BIOLOGICAL MATERIALS AND THERAPEUTIC USES THEREOF

Abstract

There is provided agents for modulation of a chronic inflammatory response wherein the agent modulates the biological activity of citrullinated tenascin-C. There is also provided methods of identifying agents modulating citrullinated tenascin-C and chronic inflammation. There are also provided therapeutic uses of such agents.

Claims

1.-67. (canceled)

68. A method of diagnosing or monitoring a subject having a chronic inflammatory condition comprising the step of testing a sample derived from the subject for the presence of: (i) citrullinated tenascin-C and/or one or more fragments of citrullinated tenascin-C; and/or (ii) autoantibodies having specificity for citrullinated tenascin-C and/or for one or more fragments of citrullinated tenascin-C.

69. The method of claim 68, wherein the sample is blood, synovial fluid and/or joint tissue.

70. The method of claim 68, wherein the citrullinated tenascin-C is citrullinated at the FBG domain.

71. The method of claim 68, wherein the citrullinated tenascin-C is citrullinated at one or more of residues 55, 209, 214, 219, 220, 50, 51, 72, 120, 169, 173 and 222 as numbered in SEQ ID NO: 70.

72. The method of claim 68, wherein the presence of citrullinated tenascin-C is tested by contacting the sample with an agent that has binding specificity for citrullinated tenascin-C and/or autoantibodies having specificity for citrullinated tenascin-C.

73. The method of claim 72, wherein the agent has binding affinity for the FBG domain of citrullinated tenascin-C.

74. The method of claim 73, wherein the agent is selected from short interfering RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides, compounds with binding affinity for citrullinated tenascin-C, antibodies or antigen-binding fragments thereof, small inhibitor compounds, a domain of citrullinated tenascin-C or variant thereof, polypeptides or proteins.

75. The method of claim 73, wherein the agent is an antibody or antigen-binding fragment thereof that is specific for the FBG domain of citrullinated tenascin-C.

76. The method of claim 68, wherein the chronic inflammatory condition is associated with rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus, asthma, fibrotic diseases, pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis and/or cardiovascular disease.

77. The method of claim 76, wherein the chronic inflammatory condition is rheumatoid arthritis (RA).

78. The method of claim 68, further comprising the step of administering an appropriate treatment to a subject diagnosed as having a chronic inflammatory condition.

79. The method of claim 78, wherein the treatment is an anti-inflammatory agent, a statin, a biological agent, an immunosuppressive agent, a salicylate or a microbicidal agent.

80. The method of claim 79, wherein the anti-inflammatory agent is selected from non-steroidal anti-inflammatories (NSAIDs), corticosteroids, disease-modifying antirheumatic drugs (DMARDs) or immunosuppressants.

81. The method of claim 80, wherein the non-steroidal anti-inflammatory is an anti-metabolite agent or an anti-inflammatory gold agent; wherein the corticosteroid is selected from cortisone, prednisolone or dexamethasone; or wherein the immunosuppressant is selected from cyclosporin, FK506, rapamycin or mycophenolic acid.

82. The method of claim 78, wherein the treatment is selected from an anti-TNF drug; an anti-IL17 therapy; a T-cell co-stimulation modulator; an interleukin-6 (IL-6) inhibitor; an anti-CD20 antibody; a B cell activating factor; an inhibitor of janus kinase (JAK); an inhibitor of spleen tyrosine kinase (Syk); an antibody specific to citrullinated tenascin-C, or an agent that modulates the biological activity of citrullinated tenascin-C.

Description

[0241] Examples embodying an aspect of the invention will now be described with reference to the following figures:

[0242] FIG. 1. Accelerated resolution of acute inflammation in tenascin-C deficient mice.

[0243] (a) Paw swelling in wild type (+/+)(white bars) and tenascin-C null (/)(black bars) mice over time after injection of zymosan. Data are shown as the mean increase in paw diameter compared to paw diameter before injection+/SEM (n=24 mice per genotype). **=p<0.01. (b-e) Representative sections of the ankle joint from wild type (b, c) and tenascin-C null (d, e) mice 4 days after zymosan injection, stained with hemotoxylin and eosin (b, d) and safranin-O (c, e). Boxes highlight the joint synovium (s) and cartilage proteoglycan (cp). Magnification 10. Quantification of joint inflammation (f) and chondrocyte death (g) in knee joints 4 days after injection with zymosan from wild type mice (white bars) and tenascin-C null mice (black bars). Data are expressed as the mean (+/SD) (n=24 mice per genotype). *=p<0.05.

