BRADYKININ B1 RECEPTOR AS MARKER FOR INFLAMMATORY DISEASES

Abstract: The present invention relates to a marker for inflammatory diseases, a tool to measure inflammatory disease activity and to a drug screening assay of therapeutical agents for the treatment of inflammatory diseases. The marker for inflammatory diseases includes bradykinin B1 receptor. The invention also relates to a method for the diagnosis of inflammatory diseases in a patient, which comprises determining the presence of bradykinin B1 receptor in a biological sample of said patient, wherein the presence of bradykinin B1 receptor is indicative of an inflammatory disease.

Description: BRADYKININ Bl RECEPTOR AS MARKER FOR INFLAMMATORY DISEASES BACKGROUND OF THE INVENTION (a) Field of the Invention The invention relates to the use of the bradykinin Bl receptor as a marker for inflammatory diseases, to a diagnosis to measure inflammatory disease activity and to a drug screening assay of therapeutical agents for the treatment of inflammatory diseases, (b) Description of Prior Art Inflammatory diseases involving target tissue injury require migration of inflammatory cells from the blood into the target tissue. This process requires interaction of the inflammatory cell with cells forming the vascular barrier. Such interaction results in changes in vascular permeability, inflammatory cells - endothelial cell interaction and movement of cells and fluid into tissues. Multiple sclerosis (MS) is an inflammatory demy- elinating disorder of the central nervous system (CNS) most often characterized by an initial pattern of cyclic exacerbations and remissions, followed by evolution into a chronic progressive course (Antel, J.P., Feb. 1995, Neurology Cl ini cs ; Mul tipl e Scl erosi s , W.B. Saunders Company Ed., 13:1). In parallel with observations made in the animal model, the experimental allergic encephalomyelitis (EAE) , migration of systemic T cells into the CNS is regarded to initiate the inflammatory lesions. EAE based studies further demonstrated that both antigen specific and non specific T cells can access the CNS with extent of such trafficking being dependent on the activation state of the T cells (Antel, J.P., Feb. 1995, Neurology Clini cs ; Mul tipl e Scl erosi s , W.B. Saunders Company Ed., 13:1). The mechanism by which systemic T cells infiltrate the CNS during MS relapses is not entirely understood but production of matrix metallo proteinase (MMP) by these cells contribute to this process. It was previously demonstrated that immediately ex vivo T lymphocytes obtained from MS patients show increased migration across membranes coated with fibronectin (FN) , a major component of the basal lamina that surrounds brain endothelium. Such migration could be inhibited with a tissue inhibitor of metallo proteinase (TIMP) . It was also demonstrated that MS patients treated with IFN-β, a current medication used for the treatment of MS, show a significantly reduced migration compared to untreated patients in this assay system. Signaling through neuropeptide receptors expressed on the surface of immune cells provide an additional mean whereby lymphocyte properties, including migration, can be modulated as demonstrated by studies using substance P, vasoactive intestinal poly- peptide (VIP) and calcitonin gene related peptide. VIP stimulated PB lymphocytes are shown to secrete MMP and to degrade FN in vi tro . The blood derived kinin family, of which the nonapeptide bradykinin (BK) is probably the best known member, are implicated as modulators of the inflammatory response (Bhoula, K.D. et al . , 1992, Pharmacol . Rev. , 44:1-92). Kinins injection increases vascular permeability, induces vascular smooth muscle cell contraction, and synthesis of eicosanoids and IL-lβ by fibroblasts and endothelial cells (Hall, J.M., 1992, Pharmac . Ther. , 56:131-190; Marceau, F., 1995, Immuno- pharmacol . , 30:1-26) . Kinins may exert their action by two distinct receptors : Bι_ and B2 receptors. The B2 receptor which is sensitive to BK and kallidin, is con- stitutively expressed in multiple cell types. The B^ receptor is not expressed under normal physiological circumstances but is up-regulated following various types of tissue injury. The B Q _ receptor can be up-regulated on smooth muscle cells by PS , oncostatin M and IFN-γ (4) . Induction of Bl receptor expression is partially regulated by NF-kb signaling The Bl receptor is selectively sensitive to the natural peptides desArg -BK (4) and desArg -kallidin generated in vivo after the cleavage of BK and Kallidin by kallikreins, and to the synthetic peptide Sar (dPhe 8 ) desArg 9 -BK. The functional role of the Bl receptor on different tissue type remains to be define. Although a thorough neurological evaluation combined to Magnetic Resonance Imaging of the CNS is the