Abstract:
CXCR3 is identified as a physiological receptor for Gliadin. Assays for determining modulators of CXCR3 signaling are provided. Fragments of gliadin which function as inhibitors of CXCR3 signaling can be determined. Methods for treating diseases relating to gluten and/or autoimmunity by targeting CXCR3 are provided. Such diseases include celiac disease, gluten sensitivity, gluten allergy, rheumatoid arthritis, multiple sclerosis, immune-mediated or type 1 diabetes mellitus, inflammatory bowel diseases, systemic lupus erythematosus, psoriasis, scleroderma, and autoimmune thyroid diseases.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. provisional patent application Ser. No. 60/653,118, filed Feb. 16, 2005, and to U.S. provisional patent application Ser. No. 60/741,998, filed Dec. 2, 2005, the contents of both of which are specifically incorporated herein by reference. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
     The subject matter of this application was in part funded by the National Institutes of Health grant no. A118797. The United States Governments has certain rights in the invention. 
    
    
     TECHNICAL FIELD OF THE INVENTION 
     This invention is related to the area of autoimmune diseases. In particular, it relates to treatment and drug screening and discovery for autoimmune diseases. 
     BACKGROUND OF THE INVENTION 
     Environmental stimuli, such as microorganisms and gluten, induce zonulin release in the intestine, brain, heart, and other organs. Zonulin release causes an increase in permeability of epithelia as measured by a decrease in trans-epithelial electrical resistance (TEER) (ex vivo) or the Lactulose/mannitol test (in vivo). Presumably, the environmental stimuli interact with the surface of cells, possibly by binding to a receptor on the cell surface. However, such a receptor has not been identified. 
     Many inflammatory diseases are thought to be autoimmune. These include rheumatoid arthritis, multiple sclerosis, immune-mediated or type 1 diabetes mellitus, inflammatory bowel diseases, systemic lupus erythematosus, psoriasis, scleroderma, and autoimmune thyroid diseases. Prolonged inflammation is often associated with these diseases, although the inflammation is thought to be a sequela rather than a primary pathological insult. 
     Chemokine (C-X-C motif) receptor 3 (CXCR3) is a G protein-coupled receptor which is known to bind to three chemokines, IP10 (interferon-γ-inducible 10 kDa protein), MIG (monokine induced by interferon-γ) and I-TAC (interferon-inducible T cell α-chemoattractant). IP10, MIG and I-TAC are termed CXC chemokines, because they contain a CXC sequence motif. CXCR3 has been linked to integrin activation, cytoskeletal changes, and chemotaxis. CXCR3 is prominently expressed in inflamed tissues. 
     There is a continuing need in the art for methods to treat autoimmune diseases more effectively and to discover or identify drugs which are suitable for treating autoimmune diseases. 
     SUMMARY OF THE INVENTION 
     One embodiment of the invention provides a method to screen for modulators of CXCR3 signaling. Gliadin or a fragment of gliadin (e.g. a fragment comprising at least six amino acid residues) is contacted with CXCR3. Binding of the gliadin or fragment of gliadin to CXCR3 is determined. A fragment of gliadin which binds to CXCR3 is identified as a modulator of CXCR3 signaling. 
     Another embodiment of the invention provides a method to screen for modulators of CXCR3 signaling. Gliadin or a fragment of gliadin comprising at least six amino acid residues is contacted with a first cell which expresses CXCR3 and with a second cell which does not express CXCR3. Binding of the gliadin or fragment to the first and second cells is determined. A fragment of gliadin which binds preferentially to the first cell relative to the second cell is identified as a modulator of CXCR3 signaling. 
     Still another embodiment of the invention provides a method to screen for modulators of CXCR3 signaling. Gliadin or fragment of gliadin comprising at least six amino acid residues is contacted with CXCR3 or another CXCR3 ligand, such as IP10, MIG, or ITAC. Inhibition of binding of ligand to CXCR3 caused by the fragment of gliadin is determined. A fragment of gliadin which inhibits binding of ligand to CXCR3 is identified as a modulator of CXCR3 signaling. 
     Yet another aspect of the invention is a method to screen for modulators of CXCR3 signaling. A fragment of gliadin comprising at least six amino acid residues is contacted with a cell which expresses CXCR3 or another CXCR3 ligand, such as IP10, MIG, or ITAC. Binding of ligand to the cell is determined. A fragment of gliadin which inhibits binding of ligand to the cell is identified as a modulator of CXCR3 signaling. 
     Also provided by the present invention is a method to screen for modulators of zonulin release. A test compound is contacted with CXCR3. Binding of the test compound to CXCR3 is determined. A test compound which binds to CXCR3 is identified as a modulator of zonulin release. 
     Another embodiment provided by the present invention is a method to screen for modulators of zonulin release. A test compound is contacted with CXCR3. Binding of the gliadin to CXCR3 in the presence and absence of the test compound is determined. A test compound which inhibits binding of gliadin to CXCR3 is identified as a modulator of zonulin release. 
     Still another embodiment of the invention is a method to screen for modulators of zonulin release. A test compound is contacted with a first cell which expresses CXCR3 and with a second cell which does not express CXCR3. Binding of the test compound to the first and second cells is determined. A test compound which binds preferentially to the first cell relative to the second cell is identified as a modulator of zonulin release. 
     Even a further embodiment is a method to screen for modulators of zonulin release. A test compound is contacted with a cell which expresses CXCR3 . Binding of gliadin to the cell is determined. A test compound which inhibits binding of gliadin to the cell is identified as a modulator of zonulin release. 
     Another embodiment of the invention is a method of treating a patient with a disease selected from the group consisting of celiac disease, gluten allergy, gluten sensitivity, and gluten ataxia. An antibody which specifically binds to CXCR3 is administered to the patient. Zonulin release is thereby inhibited. 
     A further embodiment of the invention is a method of treating a patient with an autoimmune or inflammation-associated disease. Typically, these diseases will be characterized by an undesired CXCR3 signaling. The disease is selected from the group consisting of type 1 diabetes, celiac disease, autoimmune hepatitis, multiple sclerosis, autism, dermatitis herpetiformis, IgA nephropathy, primary biliary cirrhosis, rheumatoid arthritis, systemic lupus erythematosus, Grave&#39;s disease, Hashimoto&#39;s disease, and depression. An antibody which specifically binds to CXCR3 is administered to the patient. CXCR3 signaling is thereby inhibited. 
     In some embodiments, the present invention provides methods of identifying a CXCR3 ligand comprising contacting a cell expressing CXCR3 with gliadin or a fragment thereof and a compound to be tested and determining the amount of gliadin or fragment thereof bound to the cell. For example, one or more gliadins or fragments thereof may be labeled with one or more fluorescent moieties. A CXCR3-expressing cell may then be brought into contact with the fluorescently labeled gliadin or fragment in the presence of the compound to be tested. The binding of the gliadin or fragment thereof and CXCR3 may be determined using standard techniques. Suitable techniques include, but are not limited to, fluorescence activated cell sorting (FACS), fluorescent microscopy, and fluorescence spectrophotometry. Optionally the gliadin or fragment thereof may be contacted with CXCR3-expressing cells in the absence of compound to be tested and the amount of binding of gliadin or fragment thereof to CXCR3-expressing cell may be determined. The amount of binding in the presence of compound to be tested and in the absence of compound to be tested may be compared. Other techniques known to those skilled in the art may be used to quantify the gliadin or fragment thereof binding. For example, cells expressing CXCR3 may be fixed to a solid surface, for example, a microtiter plate or a bead (e.g., a magnetic bead) and contacted with fluorescently labeled gliadin or fragment thereof. The amount of bound fluorescently labeled gliadin may be determined. In some embodiments, gliadin may be labeled with a detectable moiety other than a fluorescent moiety, for example, with biotin or digoxigenein and, detected with a suitable reagent, for example, streptavidin or anti-digoxigenin antibody. 