[0244] FIG. 2. Synovial inflammation is induced in tenascin-C deficient mice upon injection of antigen.

[0245] (a-b, g) Representative sections of the knee joint of sham injected wild type mice. (c-f, h-i) Representative sections of the knee joint of wild type (c, d, h) or tenascin-C null (e, f, i) mice 24 hours after intra-articular injection of mBSA. Inflammatory cell infiltration in the capsule, meniscus and the joint space of both wild type and tenascin-C null mice is highlighted by (cap), (M) and (J) respectively. (S) highlights the healthy synovium of sham injected mice that is no more than 1-3 cells thick along the entire bone surface and (ST) highlights the synovia of wild type and tenascin-C null mice which are both significantly thickened. Sections are stained with hemotoxylin and eosin (a, c, e, g, h, i) and safranin-O (b, d, f). Magnification 10 (a-f) or 40 (g-i). (n=5 mice per genotype).

[0246] FIG. 3. Synovial Inflammation Subsides Rapidly in Tenascin-C Deficient Mice.

[0247] Representative sections of the knee joint of wild type (a, b, f) or tenascin-C null (c, d, e) mice 3 days after intra-articular injection of mBSA. (a, c) The line highlights increased inflammation of the capsule in wild type mice compared to tenascin-C null mice. (b, d) (cp) highlights increased cartilage proteoglycan loss in wild type mice compared to tenascin-C null mice. (e, f) Significant synovial hyperplasia (line), cell and fibrin deposits in the joint space (arrow) and pannus invasion (arrow heads) are observed in wild type mice compared to tenascin-C null mice. Sections are stained with hemotoxylin and eosin (a, c, e, f) and safranin-O (b, d) Magnification 10 (a-d) or 20 (e-f). (n=5 mice per genotype).

[0248] FIG. 4. tenascin-C deficient mice are protected from tissue destruction during antigen induced arthritis.

[0249] (a-b) Representative sections of the knee joint of wild type mice 7 days after intra-articular injection of mBSA, stained with hemotoxylin and eosin (a) and safranin-O (b). Magnification 10. (n=24 mice per genotype). Arrowhead highlights area of bone erosion. Arrow highlights pannus invasion into articular cartilage. (c-d) Representative sections of the knee joint of tenascin-C null type mice 7 days after intra-articular injection of mBSA, stained with hemotoxylin and eosin (c) and safranin-O (d). Magnification 10. (n=24 mice per genotype). J highlights the joint space and AC the intact articular cartilage. (e) Histological score of knee joint inflammation 24 hours, 3 days and 7 days after injection with mBSA from wild type mice (white bars) and tenascin-C null mice (black bars). Data represent the mean+/SD (n=5 per genotype (24 h, 3 d) or 24 per genotype (7 d)). (f) Quantification of chondrocyte death, cartilage surface erosion and bone erosion after injection with mBSA in knee joints from wild type mice (white bars) and tenascin-C null mice (black bars). Chondrocyte death is shown at 24 hours, 3 days and 7 days, and cartilage surface erosion and bone erosion at 7 d. Data represent the mean+/SD (n=5 per genotype (24 h, 3 d) or 24 per genotype (7 d)).

[0250] FIG. 5. tenascin-C induces TNF-, IL-6 and IL-8 synthesis in primary human macrophages and RA synovial fibroblasts.

[0251] (a-b) Primary human macrophages (a) and RA synovial fibroblasts (b) were unstimulated (no addition) or stimulated with LPS (1 ng/ml (a) or 10 ng/ml (b)) or recombinant tenascin-C (1.0 M-1.0 nM) for 24 h. Data shown are the mean of triplicate values (+/SD) from one of three representative experiments. (c) Primary human macrophages were unstimulated (no addition) or stimulated with LPS (1 ng/ml) or recombinant tenascin-C (1.0 M) for 24 h. () indicates cells were pre-incubated with medium alone. (P) Cells were pre-incubated with 25 g/ml polymyxin B for 30 min before stimulation. (H) Cells were incubated with medium with no addition or containing LPS or tenascin-C that was boiled for 15 minutes before addition to cells. Data shown are the mean of triplicate values (+/SD) from one of three representative experiments.

[0252] FIG. 6. The FBG domain of tenascin-C mediates stimulation of cytokine synthesis in vivo and in vitro.