keystone in the diagnosis of MS, it would be important to develop a blood test to evaluate disease activity. This tool, used to measure inflammatory disease activity, could also provide a drug screening assay for therapeutical agents in MS and in any other tissue tar- geted inflammatory disease. It would be highly desirable to be provided with a marker for inflammatory diseases. " It would be highly desirable to be provided with a tool to measure inflammatory disease activity. It would be highly desirable to be provided with a drug screening assay of therapeutical agents for the treatment of inflammatory diseases. SUMMARY OF THE INVENTION One aim of the present invention is to provide a marker for inflammatory diseases. Another aim of the present invention is to provide a tool to measure inflammatory disease activity. Another aim of the present invention is to provide for a drug screening assay of therapeutical agents for the treatment of inflammatory diseases. In accordance with the present invention there is provided a marker for inflammatory diseases, which comprises bradykinin Bl receptor. In accordance with the present invention there is also provided a method for the diagnosis of inflammatory diseases in a patient, which comprises determin- ing the presence of bradykinin Bl receptor in a biological sample of the patient, wherein the presence of bradykinin Bl receptor is indicative of an inflammatory disease . In accordance with the present invention there is also provided a method to measure activity of inflammatory diseases in a patient, which comprises determining the level of bradykinin Bl receptor in a biological sample of the patient, wherein a level of bradykinin Bl receptor greater than two (2) standard deviation over a control sample level is indicative of an active inflammatory disease, and wherein the control sample is from non- inflammatory disease healthy donors. In accordance with the present invention there is also provided a method for the screening of therapeutical agents for the treatment of inflammatory diseases, which comprises the step of: a) evaluating the effect of therapeutical agents on bradykinin Bl receptor mRNA synthesis in a human cell or cell line, wherein a reduced Bl receptor mRNA synthesis is indicative an anti- inflammatory disease potent therapeutical agent. The evaluation may be effected using Northern blot, PCR or Rnase protection assay. In accordance with the present invention there is also provided a method for the screening of therapeutical agents for the treatment of inflammatory diseases, which comprises the step of: a) evaluating the effect of therapeutical agents on bradykinin Bl receptor protein expression in a human cell or cell line, wherein a reduced Bl receptor protein expression is indicative an anti- inflammatory disease potent therapeutical agen . The evaluation may be effected using Western blot, immunochernistry, flow cytometry, radioligand- binding assay. Migration of systemic T cells into the central nervous system is an initial event in the course of lesion development in multiple sclerosis (MS) . The recently cloned bradykinin Bl receptor is a membrane bound G coupled receptor shown to be up-regulated on the surface of endothelial cells and smooth muscle cells in various models of inflammation in vi tro and in vivo . In accordance with the present invention, levels of Bl receptor mRNA on peripheral blood derived T lymphocytes of MS patients were compared to controls. The receptor was up-regulated on T cells from patients with secondary progressive (SP) MS and relapsing remitting (RR) patients in active relapse. Expression was lower in RR patients in remission and not detectable in controls. In vi tro treatment of T cells from healthy controls with TNF-α and IFN-γ can also induce the expression of Bl receptors. Using a modified Boyden chamber assay we further found that the significantly higher rate of migration of MS T lymphocytes compared to controls, can be prevented by the addition of the selec- tive Bl agonist Sar- (dPhe 8) -desArg9-Bradykinin. Pre- incubation of the cells with the selective human Bl receptor antagonist R-715 abolished the anti-migratory effect of the Bl agonist. These results indicate how interactions between a neuropeptide and the immune system can regulate lymphocyte migration, an important component in the immunopathogenesis of MS. For the purpose of the present invention the following terms are defined below. The term "inflammatory disease" is intended to mean any tissue or organ targeted inflammatory disorder, including, without limitation multiple sclerosis (MS) , rheumatoid arthritis (RA) , osteoarthritis (OA) , Crohn ' s disease (CD) , ulcerative colitis (UC) or lupus erythematosum disseminated (LED) . BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1A and IB illustrate