     In some embodiments, the present invention provides methods of identifying a CXCR3 ligand comprising contacting a purified CXCR3 or fragment thereof with gliadin or a fragment thereof and a compound to be tested and determining the amount of gliadin or fragment thereof bound to the CXCR3. For example, one or more gliadins or fragments thereof may be labeled with one or more fluorescent moieties. CXCR3 may then be brought into contact with the fluorescently labeled gliadin or fragment in the presence of the compound to be tested. The binding of the gliadin or fragment thereof and CXCR3 may be determined using standard techniques. Suitable techniques include, but are not limited to, ELISA and fluorescence spectrophotometry. Optionally the gliadin or fragment thereof may be contacted with CXCR3 in the absence of compound to be tested and the amount of binding of gliadin or fragment thereof to CXCR3 cell may be determined. The amount of binding in the presence of compound to be tested and in the absence of compound to be tested may be compared. Other techniques known to those skilled in the art may be used to quantify the gliadin or fragment thereof binding. For example, CXCR3 may be fixed to a solid surface, for example, a microtiter plate or a bead (e.g., a magnetic bead) and contacted with fluorescently labeled gliadin or fragment thereof. The amount of bound fluorescently labeled gliadin may be determined. In some embodiments, gliadin may be labeled with a detectable moiety other than a fluorescent moiety, for example, with biotin or digoxigenein and, detected with a suitable reagent, for example, streptavidin or anti-digoxigenin antibody. In some embodiments, gliadin or fragment thereof may be attached to a solid support and labeled CXCR3 or fragment thereof may be detected in the presence and absence of a compound to be tested. CXCR3 may be labeled and detected using any techniques known in the art, for example, using the techniques described above for labeling and detecting gliadin. 
     These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with methods of screening for useful therapeutic agents and with methods of treating autoimmune and inflammation-associated diseases. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A and 1B  show results when jejunal intestinal fragments from wild-type mice mounted in microsnapwells were challenged with gliadin.  FIG. 1A  shows the amount of zonulin released after challenge. Gliadin challenge causes an increase in zonulin released.  FIG. 1B  shows the Trans Epithelial Electrical Resistance (TEER) after challenge. Gliadin challenge causes a decrease in TEER. 
         FIG. 2  shows the zonulin released after gliadin challenge of endoscopic jejunal biopsies from CXCR3-deficient mice. Gliadin challenge failed to lead to zonulin release. Compare to  FIG. 1A . 
         FIG. 3  shows the TEER after gliadin challenge of endoscopic jejunal biopsies from CXCR3-deficient mice. Gliadin challenge failed to decrease TEER. Compare to  FIG. 1B . 
         FIG. 4  shows the results of the protein bound to the gliadin affinity column is CXCR3, according to protein database search. 
         FIG. 5A  and  FIG. 5B , respectively, show the zonulin levels and TEER of B6 wild type mice challenged with gliadin.  FIG. 5C  and  FIG. 5D , repectively, show the zonulin levels and TEER of CXCR3 knock-out mice (n=20) challenged with gliadin. The CXCR3 knock-out mice do not respond to gliadin and do not release zonulin, and accordingly, do not exhibit an increase in intestinal permeability. In contrast, the wild-type cohort responds to gliadin, releases zonulin and, therefore, does exhibit an increase in intestinal permeability. 
         FIG. 6  shows the results of CXCR3 transfected HEK293 cells probed with anti-CXCR3 mAb (red trace) and IgG1 isotype control (blue trace).  FIG. 6A  shows cells transfected with vector.  FIG. 6B  shows cells transfected with vector expressing CXCR3. 
         FIG. 7  shows the results of fluorescence microscopy of cells transfected probed with DAPI (blue), RITC-labeled anti-CXCR3 monoclonal antibody (red), and FITC-labeled PT-gliadin (green). Panel A shows cells transfected with control vector and probed with DAPI and anti-CXCR3 monoclonal antibody, Panel B shows cells transfected with CXCR3-expressing vector and probed with anti-CXCR3 monoclonal antibody, and Panel C shows cells transfected with CXCR3-expressing vector and probed with anti-CXCR3 monoclonal antibody and FITC-labeled PT-gliadin. 
         FIG. 8  shows the results of the effect of gliadin digested with pepsin and trypsin (PT-Gliadin) on HLA-DR expression in dendritic cells from normal volunteers. 
         FIG. 9  shows the effect of PT-gliadin on TEER and zonulin release in Black 6 wild type mice small intestine.  FIG. 9A  shows the TEER measurements made in snapwells and  FIG. 9B  shows the zonulin concentrations. 
         FIG. 10  shows the effect of PT-gliadin on TEER and zonulin release on small intestine from CXCR3 knockout mice.  FIG. 10A  shows the TEER measurements made in snapwells and  FIG. 10B  shows the zonulin concentrations. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The inventors have discovered that the receptor known as CXCR3 is a physiological receptor for gliadin. This receptor not only binds to gliadin, but it also signals the release of zonulin and a decrease in trans-epithelial electrical resistance (TEER). These downstream effects indicate that the binding to gliadin is physiological. 
     Screening for modulators of CXRC3 signaling can be accomplished by a variety of techniques. Binding to CXRC3 to test compounds can be directly measured, or inhibition of binding of gliadin or another ligand to the receptor can be measured. Other ligands which can be used include IP10, MIG, and ITAC. Ligands can be labeled to facilitate measurement of binding. Assays may be in cell-free systems or in cell-based systems. Any binding assay format can be used, including formats where the receptor is attached to a solid support, either directly or indirectly. 
     Test compounds which can be tested are any compounds. The compounds may be tested as single compounds or in combinations of compounds. The compounds may be structurally identified or of unknown structure. The compounds may be novel or previously known. The compounds may be natural products or synthetic. 
     According to one embodiment of the invention the test compounds are fragments of gliadin. Gliadin is a family of proteins which are produced by wheat and other grains. Examples of gliadins are gliadin alpha, gamma, and omega. Gliadins are the aqueous alcohol-soluble storage proteins in the seed. There is great heterogeneity even within a single class of gliadins. At least six, seven, eight, nine, ten, eleven, fifteen, twenty, thirty, thirty-five, fifty, or seventy-five amino acid residues may be used in fragments of gliadin as test compounds. Fragments include any molecule which is less than full length. Fragments may be, e.g., synthesized or the result of proteolytic degradation. The following tables provide the sequences of a representative number of gliadins. 
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Amino acid sequence of alpha-gliadin from 
                   