[0253] (a) Primary human macrophages were unstimulated (no addition) or stimulated with LPS (1 ng/ml), recombinant tenascin-C (TNC) or 1.0 M tenascin-C domains (TA, EGF-L, TNIII1-5, TNIII1-3, TNIII3-5, TNIII5-7, TNIII6-8 and FBG) for 24 h. Data shown are the mean of triplicate values (+/SD) from one of three representative experiments. (b) RA synovial membrane cells were unstimulated (no addition) or stimulated with LPS (10 ng/ml) or recombinant FBG (1.0-0.01 M) for 24 h. Data shown are the mean % change in cytokine levels compared to unstimulated cells (+/SEM) from five different patients. (c-h) Representative sections of the knee joint of wild type mice 3 days after intra-articular injection of PBS (c-e) or 1 g FBG (f-h). Sections are stained with hemotoxylin and eosin (c,d,f,g) or Safranin-O (e, h). Magnification 10 (c, f) or 25 (d,e,g,h) (n=5 mice per genotype). (i) Quantification of joint inflammation, bone erosion, cartilage surface erosion and chondrocyte death in the knee joints of wild type mice 3 days after intra-articular injection of PBS (black bars) or 1 g FBG (white bars). Data represent the mean+/SD (n=5 per genotype).

[0254] FIG. 7. FBG mediated cytokine synthesis is MyD88 dependent.

[0255] (a) Human RA synovial fibroblasts were either uninfected, infected with adenovirus expressing GFP alone (AdGFP) or infected with adenovirus expressing dominant negative MyD88 (AdMyD88dn). Cells were unstimulated, stimulated with LPS (10 ng/ml) or stimulated with FBG (1 M) for 24 h. Data shown are the mean of three independent experiments (+/SEM). (b) Mouse embryonic fibroblasts isolated from wild type (+/+) or MyD88 deficient (/) mice were unstimulated () or stimulated with PAM3 (100 ng/ml), LPS (100 ng/ml), TNF (100 ng/ml), IL-1 (5 ng/ml) and FBG (1 M) for 24 h. Data shown are the mean of three independent experiments (+/SEM).

[0256] FIG. 8. FBG mediated cytokine synthesis is TLR4 dependent but does not require CD14 or MD-2.

[0257] (a) Primary human macrophages were pre-incubated with medium alone or medium containing function blocking antibodies to TLR2 (10 g/ml), TLR4 (25 g/ml) or isotype control antibodies (25 g/ml) for 30 min before stimulation. Cells were unstimulated, or stimulated with LPS (1 ng/ml), FBG (1 M) or PAM3 (10 ng/ml) for 24 h. Data shown are the mean of three independent experiments (+/SEM). (b) Mouse embryonic fibroblasts isolated from wild type, TLR2 (TLR2/) or TLR4 (TLR4/) deficient mice were unstimulated or stimulated with PAM3 (100 ng/ml), LPS (100 ng/ml), IL-1 (5 ng/ml) and FBG (1 M) for 24 h. Data shown are the mean of three independent experiments (+/SEM). (c) Bone marrow derived macrophages isolated from wild type, TLR2 (TLR2/) or TLR4 (TLR4/) deficient mice were unstimulated or stimulated with PAM3 (100 ng/ml), LPS (100 ng/ml) or FBG (1 M) for 24 h. Data shown are the mean of three independent experiments (+/SEM). (d) Human macrophages were pre-incubated with no inhibitor, 1 g/ml msbB LPS or 10 g/ml anti-CD14 antibody for 30 min before stimulation with LPS (1 ng/ml), FBG (1 M) or PAM3 (10 ng/ml) for 24 h. Data shown are the mean of three independent experiments (+/SEM).

[0258] FIG. 9. Paw swelling over time after injection of zymosan.

[0259] Representative images of the paws of non-injected tenascin-C null mice (a, e) (diameter 1.6 mm), tenascin-C null mice 24 h (d, f) (diameter 2.5 mm) and 4 d (b, h) (diameter 1.7 mm) after zymosan injection and from wild type mice 4 d after zymosan injection (c, g) (diameter 2.1 mm).

[0260] FIG. 10. Synthesis of recombinant proteins.

[0261] (a) Domain structure of the tenascin-C monomer comprising different domains, including the assembly domain (TA), 14 and a half EGF-like repeats (EGF-L), 17 fibronectin type III-like repeats (TNIII) (8 constitutively expressed (1-8) and 9 that can be alternatively spliced, and a fibrinogen-like globe (FBG). (b) The regions covered by the recombinant proteins that were synthesized, the corresponding amino acid residues and the molecular weight of each protein.