expression of bradykinin Bl receptor mRNA in PB T cells obtained from MS SP and healthy controls; Fig. 1C illustrates the quantification of radioactivity incorporation; Figs. 2A to 2C illustrate expression of brady- kinin Bl receptor mRNA in PB T cells obtained from MS AR, MS remitting and healthy controls; Figs. 3A to 3C illustrate induction of Bl receptor mRNA expression in healthy controls PB T cells by TNF-α and IFN-γ; and Fig. 4 illustrates Boyden Chamber Assay: effect of Sar (dPhe 8 ) desArg 9 -BK and R 715 on the total number of T cells that have migrated into the lower chamber. DETAILED DESCRIPTION OF THE INVENTION The overall objectives of the present invention were to determine whether expression of the Bη_ receptor was induced in human T lymphocytes at any time during the course of MS and whether this receptor could be used to modulate functional lymphocytes properties relevant to the MS disease process. To address these aims, we investigated whether the Bl receptor mRNA could be detected in peripheral .blood T lymphocytes from MS patients in different phase of their disease course and whether the migratory ability of T cells through FN could be modulated by selective human Bl receptor agonist and antagonist. METHODS Patient selection MS patients followed at the Montreal Neurological Hospital (Montreal, QC) or at Notre-Dame Hospital (Montreal, QC) MS clinics were selected according to the following criteria: RR patients- presence of at least 2 relapses during the previous 2 years with MRI confirmation of disease. Patients were sub-grouped based on presence (n=12) or absence (n=4) of an acute relapse (AR) within one week of blood collection; SP patients: secondary chronic progression of neurological deficit over the past year in patients with previously relapsing form of disease. None of the patients selected were on immuno-suppressive therapy or anti- inflammatory treatment. 20 healthy controls subjects were also included in this study. The mean age of patients with MS-RR, MS-SP and of healthy controls was 30.5, 37.5 and 30 years. Lymphocyte isolation Mononuclear cells (MNC) were isolated from hepa- rinized PB samples using gradient centrifugation through Ficol Hypaque (Pharmacia) , washed with phosphate buffer saline (PBS) pH 7.4, counted and resus- pended at a density of 10 6 cells/ml in 1640 RPMI containing 10% fetal calf serum (FCS) , 2.5 mg/ml penicillin and streptomycin and 2.5 mM glutamine . Adherent cells were removed by a one hour incubation of the MNC in 75 ml tissue culture dishes (Falcon) at 37°C. Non adherent cells were collected, washed with PBS, counted and resuspended at a density of 10 6 cells/ml in 10% FCS RPMI . Aliquots of those cells were stained with anti- CD3 , anti-CD14 and anti-CD19 monoclonal antibody (mAb) (Becton Dickinson) and analyzed on a Becton Dickinson™ FACScan. Non adherent cell fraction consisted of >95% of T lymphocytes in all MS and control groups. This cell population was subsequently used in the Boyden chamber assay. Highly enriched T cell fractions to be used for mRNA isolation were obtained by para magnetic beads coupled to anti-CD3 monoclonal antibody (mAb) used according to the manufacturer instructions. Briefly, the non adherent cells were incubated on ice for one hour with anti-CD3 mAb bound to paramagnetic beads (Dynal) at a ratio of 5 beads/cell. Cells were then washed in 2% FCS in PBS, collected with a magnet and dissolved in Trizol reagent (GIBCO BRL) for RNA extraction. RNA extraction, cDNA synthesis and multiplex PCR amplification RNA was extracted according to the protocol provided with TRIZOL™ (Gibco) . To transcribe into cDNA, 5 μg of RNA, 3.3 mM hexamer primers (Boehringer Mannheim, Germany) , RT buffer, 10 mM dNTPs, 400 U MMLV-RT (all from GIBCO), 0.6 ml RNA guard and 3mM DTT (Pharmacia) were added to a total volume of 30 ml. The reaction mixture was incubated at 42°C for one hour followed by 10 min at 75°C. G3PDH primers (Clonetech) for the PCR amplification have been designed to span 6 introns (G3PDH 5 'primer TGAAGGTCGGAGTCAACGGATTTGGT (SEQ ID N0:1); G3PDH 3 ' primer CATGTGGGCCATGAGGTCCACCAC (SEQ ID NO: 2) ; predicted amplified PCR fragment 983 bp) . Brady- kinin Bl receptor mRNA sequence (accession number #U12512) was used to design 5' and 3' primers using the Primer 3 Output™ software (Bl 5' primer ATCTGGTGTTTGTCTTGGGC (SEQ ID NO : 3 ) ; Bl 3' primer AGGCCAGGATGTGGTAGTTG (SEQ ID NO: 4) ; predicted amplified PCR fragment 435 bp) . 