               
               
                   Triticum aestivum  (NCBI accession no. CAB76964, 
               
               
                 (SEQ ID NO:1)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MVRVPVPQLQ PQNPSQQQPQ EQVPLVQQQQ FPGQQQPFPP 
                   
               
               
                   
                 QQPYPQPQPF PSQQPYLQLQ 
               
               
                   
               
               
                 61 
                 PFPQPQLPYP QPQLPYPQPQ LPYPQPQPFR PQQPYPQSQP 
               
               
                   
                 QYSQPQQPIS QQQQQQQQQQ 
               
               
                   
               
               
                 121 
                 QQKQQQQQQQ QILQQILQQQ LIPCRDVVLQ QHSIAYGSSQ 
               
               
                   
                 VLQQSTYQLV QQLCCQQLWQ 
               
               
                   
               
               
                 181 
                 IPEQSRCQAI HNVVHAIILH QQQQQQQQQQ QQPLSQVSFQ 
               
               
                   
                 QPQQQYPSGQ GSFQPSQQNP 
               
               
                   
               
               
                 241 
                 QAQGSVQPQQ LPQFEEIRNL ALETLPAMCN VYIPPYCTIA 
               
               
                   
                 PVGIFGTNYR 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Amino acid sequence of alpha-gliadin precursor 
                   
               
               
                 from  Triticum turgidum  subsp. durum (NCBI 
               
               
                 accession no. CAI35909, (SEQ ID NO:2)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTFLILALL AIVATTATTA VRVPVPQLQR QNPSQQQPQE 
                   
               
               
                   
                 QVPLVQQQQF LGQQQPFPPQ 
               
               
                   
               
               
                 61 
                 QPYPQPQPFP SQQPYLQLQP FPQPQLPYSQ PQPFRPQQPY 
               
               
                   
                 PQPQPRYSQP QQPISQQQQQ 
               
               
                   
               
               
                 121 
                 QHQQHQQHHQ EQQILQQILQ QQLIPCMDVV LQQHNIAHRR 
               
               
                   
                 SQVLQQSTYQ LLQELCCQHL 
               
               
                   
               
               
                 181 
                 WQIPEQSQCQ AIHNVVHAII PHQQQKQQQQ PSSQFSFQQP 
               
               
                   
                 LQQYPLGQGS FRPSQQNPQA 
               
               
                   
               
               
                 241 
                 QGSVQPQQLP QFEEIRNLAL QTLPAMCNVY IPPYCTIAPF 
               
               
                   
                 GIFGTN 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Amino acid sequence of alpha/beta-gliadin 
                   
               
               
                 precursor from  Triticum aestivum  (NCBI 
               
               
                 accession no. AAA34280, (SEQ ID NO:3)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTFLILVLL AIVATTATTA VRFPVPQLQP QNPSQQQPQE 
                   
               
               
                   
                 QVPLVQQQQF LGQQQPFPPQ 
               
               
                   
               
               
                 61 
                 QPYPQPQPFP SQLPYLQLQP FPQPQLPYSQ PQPFRPQQPY 
               
               
                   
                 PQPQPQYSQP QQPISQQQQQ 
               
               
                   
               
               
                 121 
                 QQQQQQQQQQ QQQILQQILQ QQLIPCMDVV LQQHNIAHGR 
               
               
                   
                 SQVLQQSTYQ LLQELCCQHL 
               
               
                   
               
               
                 181 
                 WQIPEQSQCQ AIHNVVHAII LHQQQKQQQQ PSSQVSFQQP 
               
               
                   
                 LQQYPLGQGS FRPSQQNPQA 
               
               
                   
               
               
                 241 
                 QGSVQPQQLP QFEEIRNLAL QTLPAMCNVY IPPYCTIAPF 
               
               
                   
                 GIFGTN 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 Amino acid sequence of Gamma-gliadin 
                   
               
               
                 precursor from  Triticum aestivum  (NCBI accession 
               
               
                 no. P21292, (SEQ ID NO:4)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTLLILTIL AMATTIATAN MQVDPSGQVQ WPQQQPFPQP 
                   
               
               
                   
                 QQPFCQQPQR TIPQPHQTFH 
               
               
                   
               
               
                 61 
                 HQPQQTFPQP QQTYPHQPQQ QFPQTQQPQQ PFPQPQQTFP 
               
               
                   
                 QQPQLPFPQQ PQQPFPQPQQ 
               
               
                   
               
               
                 121 
                 PQQPFPQSQQ PQQPFPQPQQ QFPQPQQPQQ SFPQQQQPAI 
               
               
                   
                 QSFLQQQMNP CKNFLLQQCN 
               
               
                   
               
               
                 181 
                 HVSLVSSLVS IILPRSDCQV MQQQCCQQLA QIPQQLQCAA 
               
               
                   
                 IHSVAHSIIM QQEQQQGVPI 
               
               
                   
               
               
                 241 
                 LRPLFQLAQG LGIIQPQQPA QLEGIRSLVL KTLPTMCNVY 
               
               
                   
                 VPPDCSTINV PYANIDAGIG 
               
               
                   
               
               
                 301 
                 GQ 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 Amino acid sequence of Gamma-gliadin B precursor 
                   
               
               
                 from  Triticum aestivum  (NCBI accession no. 
               
               
                 P06659, (SEQ ID NO:5)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTLLILTIL AMAITIATAN MQADPSGQVQ WPQQQPFLQP 
                   
               
               
                   
                 HQPFSQQPQQ IFPQPQQTFP 
               
               
                   
               
               
                 61 
                 HQPQQQFPQP QQPQQQFLQP RQPFPQQPQQ PYPQQPQQPF 
               
               
                   
                 PQTQQPQQPF PQSKQPQQPF 
               
               
                   
               
               
                 121 
                 PQPQQPQQSF PQQQPSLIQQ SLQQQLNPCK NFLLQQCKPV 
               
               
                   
                 SLVSSLWSII LPPSDCQVMR 
               
               
                   
               
               
                 181 
                 QQCCQQLAQI PQQLQCAAIH SVVHSIIMQQ EQQEQLQGVQ 
               
               
                   
                 ILVPLSQQQQ VGQGILVQGQ 
               
               
                   
               
               
                 241 
                 GIIQPQQPAQ LEVIRSLVLQ TLPTMCNVYV PPYCSTIRAP 
               
               
                   
                 FASIVASIGG Q 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Amino acid sequence of Gamma-gliadin (Gliadin 
                   
               
               
                 B-III) from  Triticum aestivum  (NCBI accession no. 
               
               
                 P04730, (SEQ ID NO:6)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 PQQPFPLQPQ QSFLWQSQQP FLQQPQQPSP QPQQVVQIIS 
                   
               
               
                   
                 PATPTTIPSA GKPTSAPFPQ 
               
               
                   
               
               
                 61 
                 QQQQHQQLAQ QQIPVVQPSI LQQLNPCKVF LQQQCSPVAM 
               
               
                   
                 PQRLARSQML QQSSCHVMQQ 
               
               
                   
               
               
                 121 
                 QCCQQLPQIP QQSRYQAIRA IIYSIILQEQ QQVQGSIQSQ 
               
               
                   
                 QQQPQQLGQC VSQPQQQSQQ 
               
               
                   
               
               
                 181 
                 QLGQQPQQQQ LAQGTFLQPH QIAQLEVMTS IALRILPTMC 
               
               
                   
                 SVNVPLYRTT TSVPFGVGTG 
               
               
                   
               
               
                 241 
                 VGAY 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 Amino acid sequence of Gamma-gliadin precursor 
                   
               
               
                 from  Triticum aestivum  (NCBI accession no. 
               
