient. 25. Use of a compound chosen from: - a monoclonal or polyclonal antibody, where appropriate labeled, directed against the PPARγ receptor; or a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor, for the preparation of a composition intended for the diagnosis or prognosis of ulcerative colitis (UC). in a patient. 26. Kit or kit for the diagnosis or prognosis of inflammatory bowel disease in a patient characterized in that it comprises: a) at least one of the compounds chosen from the following compounds: - a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor; - a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor, or - a nucleic sequence, if necessary labeled, capable of hybridizing specifically with a fragment of the mRNA coding for the PPARγ receptor; b) where appropriate, the reagents for constituting the medium conducive to the formation of a complex between said compound as defined in a) and an expression product of the PPARγ gene; c) where appropriate, the reagents allowing the quantification or visualization of complexes possibly formed between said compound as defined in a) and an expression product of the PPARγ gene; d) if appropriate, a control sample, preferably an extract of total proteins from a sample of intestinal tissue or a section of intestinal tissue taken from a healthy patient and / or from a patient suffering from an inflammatory disease of the intestine of known type. 27. Kit or kit for the diagnosis by immunohistochemistry of ulcerative colitis (UC) in a patient likely to have or progress to an inflammatory bowel disease, characterized in that it comprises: a) at least one of the compounds chosen from the following compounds: - a monoclonal or polyclonal antibody, if appropriate labeled, directed against the PPARγ receptor; or - a natural or synthetic agonist or antagonist ligand, if appropriate labeled, of the PPARγ receptor; and b) where appropriate, the reagents for constituting the medium suitable for the formation of a complex between said compound as defined in a) and the translation product of the PPARγ gene; c) where appropriate, the reagents allowing the quantification or visualization of complexes possibly formed between said compound as defined in a) and the translation product of the PPARγ gene; d) where appropriate, a control histological section of intestinal tissue removed from a healthy patient and / or from a patient suffering from an inflammatory bowel disease of known type, on which or which control histological sections, labeling by immunohistochemistry translation product of the PPARγ gene was previously produced; e) if applicable, an explanatory note showing images of histological sections obtained after immunohistochemical labeling of the translation product of the PPARγ gene for histological sections of intestinal tissue obtained from samples of healthy patient, of patient suffering from ulcerative colitis and / or patient with Crohn's disease.