300 ng cDNA was added to a reac- tion mix containing PCR buffer, 4mM dNTPs (GIBCO) , 20 μCi dCTP-a[P 32 ] (NEN Du Pont, Mississauga, ON) , 50 pmol of each primer pair (G3PDH and Bl) and 2.5 U of Taq polymerase (GIBCO) . The reaction mix was completed with distilled water to a volume of 50 ml and placed in a PTC-100™ thermal cycler (MJ Research Inc.) for 25 cycles of 94°C (1 min) , 62°C (35 sec) and 72°C (1 min) . Following amplification, 20 ml of each sample was elec- trophoresed on a 1.5% agarose gel (GIBCO) stained with ethidium bromide. For quantification purposes, agarose gels were dried and analyzed using a PHOSPHORIMAGER™ and ImageQuant™ software (Molecular Dynamic, Sunnyvale, CA) . Boyden Chamber assay To assess lymphocytes migration we used a Boyden chamber assay as previously described. Boyden chambers (Collaborative Biomedical Products, Bedford, MD) consist of two plastic compartments " separated by a polycarbonate membrane (3 mm pore size) precoated with FN. A total of 7xl0 5 T lymphocytes suspended in 2.5% FCS RPMI and treated as described in the results section was added to the upper chamber. The lower chamber was filled with RPMI plus 10% FCS to provide a chemoattrac- tant FCS gradient between the two compartments. Follow- ing incubation for 6 hours at 37°C cells were collected from the lower chamber and counted with a hemocytome- ter . T cell proliferation assay 10 MNC were plated in triplicate in 96 well microtiter culture plate (Falcon) and activated for 72 10 hours with anti-CD3 mAb and TNF-α (100 U/ml) . Sar (dPhe ) desArg -BK was added to some wells for 6 hours. All cultures were then pulsed overnight with mCi of [H3] thymidine and harvested the following day with a SKATRON™ cell harvester. Filters were counted in a BECKMAN™ β counter. RNA was also collected from these cells in order to assess the expression of the Bl receptor gene . Data and statistical analysis In order to compare the level of Bl receptor mRNA expression in MS and non MS T cells, the ratio of Bl receptor phosphorimager unit/G3PDH phosphorimager unit was calculated for each sample. Data are presented as percentage of increased Bl receptor expression compared to controls. An unpaired Student t test was used to evaluate differences between the groups, both in the Boyden chamber assay and in the Bl gene expression experiment . RESULTS Expression of Bl receptor mRNA by T cells of MS patients Figs. 1A and IB indicate that T cells obtained from MS-SP groups express a high level of Bl receptor whereas very low levels of Bl receptor were expressed in T cells of healthy donors. Multiplex PCR amplification of G3PDH and Bl mRNA from 8 MS SP and 4 controls was visualized by Phosphorimaging technique (A) or ethidium bromide staining (B) . The quantification data (B1/G3PDH) obtained on the phosphorimager (Fig. 1C) and mean B1/G3PDH ratio show an increased Bl receptor gene expression of 6.5 times in the MS-SP group compared to healthy controls (p<0.001) (***, P<0.001; n= number of patients or controls tested) . Similar results were obtained from the 11 PCR of mRNA obtained from the MS-AR group (Figs. 2A, 2B and 2C) ; mean mRNA expression was 4.9 times that of controls (p<0.001). Multiplex PCR amplification of G3PDH and Bl mRNA from 3 MS AR, 2 MS remitting and 3 controls was visualized by phosphorimaging (Fig. 2A) or ethidium bromide (Fig. 2B) . Total, radioactivity incorporated in G3PDH and Bl amplimer from a total of 12 MS AR, 20 controls and 4 MS remitting are compared in (Fig. 2C) . (MS AR are compared to controls: * and ***, p<0.05 and p<0.001 respectively; MS AR are compared to MS remitting: ++, p<0.01) . T cells from the MS-remitting group were also positive for Bl receptor gene expression, but quantification data showed that the Bl mRNA was significantly lower in these remitting patients compared to AR. Induction of Bl receptor gene transcription by TNF-α and IFN-γ T cells from healthy donors were used to deter- mine whether the cytokines TNF-α and IFN-γ could induce Bl receptor gene expression. In each of two different experiment, T cells from 3 donors incubated with TNF-α (100 U/ml) or IFN-γ (100 U/ml) for 24 hours were found to show a 5 to 6 fold increase in Bl receptor mRNA com- pared to non treated cells (controls; p<0.001) as assessed by multiplex PCR (Figs. 3A to 3C) . T cells from 3 different controls were cultured for 24 hours in absence or presence of TNFα or IFNγ (100 U/ml) . Multiplex PCR amplification of G3PDH and Bl mRNA was revealed by Phosphorimaging (Fig. 3A) or ethidium bromide staining (Fig. 3B) . Total radioactivity incorporation in Bl and GePDH amplimers was quantified using a phosphor screen in 2 consecutive experiments (n = 6 controls) , and expressed as the mean of numerical val- ues in (Fig. 3C) . (controls are compared to IFNγ or TNFα treated cells: ***, p<0.001). 