               
                 P08453, (SEQ ID NO:7)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTLLILTIL AMAITIGTAN IQVDPSGQVQ WLQQQLVPQL 
                   
               
               
                   
                 QQPLSQQPQQ TFPQPQQTFP 
               
               
                   
               
               
                 61 
                 HQPQQQVPQP QQPQQPFLQP QQPFPQQPQQ PFPQTQQPQQ 
               
               
                   
                 PFPQQPQQPF PQTQQPQQPF 
               
               
                   
               
               
                 121 
                 PQQPQQPFPQ TQQPQQPFPQ LQQPQQPFPQ PQQQLPQPQQ 
               
               
                   
                 PQQSFPQQQR PFIQPSLQQQ 
               
               
                   
               
               
                 181 
                 LNPCKNILLQ QSKPASLVSS LWSIIWPQSD CQVMRQQCCQ 
               
               
                   
                 QLAQIPQQLQ CAAIHSVVHS 
               
               
                   
               
               
                 241 
                 IIMQQQQQQQ QQQGIDIFLP LSQHEQVGQG SLVQGQGIIQ 
               
               
                   
                 PQQPAQLEAI RSLVLQTLPS 
               
               
                   
               
               
                 301 
                 MCNVYVPPEC SIMRAPFASI VAGIGGQ 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 Amino acid sequence of Gamma-gliadin B-I 
                   
               
               
                 precursor from  Triticum aestivum  (NCBI accession 
               
               
                 no. P04729, (SEQ ID NO:8)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTFLVFALI AVVATSAIAQ METSCISGLE RPWQQQPLPP 
                   
               
               
                   
                 QQSFSQQPPF SQQQQQPLPQ 
               
               
                   
               
               
                 61 
                 QPSFSQQQPP FSQQQPILSQ QPPFSQQQQP VLPQQSPFSQ 
               
               
                   
                 QQQLVLPPQQ QQQQLVQQQI 
               
               
                   
               
               
                 121 
                 PIVQPSVLQQ LNPCKVELQQ QCSPVAMPQR LARSQMWQQS 
               
               
                   
                 SCHVMQQQCC QQLQQIPEQS 
               
               
                   
               
               
                 181 
                 RYEAIRAIIY SIILQEQQQG FVQPQQQQPQ QSGQGVSQSQ 
               
               
                   
                 QQSQQQLGQC SFQQPQQQLG 
               
               
                   
               
               
                 241 
                 QQPQQQQQQQ VLQGTFLQPH QIAHLEAVTS IALRTLPTMC 
               
               
                   
                 SVNVPLYSAT TSVPFGVGTG 
               
               
                   
               
               
                 301 
                 VGAY 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 Amino acid sequence of Gamma-gliadin precursor 
                   
               
               
                 from  Triticum aestivum  (NCBI accession no. 
               
               
                 P08079, (SEQ ID NO:9)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTLLILTIL AMAITIGTAN MQVDPSSQVQ WPQQQPVPQP 
                   
               
               
                   
                 HQPFSQQPQQ TFPQPQQTFP 
               
               
                   
               
               
                 61 
                 HQPQQQFPQP QQPQQQFLQP QQPFPQQPQQ PYPQQPQQPF 
               
               
                   
                 PQTQQPQQLF PQSQQPQQQF 
               
               
                   
               
               
                 121 
                 SQPQQQFPQP QQPQQSFPQQ QPPFIQPSLQ QQVNPCKNFL 
               
               
                   
                 LQQCKPVSLV SSLWSMIWPQ 
               
               
                   
               
               
                 181 
                 SDCQVMRQQC CQQLAQIPQQ LQCAAIHTII HSIIMQQEQQ 
               
               
                   
                 EQQQGMHILL PLYQQQQVGQ 
               
               
                   
               
               
                 241 
                 GTLVQGQGII Q 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                 Amino acid sequence of Alpha/beta-gliadin MM1 
                   
               
               
                 precursor (Prolamin) from  Triticum aestivum  (NCBI 
               
               
                 accession no. P18573, (SEQ ID NO:10)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTFLILALL AIVATTARIA VRVPVPQLQP QNPSQQQPQE 
                   
               
               
                   
                 QVPLVQQQQF PGQQQPFPPQ 
               
               
                   
               
               
                 61 
                 QPYPQPQPFP SQQPYLQLQP FPQPQLPYPQ PQLPYPQPQL 
               
               
                   
                 PYPQPQPFRP QQPYPQSQPQ 
               
               
                   
               
               
                 121 
                 YSQPQQPISQ QQQQQQQQQQ QKQQQQQQQQ ILQQILQQQL 
               
               
                   
                 IPCRDVVLQQ HSIAYGSSQV 
               
               
                   
               
               
                 181 
                 LQQSTYQLVQ QLCCQQLWQI PEQSRCQAIH NVVHAIILHQ 
               
               
                   
                 QQQQQQQQQQ QPLSQVSFQQ 
               
               
                   
               
               
                 241 
                 PQQQYPSGQG SFQPSQQNPQ AQGSVQPQQL PQFEEIRNLA 
               
               
                   
                 LETLPAMCNV YIPPYCTIAP 
               
               
                   
               
               
                 301 
                 VGIFGTN 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                 Amino acid sequence of Alpha/beta-gliadin clone 
                   
               
               
                 PTO-A10 (Prolamin) from  Triticum aestivum  (NCBI 
               
               
                 accession no. P04728, (SEQ ID NO:11)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 PQPQPQYSQP QQPISQQQQQ QQQQQQQQQQ EQQILQQILQ 
                   
               
               
                   
                 QQLIPCMDVV LQQHNIAHGR 
               
               
                   
               
               
                 61 
                 SQVLQQSTYQ LLQELCCQHL WQIPEQSQCQ AIHNVVHAII 
               
               
                   
                 LHQQQQKQQQ QPSSQFSFQQ 
               
               
                   
               
               
                 121 
                 PLQQYPLGQG SFRPSQQNPQ AQGSVQPQQL PQFEIRNLAL 
               
               
                   
                 QTLPAMCNVY IPPYCTIAPF 
               
               
                   
               
               
                 181 
                 GIFGTN 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                 Amino acid sequence of Alpha/beta-gliadin clone 
                   
               
               
                 PW8142 precursor (Prolamin) from  Triticum aestivum   
               
               
                 (NCBI accession no. P04727, (SEQ ID NO:12)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTFLILALV ATTATTAVRV PVPQLQPKNP SQQQPQEQVP 
                   
               
               
                   
                 LVQQQQFPGQ QQQFPPQQPY 
               
               
                   
               
               
                 61 
                 PQPQPFPSQQ PYLQLQPFPQ PQPFLPQLPY PQPQSFPPQQ 
               
               
                   