[0262] FIG. 11. Analysis of protein purity.

[0263] Silver stained gel. showing 1 g of each recombinant protein analysed by SDS-PAGE under reducing conditions. Lanes: 1 (TA), 2 (EGF-L), 3 (TNIIII-5), 4 (TNIII5-7), 5 (TNIII6-8), 6 (TNIIII-3), 7 (TNIII3-5) and 8 (FBG).

[0264] FIG. 12. FBG-mediated joint inflammation in vivo requires expression of TLR4.

[0265] Representative sections of the knee joint of TLR2 (a) and TLR4 (b) null mice 3 days after intra-articular injection of 1 g FBG. Sections are stained with hemotoxylin and eosin. Magnification 10 (n=5 mice per genotype). (c) Quantification of joint inflammation, bone erosion, cartilage surface erosion and chondrocyte death in the knee joints of TLR2 (white bars) and TLR4 (black bars) null mice 3 days after intra-articular injection of 1 g FBG. Data represent the mean+/SD (n=5 per genotype).

[0266] FIG. 13. Amino acid sequence of human tenascin-C and its domains

[0267] FIG. 14. Nucleotide sequence of human tenascin-C FIG. 15. TNF synthesis in response to specific FBG peptides.

[0268] TNF synthesis by RA membrane cultures incubated for 24 h with no addition or 100 M of each FBG peptide (P1, P3-P9).

[0269] FIG. 16. TNF and IL8 synthesis in response to varying concentrations of specific FBG peptides.

[0270] TNF and IL8 synthesis by RA membrane cultures incubated for 24 h with no addition or 25, 100 or 250 M of FBG peptide.

[0271] FIG. 17. IL8 synthesis in response to LPS, whole FBG domain or specific FBG peptides.

[0272] IL8 synthesis by macrophages after 24 h incubation with no addition, 1 ng/ml LPS, 1 M whole FBG domain (FBG) or 1 or 20 M of FBG peptides (P1, P3-P9).

[0273] FIG. 18. IL8 and TNF synthesis in response to LPS and FBG following pre-incubation with FBG peptides.

[0274] TNF and IL8 synthesis by macrophages after 24 h incubation with no addition, 1 ng/ml LPS or 1 M whole FBG domain (FBG), either with or without pre-incubation with 20 M of FBG peptides.

[0275] FIG. 19. 1L8 and TNF synthesis in response to tenascin-C targeted siRNAs.

[0276] Tenascin-C mRNA levels in RA fibroblasts transfected with luciferase specific siRNA (control), or with tenascin-C targeted siRNAs: oligo 1 (si 1), oligo 2 (si 2) or a combination of oligos 1+2 (si 1+2). IL6 synthesis in RA fibroblasts transfected with luciferase siRNA (control) or with a combination of tenascin-C targeted oligos 1+2 (siRNA) in the presence or absence of 10 ng/ml LPS for 24 h

[0277] FIG. 20. Purified fibrinogen and FBG can be citrullinated in vitro

[0278] Coomassie stained gel showing purified fibrinogen (lanes 2-5) and FBG (lanes 6-9) that have been left unmodified (lanes 2, 6) or citrullinated by incubation with citrullination buffer, PAD and CaCl (lanes 3, 7). Citrullination of fibrinogen was confirmed by the observation of an increase in MW of this protein. However, changes in the size of FBG were less apparent. Incubation of proteins with PAD in the absence of CaCl or with CaCl in the absence of PAD had no effect on protein size (lanes 4, 5 and 8, 9).

[0279] FIG. 21. Purified full length tenascin-C can be citrullinated in vitro

[0280] Coomassie stained gel showing purified full length tenascin-C that has been left unmodified (lane 9) or citrullinated by incubation with citrullination buffer, PAD and CaCl (lane 6). Citrullination of tenascin-C was confirmed by the observation of an increase in MW of this protein. Incubation of tenascin with PAD in the absence of CaCl or with CaCl in the absence of PAD had no effect on protein size (lanes 7,8). Purified fibronectin was included as a loading control (lane 2).