12 In contrast, MNC's from these donors cultured with anti-CD3 mAb for 72 hours induced T cell proliferation but did not express Bl gene transcription. Effect of Bl receptor agonist on T cell migration As shown in Fig. 4, we found that both SP and AR patient groups had a significantly higher (5 to 6 fold) number of T cells migrated through the FN membrane compared to controls (footnotes: (**) MS AR or SP T cells treated with Sar (dPhe 8 ) desArg 9 -BK compared to untreated cells, p<0.01. (++) MS AR or SP T cells treated with Sar (dPhe 8 ) desArg 9 -BK plus R 715 compared to Sar (dPhe 8 ) desArg 9 -BK alone, p<0.01). The migration of MS-AR and MS-CP T cells was inhibited by 50-60% with the Bl agonist treatment (p<0.01) . The anti -migratory effect of Sar (dPhe ) desArg -BK (10 " and 10 " M) could be prevented by co- incubation of the T cells with the Bl receptor antagonist R-715 (10 "7 M) , showing that the effect of the Bl agonist is more likely to be mediated by a specific interaction between the agonist and the Bl receptor. This Bl receptor antagonist did not modify the migration of MS T cells in absence of Bl agonist thus showing very little intrinsic agonist activity for the human Bl receptor. The net migration of control cells which do not express Bl receptor was not influenced by the Bl agonist Sar (dPhe ) desArg -BK at either 10 "8 or 10 "10 M. Addition of Sar (dPhe 8 ) desArg 9 -BK did not change the rate of proliferation of T cells. In accordance with the present invention human ex vivo T cells were immediately used to assess the expression of kinin Bl receptor mRNA during the course of MS and its role on T cell migration. Bl receptor mRNA in T lymphocytes were quantified using a combina- tion of radioactive PCR and phosphorimaging techniques. The results demonstrate that Bl receptor mRNA is 13 expressed in high levels in T cells obtained from the blood of SP as well as AR MS patients. The expression of Bl receptor by T lymphocytes was however much lower in MS-RR patient with no clinical evidence of disease activity. Furthermore Bl receptor mRNA was virtually absent from the 20 healthy controls. We found that TNF-α and IFN-γ are potent induc- ers of Bl receptor gene expression in freshly isolated human T lymphocytes, while anti-CD3 Ab cross linking of the T cell receptor, which induces T cells proliferation, do not provide a significant increase in Bl receptor mRNA. Levels of TNF-α mRNA in blood monocytes correlate with disease activity in RR MS patients. RR MS patients are also reported to have increased numbers of IFN-γ secreting cells in the blood. Our recent intra- cellular cytokine data indicates no increase in stable patients whereas levels are increased in SP MS. We addressed a potential functional effect of Bl receptor mediated signaling on lymphocytes using an in vitro lymphocytes migration assay. It was previously found that T cells of RR MS patients had an increased migration rate compared to controls. In accordance with the present invention, it is demonstrated that SP and AR groups show increased migration compared to controls and that migration was significantly inhibited by the Bl agonist Sar (dPhe 8 ) desArg 9 -BK. The agonist did not modify the migration of T cells which did not express the Bl receptor. It is showed that this Bl receptor mediated effect is specific because it can be prevented by a treatment with R-715. These functional studies with the Boyden chamber assay provide evidences that MS T lymphocytes expressing Bl receptor m RNA also have Bl binding sites on their surface. Currently no specific antibody to this human Bl receptor is available for studying Bl receptor at the protein level. 14 We demonstrated that Bl receptors are up regulated on the surface of human T lymphocytes during the course of MS and that Bl agonist can negatively regulate T cell migration in vi tro . This effect is unique in the overall concept of kinins being molecules which promotes inflammation. This effect of Bl receptors on PB immune cells may provide a mean to modulate lymphocytes infiltration in tissues, as preventing these cells from entering the CNS. The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope. EXAMPLE I Current inflammatory disease activity is measured clinically or by radio-imaging. The latter indicate that disease activity in the CNS can be ongoing without apparent clinical manifestation. Imaging techniques (MRI) cannot be applied in a regular serial fashion to MS patients in a non research setting because of the price and of the time commitment. A blood test indicating disease activity would not face these limitations. Our test also could be used by pharmaceutical industry as a drug screening assay. We showed that the level of bradykinin Bl receptor mRNA in human T cells correlates with disease activity and can be dose-dependently induced by TNF-α and IFN-γ. T cell lines which can be induced to express bradykinin Bl receptor, could be used for large scale in vi tro screening of drug effect. This could identify drugs with anti-inflammatory properties. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia- 15 tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims .

Claims: 16 The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows : 1. A marker for inflammatory diseases, which comprises bradykinin Bl receptor. 2. A method for the diagnosis of inflammatory diseases in a patient, which comprises determining the presence of bradykinin Bl receptor in a biological sample of said patient, wherein the presence of bradykinin Bl receptor is indicative of an inflammatory disease. 3. The method of claim 2 , wherein the inflammatory disease is selected from the group " consisting of multiple sclerosis (MS) , rheumatoid arthritis (RA) , osteoar- thritis (OA) , Crohn's disease (CD), ulcerative colitis (UC) or lupus erythematosum disseminated (LED) . 4. A method to measure activity of inflammatory diseases in a patient, which comprises determining the level of bradykinin Bl receptor in a biological sample of said patient, wherein a level of bradykinin Bl receptor greater than two (2) standard deviation over a control sample level is indicative of an active inflammatory disease, and wherein said control sample is from non- inflammatory disease healthy donors. 5. The method of claim 4, wherein the inflammatory disease is selected from the group consisting of multiple sclerosis (MS) , rheumatoid arthritis (RA) , osteoar- thritis (OA) , Crohn's disease (CD), ulcerative colitis (UC) or lupus erythematosum disseminated (LED) . 17 6. A method for the screening of therapeutical agents for the treatment of inflammatory diseases, which comprises the step of : a) evaluating the effect of therapeutical agents on bradykinin Bl receptor mRNA synthesis in a human cell or cell line, wherein a reduced Bl receptor mRNA synthesis is indicative an anti -inflammatory disease potent therapeutical agent . 7. The method of claim 6, wherein the evaluation is effected using Northern blot, PCR or Rnase protection assay. 8. The method of claim 6 or 7 , wherein the inflammatory disease is selected from the group consisting of multiple sclerosis (MS) , rheumatoid arthritis (RA) , osteoarthritis (OA) , Crohn's disease (CD), ulcerative colitis (UC) or lupus erythematosum disseminated (LED) . 9. A method for the screening of therapeutical agents for the treatment of inflammatory diseases, which comprises the step of: a) evaluating the effect of therapeutical agents on bradykinin Bl receptor protein expression in a human cell or cell line, wherein a reduced Bl receptor protein expression is indicative an anti-inflammatory disease potent therapeutical agent . 10. The method of claim 9, wherein the evaluation is effected using Western blot, immunochemistry, flow cytometry, radioligand-binding assay. 11. The method of claim 9 or 10, wherein the inflammatory disease is selected from the group consisting of multiple sclerosis (MS) , rheumatoid arthritis (RA) , osteoarthritis (OA) , Crohn's disease (CD), ulcerative colitis (UC) or lupus erythematosum disseminated (LED) .

Inventors: ANTEL JACK P, PRAT ALEXANDRE, ANTEL, JACK, P., PRAT, ALEXANDRE
Applicants: ANTEL JACK P, PRAT ALEXANDRE, UNIV MCGILL, ANTEL, JACK, P., MCGILL UNIVERSITY, PRAT, ALEXANDRE
Application year: 1999
Main classification: G01N33
IPC codes: G01N33