                 PYPQQRPKYL QPQQPISQQQ 
               
               
                   
               
               
                 121 
                 AQQQQQQQQQ QQQQQQQQIL QQILQQQLIP CRDVVLQQHN 
               
               
                   
                 IAHASSQVLQ QSTYQLLQQL 
               
               
                   
               
               
                 181 
                 CCQQLLQIPE QSRCQAIHNV VHAIIMHQQE QQQQLQQQQQ 
               
               
                   
                 QQLQQQQQQQ QQQQQPSSQV 
               
               
                   
               
               
                 241 
                 SFQQPQQQYP SSQGSFQPSQ QNPQAQGSVQ PQQLPQFAEI 
               
               
                   
                 RNLALQTLPA MCNVYIPPHC 
               
               
                   
               
               
                 301 
                 STTIAPFGIF GTN 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 13 
               
               
                   
               
               
                 Amino acid sequence of Alpha/beta-gliadin clone 
                   
               
               
                 PW1215 precursor (Prolamin) from  Triticum aestivum   
               
               
                 (NCBI accession no. P04726, (SEQ ID NO:13)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTFLILALL AIVATTATTA VRVPVPQPQP QNPSQPQPQG 
                   
               
               
                   
                 QVPLVQQQQF PGQQQQFPPQ 
               
               
                   
               
               
                 61 
                 QPYPQPQPFP SQQPYLQLQP FPQPQPFPPQ LPYPQPPPFS 
               
               
                   
                 PQQPYPQPQP QYPQPQQPIS 
               
               
                   
               
               
                 121 
                 QQQAQQQQQQ QQQQQQQQQQ QQILQQILQQ QLIPCRDVVL 
               
               
                   
                 QQHNIAHARS QVLQQSTYQP 
               
               
                   
               
               
                 181 
                 LQQLCCQQLW QIPEQSRCQA IHNVVHAIIL HQQQRQQQPS 
               
               
                   
                 SQVSLQQPQQ QYPSGQGFFQ 
               
               
                   
               
               
                 241 
                 PSQQNPQAQG SVQPQQLPQF EEIRNLALQT LPRMCNVYIP 
               
               
                   
                 PYCSTTIAPF GIFGTN 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 14 
               
               
                   
               
               
                 Amino acid sequence of Alpha/beta-gliadin A-IV 
                   
               
               
                 precursor (Prolamin) from  Triticum aestivum  (NCBI 
               
               
                 accession no. P04724, (SEQ ID NO:14)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTFLILALR AIVATTATIA VRVPVPQLQP QNPSQQQPQK 
                   
               
               
                   
                 QVPLVQQQQF PGQQQPFPPQ 
               
               
                   
               
               
                 61 
                 QPYPQQQPFP SQQPYMQLQP FPQPQLPYPQ PQLPYPQPQP 
               
               
                   
                 FRPQQSYPQP QPQYSQPQQP 
               
               
                   
               
               
                 121 
                 ISQQQQQQQQ QQQQQQQILQ QILQQQLIPC RDVVLQQHSI 
               
               
                   
                 AHGSSQVLQQ STYQLVQQFC 
               
               
                   
               
               
                 181 
                 CQQLWQIPEQ SRCQAIHNVV HAIILHQQQQ QQQQQQQQQQ 
               
               
                   
                 QPLSQVCFQQ SQQQYPSGQG 
               
               
                   
               
               
                 241 
                 SFQPSQQNPQ AQGSVQPQQL PQFEEIRNLA LETLPAMCNV 
               
               
                   
                 YIPPYCTIAP VGIFGTN 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 15 
               
               
                   
               
               
                 Amino acid sequence of Alpha/beta-gliadin A-III 
                   
               
               
                 precursor (Prolamin) from  Triticum aestivum  (NCBI 
               
               
                 accession no. P04723, (SEQ ID NO:15)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTFLILALL AIVATTATSA VRVPVPQLQP QNPSQQQPQE 
                   
               
               
                   
                 QVPLMQQQQQ FPGQQEQFPP 
               
               
                   
               
               
                 61 
                 QQPYPHQQPF PSQQPYPQPQ PFPPQLPYPQ TQPFPPQQPY 
               
               
                   
                 PQPQPQYPQP QQPISQQQAQ 
               
               
                   
               
               
                 121 
                 QQQQQQQTLQ QILQQQLIPC RDVVLQQHNI AHASSQVLQQ 
               
               
                   
                 SSYQQLQQLC CQQLFQIPEQ 
               
               
                   
               
               
                 181 
                 SRCQAIHNVV HAIILHHHQQ QQQQPSSQVS YQQPQEQYPS 
               
               
                   
                 GQVSFQSSQQ NPQAQGSVQP 
               
               
                   
               
               
                 241 
                 QQLPQFQEIR NLALQTLPAM CNVYIPPYCS TTIAPFGIFG 
               
               
                   
                 TN 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 16 
               
               
                   
               
               
                 Amino acid sequence of Alpha/beta-gliadin A-II 
                   
               
               
                 precursor (Prolamin) from  Triticum aestivum  (NCBI 
               
               
                 accession no. P04722, (SEQ ID NO:16)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTFPILALL AIVATTATTA VRVPVPQLQL QNPSQQQPQE 
                   
               
               
                   
                 QVPLVQEQQF QGQQQPFPPQ 
               
               
                   
               
               
                 61 
                 QPYPQPQPFP SQQPYLQLQP FPQPQLPYPQ PQPFRPQQPY 
               
               
                   
                 PQPQPQYSQP QQPISQQQQQ 
               
               
                   
               
               
                 121 
                 QQQQQQQQQQ ILQQILQQQL IPCRDVVLQQ HNIAHGSSQV 
               
               
                   
                 LQESTYQLVQ QLCCQQLWQI 
               
               
                   
               
               
                 181 
                 PEQSRCQAIH NVVHAIILHQ QHHHHQQQQQ QQQQQPLSQV 
               
               
                   
                 SFQQPQQQYP SGQGFFQPSQ 
               
               
                   
               
               
                 241 
                 QNPQAQGSFQ PQQLPQFEEI RNLALQTLPA MCNVYIPPYC 
               
               
                   
                 TIAPFGIFGT N 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 17 
               
               
                   
               
               
                 Amino acid sequence of Alpha/beta-gliadin A-I 
                   
               
               
                 precursor (Prolamin) from  Triticum aestivum  (NCBI 
               
               
                 accession no. P04721, (SEQ ID NO:17)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MKTFLILALL AIVATTATTA VRVPVPQLQP QNPSQQQPQE 
                   
               
               
                   
                 QVPLVQQQQF LGQQQPFPPQ 
               
               
                   
               
               
                 61 
                 QPYPQPQPFP SQQPYLQLQP FLQPQLPYSQ PQPFRPQQPY 
               
               
                   
                 PQPQPQYSQP QQPISQQQQQ 
               
               
                   
               
               
                 121 
                 QQQQQQQQQQ QQQQIIQQIL QQQLIPCMDV VLQQHNIVHG 
               
               
                   
                 KSQVLQQSTY QLLQELCCQH 
               
               
                   