[0281] FIG. 22. Western blot confirmation of citrullination of FBG and TNC

[0282] Western blot of native and citrullinated TNC (lanes 8, 9) and native and citrullinated FBG (lanes 4, 5) probed using the AMC detection kit. Native and citrullinated fibronectin (lanes 2, 3), native and citrullinated fibrinogen (lanes 6,7) and native and citrullinated enolase (lanes 11,10) were included as positive controls known to be citrullinated.

[0283] FIG. 23 FBG is citrullinated by PAD in vitro

[0284] Purified recombinant FBG was citrullinated in vitro by incubating with different concentrations (2, 7 and 20 Units per mg protein) of rabbit PAD2, human PAD2 and human PAD4 in citrullination buffer (100 mM Tris pH 7.4, 10 mM CaCl.sub.2, 5 mM DTT) for 3 h, 8 h and 24 h at 3TC. As a negative control FBG-C was incubated in citrullination buffer without Calcium (Ca.sup.2+) or without enzyme (-PAD). (A) 1 ug of each sample were resolved on SDS-PAGE and stained with Coornassie Blue. FBG citrullinated by PAD migrates at a slightly higher molecular weight than non-citrullinated FBG. (B) Proteins were transferred on nitrocellulose membranes and incubated in a chemical modification mix (0.0125% FeCl.sub.3 2.3 M H.sub.2SO.sub.4, 1.52 M H.sub.3PO.sub.4, 0.25 M Acetic Acid, 0.25% 2, 3-butanedione monoxime, 0.125% antipyrine). Citrullinated proteins were detected with an anti-modified citrulline specific antibody.

[0285] FIG. 24. Citrullination enhances cytokine production stimulated by FBG

[0286] TNF synthesis in primary human macrophages left unstimulated (UN) or stimulated with 0.1-1.0 M non-citrullinated (nFBG) or citrullinated (cFBG) FBG. Citrullination buffer alone (CIT) and buffer with PAD (CIT+PAD) were included as controls. FBG incubated with citrullination buffer in the absence of PAD or with PAD in the absence of calcium was not citrullinated and exhibited no enhancement of cytokine synthesis (not shown).

[0287] FIG. 25. Ten of 50 RA patients (20%) and none of 50 controls react with citrullinated tenascin-C by Western blot.

[0288] (A) Coomassie stained gel of native (nTNC) and citrullinated (cTNC) purified human recombinant tenascin-C (top panel). Citrullination of tenascin-C demonstated by western blot with the AMC kit (bottom panel). (B) Representative western blot of cTNC probed with serum from RA patients (RA) or normal healthy controls (NH) (n=50). No reactivity was observed with any sera in blots of nTNC (not shown).

[0289] FIG. 26. Serum from a subset of RA patients exhibits reactivity with citTNC

[0290] Western blot of cTNC probed with serum from 7 different RA patients showing one positive patient (lane 4).

[0291] FIG. 27. Serum from a subset of RA patients exhibits reactivity with citTNC

[0292] Western blot of cTNC probed with serum from 8 further RA patients showing one positive patient (lane 4).

[0293] FIG. 28. Serum from normal healthy controls exhibit no reactivity with citTNC

[0294] Western blot of cTNC probed with serum from 8 different controls showing no positive patients.

[0295] FIG. 29. Shows RA serum against citrullinated TNC plus citrullinated FBG

[0296] Western blot of cTNC plus cFBG run together in the same well, probed with serum from 7 different RA patients. Patient subsets were observed that reacted with full length TNC (320 kD) but not FBG (lanes 4, 5) or patients that reacted with cit FBG (27 kd) but not full length TNC (lane 6).

[0297] FIG. 30. Defining the sites of citrullination by LC-MS/MS

[0298] (A) Arginine residues citrullinated by rPAD2 (circle), hPAD2 (rectangle) and hPAD4 (triangle) were determined by LC-MS/MS. Citrullinated sites are underlined, non-citrullinated sites are dashed-underlined, and sites that were not covered by the LC-MS/MS analysis are marked with *. (B) Sequence of FBG domain. All arginine residues are shown, arginine residues that were modified to citrulline residues are underlined. Arginines marked with * were not covered by the LC-MS/MS analysis.

[0299] FIG. 31. Identifying the citrullinated antibody epitope

[0300] (A) Peptides with sequences corresponding to the amino acid sequence of FBG were designed, with the addition of cysteine residues at the amino and carboxy termini and the exchange of arginine for citrulline residues at positions identified by LC-MS/MS. (B) IgG response to citrullinated FBG peptides and arginine containing control peptides in patients with rheumatoid arthritis (RA; n=20) and healthy controls (n=20). The 95th percentile of the control sera was used to determine positivity (dashed line). Mann-Whitney U test was used to calculate p values for differences between groups (n.s.=no significant difference, *=p<0.05 and **=p<0.01, ***=p<0.001, ****=p<0.001).