               
               
                 181 
                 LWQIPEQSQC QAIHNVVHAI ILHQQQKQQQ QPSSQVSFQQ 
               
               
                   
                 PLQQYPLGQG SFRPSQQNPQ 
               
               
                   
               
               
                 241 
                 AQGSVQPQQL PQFEEIRNLA RK 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 18 
               
               
                   
               
               
                 Amino acid sequence of gamma gliadin from 
                   
               
               
                   Triticum aestivum  (NCBI accession no. AAQ63860, 
               
               
                 (SEQ ID NO:18)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MNIQVDPSSQ VPWPQQQPFP QPHQPFSQQP QQTFPQPQQT 
                   
               
               
                   
                 FPHQPQQQFS QPQQPQQQFI 
               
               
                   
               
               
                 61 
                 QPQQPFPQQP QQTYPQRPQQ PFPQTQQPQQ PFPQSQQPQQ 
               
               
                   
                 PFPQPQQQFP QPQQPQQSFP 
               
               
                   
               
               
                 121 
                 QQQPSLIQQS LQQQLNPCKN FLLQQCKPVS LVSSLWSMIL 
               
               
                   
                 PRSDCQVMRQ QCCQQLAQIP 
               
               
                   
               
               
                 181 
                 QQLQCAAIHS IVHSIIMQQE QQEQRQGVQI LVPLSQQQQV 
               
               
                   
                 GQGTLVQGQG IIQPQQPAQL 
               
               
                   
               
               
                 241 
                 EVIRSLVLQT LATMCNVYVP PYCSTIRAPF ASIVAGIGGQ 
               
               
                   
                 YR 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 19 
               
               
                   
               
               
                 Amino acid sequence of Omega-gliadin from 
                   
               
               
                   Triticum monococcum  (NCBI accession no. P02865, 
               
               
                 (SEQ ID NO:19)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 1 
                 ARQLNPSDQE LQSPQQLYPQ QPYPQQPY 
               
               
                   
                   
               
             
          
         
       
     
     Fragments of gliadin that may be used in the practice of the invention include, but are not limited to, Leu-Gln-Leu-Gln-Pro-Phe-Pro-Gln-Pro-Gln-Leu-Pro-Tyr-Pro-Gln-Pro-Gln-Leu-Pro-Tyr-Pro-Gln-Pro-Gln-Leu-Pro-Tyr-Pro-Gln-Pro-Gln-Pro-Phe, which corresponds to amino acids 57-89 of the alpha-gliadin sequence of Table 1, and Leu-Gly-Gln-Gln-Gln-Pro-Phe-Pro-Pro-Gln-Gln-Pro-Tyr (SEQ ID NO:20), which corresponds to amino acids 32-44 of the alpha-gliadin sequence of Table 1 with the proline at position 32 of the wildtype alpha-gliadin sequence mutated to a leucine. Other suitable fragments of gliadin may be prepared, for example, by digesting a purified gliadin with proteolytic enzymes (e.g., pepsin, trypsin or mixtures thereof) and isolating peptides. Peptides may be isolated using any technique known in the art such as reverse phase high pressure liquid chromatography (RP-HPLC). 
     Modulators of CXCR3 signaling may be inhibitors, enhancers, or agonists. Inhibitors are useful for treating diseases characterized by inflammation, including autoimmune diseases and particularly including celiac disease. Enhancers or agonists can be used for increasing permeability of a tissue to a desired agent, e.g., a therapeutic agent which is less than optimally absorbed. 
     Antibodies to CXRC3 can be therapeutically by administration to patients in need thereof. Such patients include those with gluten-related diseases as well as diseases associated with inflammation and autoimmunity. Administration can be by any means known in the art for administration of antibodies. Such methods include, but are not limited to intravenous, intramuscular, and subcutaneous administration. Any form of antibodies known in the art can be used. The antibodies can be polyclonal or monoclonal. They can be, e.g., humanized or human or chimeric or recombinant. The antibodies can be of any isotype. They may be single chain antibodies, or fragments of antibodies such as F(ab′) 2 . 
     Signaling by CXCR3 can be measured by any means known in the art. Signaling events which can be determined include decrease in TEER, increase in zonulin release, microglia recruitment, tyrosine kinase phosphorylation and chemotaxis, and increase in MMP-2 and MMP-9 gelatinolytic activity in cell-conditioned media. 
     The invention provides methods of identifying agents, compounds or lead compounds for agents active at the level of CXCR3-ligand interaction. Generally, screening methods of the invention involve assaying for compounds which modulate the interaction of CXCR3 and ligand (e.g., gliadin or fragment thereof). A wide variety of assays for binding agents is provided including labeled in vitro protein-ligand binding assays, cell based assays, immunoassays, etc. A wide variety of formats may be used, including co-immunoprecipitation, 2-hybrid transactivation, fluorescent polarization, NMR, fluorescent resonance energy transfer (FRET), transcriptional activation, etc. For example, a wide variety of NMR-based methods are available to rapidly screen libraries of small compounds for binding to protein targets (Hajduk, P. J., et al. Quarterly Reviews of Biophysics, 1999, 32 (3): 211-40). In some embodiments, methods of the invention may be automated (e.g., high throughput screening) and may be used to screen chemical libraries for lead compounds. Identified compounds may be used to treat diseases involving CXCR3 signaling, for example, autoimmune diseases. Compounds identified by the methods of the invention may be further optimized to modulate CXCR3 signaling, for example, may derivatized. Multiple iterations of screening and derivatization may be employed to optimize the modulation of CXCR3 signaling. 
     In vitro ligand binding assays employ a mixture of components including CXCR3 or fragment thereof and ligand (e.g., gliadin or fragment thereof). CXCR3 and/or gliadin may be provided as fusion proteins (e.g., with purification tags such as 6-His). Assay mixtures typically further comprise a compound to be tested for CXCR3 modulating activity. Compounds to be tested may be of any kind known to those skilled in the art, for example, may be organic compounds, peptides, proteins, nucleic acids, lipids, carbohydrates and mixtures thereof. A variety of other reagents may also be included in the mixture including, but not limited to, salts, buffers, neutral proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. 
     In general, assay mixtures may be incubated under conditions in which, but for the presence of the compound to be tested, CXCR3 specifically binds the ligand (e.g., gliadin or fragment thereof) with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding. In some embodiments, incubation periods may be minimized to facilitate rapid, high-throughput screening. 
     After incubation, the effect of the compound to be tested on the CXCR3-ligand binding may be detected by any convenient way. For example, CXCR3 or ligand may be immobilized, and the other labeled; then in a solid-phase format, any of a variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc. 
     A difference in the binding affinity of CXCR3 and the ligand in the absence of the compound to be tested as compared with the binding affinity in the presence of the compound to be tested indicates that the compound modulates the binding of CXCR3 to the ligand. A difference, as used herein, is statistically significant and preferably represents at least a 50%, 60%, 70%, 80%, or 90% difference. 
     The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention. 
     EXAMPLE 1 
     In order to identify the putative receptor activated by gliadin, we performed experiments using a gliadin affinity column through which intestinal cell lysates were loaded. We eluted proteins with a step salt gradient. Three clear protein bands were observed on SDS-polyacrylamide gels with molecular weights of 97, 90, and 83 kDa. The observed proteins eluted at 0.2 M and 0.3 M NaCl off the affinity column. Mass spectrometry analysis of proteins that bound to the column identified XP — 125429 in the NCBI sequence database (see Table 20). This sequence includes a precursor of the CXCR3 receptor and implicates the CXCR3 receptor as one of the proteins engaged by gladin (see  FIG. 4 ). 
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 20 
               
               
                   
               
               
                 Amino acid sequence of the protein identified by 
                   
               
               
                 fragment sequencing (NCBI accession non. 
               
               
                 XP_125249 (SEQ ID NO:21)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MASGADSKGD DLSTAILKQK NRPNRLIVDE AINEDNSVVS 
                   
               
               
                   
                 LSQPKMDELQ LFRGDTVLLK 
               
               
                   
               
               
                 61 
                 GKKRREAVCI VLSDDTCSDE KIRMNRVVRN NLRVRLGDVI 
               
               
                   
                 SIQPCPDVKY GKRIHVLPID 
               
               
                   
               
               
                 121 
                 DTVEGITGNL FEVYLKPYFL EAYRPIRKGD IFLVRGGMRA 
               
               
                   
                 VEFKVVETDP SPYCIVAPDT 
               
               
                   
               
               
                 181 
                 VIHCEGEPIK REDEEESLNE VGYDDIGGCR KQLAQIKEMV 
               
               
                   
                 ELPLRHPALF KAIGVKPPRG 
               
               
                   
               
               
                 241 
                 ILLYGPPGTG KTLIARAVAN ETGAFFFLIN GPEIMSKLAG 
               
               
                   
                 ESESNLRKAF EEAEKNAPAI 
               
               
                   
               
               
                 301 
                 IFIDELDAIA PKREKTHGEV ERRIVSQLLT LMDGLKQRAH 
               
               
                   
                 VIVMAATNRP NSIDPALRRF 
               
               
                   
               
               
                 361 
                 GRFDREVDIG IPDATGRLEI LQIHTKNMKL ADDVDLEQVA 
               
               
                   
                 NETHGHVGAD LAALCSEAAL 
               
               
                   
               
               
                 421 
                 QAIRKKMDLI DLEDETIDAE VMNSLAVTMD DFRWALSQSN 
               
               
                   
                 PSALRETVVE VPQVTWEDIG 
               
               
                   
               
               
                 481 
                 GLEDVKRELQ ELVQYPVEHP DKFLKFGMTP SKGVLFYGPP 
               
               
                   
                 GCGKTLLAKA IANECQANFI 
               
               
                   
               
               
                 541 
                 SIKGPELLTM WFGESEANVR EIFDKARVLF FDELDSIAKA 
               
               
                   
                 RGGNIGDGGG AADRVINQIL 
               
               
                   
               
               
                 601 
                 TEMDGMSTKK NVFIIGATNR PDIIDPAILR PGRLDQLIYI 
               
               
                   
                 PLPDEKSRVA ILKANLRKSP 
               
               
                   
               
               
                 661 
                 VAKDVDLEFL AKMTNGFSGA DLTEICQRAC KLAIRESIES 
               
               
                   
                 EIRRERERQT NPSAMEVEED 
               
               
                   
               
               
                 721 
                 DPVPEIRRDH FEEAMRFARR SVSDNDIRKY EMFAQTLQQS 
               
               
                   
                 RGFGSFRFPS GNQGGAGPSQ 
               
               
                   
               
               
                 781 
                 GSGGGTGGSV YTEDNDDDLY G 
               
               
                   
               
             
          
         
       
     
     Human CXCR3 has 368 amino acid residues and a calculated molecular weight of 40,459. The sequences of human CXCR3 and mouse CXCR3 are provided in the following tables. 
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 21 
               
               
                   
               
               
                 Amino acid sequence of CXCR3 from  Homo sapiens   
                   
               
               
                 (NCBI accession no. AAH34403, (SEQ ID NO:22)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MVLEVSDHQV LNDAEVAALL ENFSSSYDYG ENESDSCCTS 
                   
               
               
                   
                 PPCPQDFSLN FDRAFLPALY 
               
               
                   
               
               
                 61 
                 SLLFLLGLLG NGAVAAVLLS RRTALSSTDT FLLHLAVADT 
               
               
                   
                 LLVLTLPLWA VDAAVQWVFG 
               
               
                   
               
               
                 121 
                 SGLCKVAGAL FNINFYAGAL LLACISFDRY LNIVHATQLY 
               
               
                   
                 RRGPPARVTL TCLAVWGLCL 
               
               
                   
               
               
                 181 
                 LFALPDFIFL SAHHDERLNA THCQYNFPQV GRTALRVLQL 
               
               
                   
                 VAGFLLPLLV MAYCYAHILA 
               
               
                   
               
               
                 241 
                 VLLVSRGQRR LRAMRLVVVV VVAFALCWTP YHLVVLVDIL 
               
               
                   
                 MDLGALARNC GRESRVDVAK 
               
               
                   
               
               
                 301 
                 SVTSGLGYMH CCLNPLLYAF VGVKFRERMW MLLLRLGCPN 
               
               
                   
                 QRGLQRQPSS SRRDSSWSET 
               
               
                   
               
               
                 361 
                 SEASYSGL 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 21 
               
               
                   
               
               
                 Amino acid sequence of CXCR3 from  Mus musculus   
                   
               
               
                 (NCBI accession no. NP_034040, (SEQ ID NO:23)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 MYLEVSERQV LDASDFAFLL ENSTSPYDYG ENESDFSDSP 
                   
               
               
                   
                 PCPQDFSLNF DRTFLPALYS 
               
               
                   
               
               
                 61 
                 LLFLLGLLGN GAVAAVLLSQ RTALSSTDTF LLHLAVADVL 
               
               
                   
                 LVLTLPLWAV DAAVQWVFGP 
               
               
                   
               
               
                 121 
                 GLCKVAGALF NINFYAGAFL LACISFDRYL SIVHATQIYR 
               
               
                   
                 RDPRVRVALT CIVVWGLCLL 
               
               
                   
               
               
                 181 
                 FALPDEIYLS ANYDQRLNAT HCQYNFPQVG RTALRVLQLV 
               
               
                   
                 AGFLLPLLVM AYCYAHILAV 
               
               
                   
               
               
                 241 
                 LLVSRGQRRF RAMRLVVVVV AAFAVCWTPY HLVVLVDILM 
               
               
                   
                 DVGVLARNCG RESHVDVAKS 
               
               
                   
               
               
                 301 
                 VTSGMGYMHC CLNPLLYAFV GVKFREQMWM LFTRLGRSDQ 
               
               
                   
                 RGPQRQPSSS RRESSWSETT 
               
               
                   
               
               
                 361 
                 EASYLGL 
               
               
                   
               
             
          
         
       
     
     CXCR3 is a G-protein coupled receptor which is known to function as a receptor of SCYB9, SCYB10, and SCYB11, also known as MIG, IP10, and ITAC, cytokines implicated in inflammation. The receptor is also identified as CD183, GPR9, CKR-L2. The amino acid sequence of the receptor is shown as SEQ ID NO: 23. Human variants are known such as a R292Q and an A363T polymorphisms see SEQ ID NO:22. 
     Methods 
     We linked α-gliadin (a gift from Dr. Donald D. Kasarda) to CARBOXYLINK™. (Pierce Biotechnology, Rockford, Ill.) coupling gel to form an affinity column. 
     We prepared human intestine mucous membranes using protease inhibitors and a standard protocol. 
     One protocol which can be used involves the following steps: Tissues are washed with buffer D (20 mmol·L −1  Tris-HCl, 20 mmol·L −1  EDTA, 250 mmol·L −1  sucrose, pH 7.5) homogenized in buffer E (buffer D containing 5 mg·L −1  leupeptin, 2 mg·L −1  aprotinin, 1 mg·L −1  pepstatin, 10 mg·L −1  phenylmethylsulfonylfluoride (PMSF), and centrifuged at 5000×g, 4° C. for 10 min. Supernatants are centrifuged at 12000×g, 4° C. for 45 min. Precipitates are discarded and supernatants are centrifuged at 30000×g, 4° C. for an additional 90 min. Precipitates are dissolved in buffer E with 5 g·L −1  3[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), sitting on ice for 60 min. with gentle mixing every five minutes. 
     EXAMPLE 2 
     Intestinal fragments isolated from normal mice, mounted on microsnapwells, and exposed to gliadin react by releasing zonulin.  FIG. 1A . Following zonulin release, the intestinal permeability increases, suggesting a loss of the mucosal barrier function.  FIG. 1B . This so-called “gluten effect” is detectable only when the protein is added to the surface (luminal side) of the intestine, suggesting that gliadin interacts with a receptor present on the enterocyte brush border. 
     To confirm the hypothesis that CXCR3 is the gliadin target receptor that needs to be activated in order to release zonulin, experiments were conducted using a CXCR3 knock out mouse model. Intestinal tissues isolated from these animals, mounted in the microsnapwell system, and exposed to gliadin failed to release zonulin and, consequently, no changes in intestinal permeability were detected.  FIGS. 2 and 3 . These results confirm the hypothesis that CXCR3 is a gliadin target receptor involved in zonulin release. 
     The “microsnapwell system,” a polarized model, is used to study the intestinal barrier function using human intestinal biopsies. The system evaluates the intestinal permeability of endoscopic jejunal biopsies by measuring the Trans Epithelial Electrical Resistance (TEER). 
     EXAMPLE 3 
     In vitro experiments using HEK cells transfected with CXCR3 were performed to study gliadin binding to the receptor by immunofluorescence (IF) microscopy. The in vitro IF experiments showed that gliadin bound on cells transfected with CXCR3 but not on cells transfected with vector alone. 
     As shown in  FIG. 6 , HEK293 cells transfected with vector expressing CXCR3 were specifically labeled with anti-CXCR3 mAb. The human CXCR3 sequence was inserted into pc DNA 3.1 (Invitrogen Corporation, Carlsbad, Calif.) under the control of a CMV1 promoter. Red trace shows results obtained with anti-CXCR3 mAb (marked with an arrow in 6B), blue trace shows results obtained with IgG1 isotype control.  FIG. 6A  shows the control transfection with vector alone while  FIG. 6B  shows the results obtained with vector expressing CXCR3. With vector alone, CXCR3 expression was 4.42%, mean 11.9 ( FIG. 6A ). In contrast, with cells transfected with vector expressing CXCR3, CXCR3 expression was 61.78%, mean 95.5. 
     When cells transfected with vector expressing CXCR3 were contacted with fluorescently labeled gliadin, the gliadin bound to the cells and not to the control cells that did not express CXCR3, thus PT-Gliadin co-localizes with CXCR3 in HEK293 transfected cells. In  FIG. 7 , nuclei were stained with DAPI (blue), CXCR3 were stained with monoclonal antibody labeled with RITC (red), and PT-Gliadin was labeled with FITC (green). Panel A shows nuclear staining only with cells transfected with control vector. Panel B shows the staining on the outside of the cells transfected with vector expressing CXCR3 and contacted with RITC-labeled monoclonal antibody specific for CXCR3. When the cells in Panel B were contacted with FITC-labeled gliadin, the gliadin co-localized with CXCR3. 
     EXAMPLE 4 
     Finally, the expression of co-stimulatory markers on peripheral blood mononuclear cells (PBMC) was studied in both normal subjects and patients affected by autoimmunity (celiac disease and type 1 diabetes). PBMC from autoimmune patients exposed to gliadin showed increase expression of co-stimulatory markers CD40, CD80, and CD86, and DR. The stimulation of DR expression (but not of the other markers) was prevented by blocking the CXCR3 receptor using specific antibodies.  FIG. 8  shows the effect of PT-gliadin on HLA-DR expression in dendritic cells from normal volunteers is CXCR3-dependent. 
     The antibodies used to measure costimulatory markers were all commercially available and were purchased from BD Biosciences and R&amp;D Systems. From BD Biosciences: CD80 R-phycoerythrin (r-PE)-conjugated mouse anti-human monoclonal antibody (CD80 r-PE, cat.no. 557227), CD40 and CD86 fluorescein isothiocyanate (FITC)-conjugated mouse anti-human monoclonal antibodies (CD40 FITC, cat.no. 555588; CD86 FITC, cat.no 555657), HLA-DR PE-cy5-conjugated mouse anti-human monoclonal antibody (HLA-DR-cy5, cat.no. 555813). From R&amp;D Systems: allophycocyanin-conjugated mouse monoclonal anti-human CXCR3 (CXCR3 APC, cat.no FAB160A). The antibodies used for blocking studies were: monoclonal anti-human CXCR3 antibody (cat.no.MAB160) and mouse IgG1 isotype control (cat.no.MAB002). 
     EXAMPLE 5 
     Ex vivo experiments to measure zonulin release and intestinal transepithelial electrical resistance (TEER) changes in response to gliadin exposure were performed using mouse small intestine mounted in microsnapwell chambers. The ex vivo experiments were conducted on both CXCR3 knock out (KO) and C57BL/6 wild-type (WT) mouse intestinal tissues mounted in microsnapwells. When exposed to PT-gliadin, intestinal segments obtained from WT mice (n=10) released zonulin (0.33±0.06 vs. 0.61±0.13 ng/mg protein, baseline vs. post-gliadin exposure, respectively; p&lt;0.04, see  FIG. 9B ) and showed a significant TEER decrement (24.1±4.5 Ω/cm 2  vs. 14.7±3.2 baseline vs. post-gliadin exposure, respectively; p&lt;0.02, see  FIG. 9A ). Conversely, intestinal segments obtained from CXCR3 KO mice (n=18) exposed to PT-gliadin failed to release zonulin (0.56±0.15 vs. 0.45±0.13 ng/mg protein, baseline vs. post-gliadin exposure, respectively; p=N.S., see  FIG. 10B ) and showed no TEER changes (20.0±4.8 Ω/cm 2  vs. 16.5±4.9, baseline vs. post-gliadin exposure, respectively; p N.S., see  FIG. 10A ). 
     Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims. All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.