[0301] FIG. 32. Citrullination of FBG reduces cell adhesion of HDF and RAF

[0302] (A) The sequence identified to bind integrin v3 is shown in white (Yokoyama et al., 2000). Within this sequence two Arginines (shown in black) were identified to be citrullinated. (B) Wells of a 96 well plate were coated with different concentrations of FBG, citrullinated FBG (cFBG), FBG incubated in buffer without Calcium (FBG Ca.sup.2+), or FBG incubated without PAD (FBG -PAD). As a positive control wells were coated with Fibronectin (FN) (1 ug/ml), and coating with BSA (10 mg/ml) served as negative control. Plates were incubated with human dermal fibroblasts (HDF) or synovial fibroblasts from RA patients (RAF) for 45 minutes and adhesion was measured by determining absorbance of attached cells after staining with crystal violet (0.1%). Data are shown as the mean of at least four independent experiments+s.e.m., *=p<0.05, **=p<0.01.

EXAMPLE 1-GENERAL METHODS

[0303] Reagents

[0304] Zymosan, methylated BSA and Freund's complete adjuvant, anti-FLAG M2 antibody (mouse monoclonal antibody), blasticidin, and isotype control antibodies (Mouse IgG2a, IgG1) were from Sigma-Aldrich (Dorset, UK). Hypnorm was from VetaPharma Ltd. (Leeds, UK). The Limulus amaebocyte lysate assay was from Associates of Cape Cod (Liverpool, UK). Wild type human embryonic kidney (HEK293-EBNA) cells were from Invitrogen (Groningen, Netherlands). M-CSF and murine IL-1 were from PeproTech (Neuilly-Sur-Seine, France). DMEM, RPMI 1640, fetal bovine serum (FBS), penicillin/streptomycin, antibiotic-antimycotic solution PSA and -Mercaptoethanol were from PAA Laboratories (Yeovil, UK). HEK293 cell lines stably expressing human TLR2 and TLR4/CD14/MD-2, polymyxin B, msbB LPS and the function blocking TLR2 (Clone: TL2.1 Isotype: Mouse IgG2a) and TLR4 antibodies (Clone: HTA125 Isotype: Mouse IgG2a) were from Invivogen (Caine, UK). Phenol-chloroform-purified Escherichia coli LPS (rough and smooth) and Pam3Cys-Ser-Lys4 (Pam3C) were from Alexis (Birmingham, UK). Murine TNF- and IL-1 receptor antagonist (IL-1ra-IL-1F3) were from R&D Systems (Abingdon, UK). Function blocking anti-CD14 antibodies (Isotype: Mouse IgG1) were from Abcam (Cambridge, UK). Human and murine TNF-, IL-6, and IL-8 ELISAs were from Pharmingen (Oxford, UK).

Purification of Full-Length Tenascin-C

[0305] To ensure that cytokine production was not attributed to bacterial contaminants such as LPS and LPS-associated molecules we purified recombinant full-length human tenascin-C from the conditioned medium of the mammalian cell line HEK293 transfected with his-tagged human tenascin-C in the pCEP-pu vector as described (Lange (2007)). tenascin-C was purified to homogeneity as described (Lange (2007) and determined to be free of LPS contamination using the Limulus amaebocyte lysate assay according to the manufacturer's instructions.

Synthesis of Recombinant Proteins

[0306] Proteins corresponding to each domain of tenascin-C were synthesized (TA, EGF-L, various TNIII repeats and FBG) and purified. See Example 2.

Measurement of LPS Contamination in Recombinant Proteins

[0307] To ascertain the levels of LPS in each recombinant protein the Limulus amaebocyte lysate assay was used according to the manufacturer's instructions (sensitivity 0.70.5 pg LPS per mg protein). All recombinant proteins used in this study had levels of LPS that were less than 10 pg/ml.

Adenoviral Vectors and their Propagation

[0308] Recombinant, replication-deficient adenoviral vectors encoding wild type MyD88 (AdMyD88 wt), dominant-negative forms of MyD88 (AdMyD88dn) and the GFP control (AdGFP) were constructed in-house. A description of the synthesis of these viruses is in Andreakos (2004). All viruses used in this study are E1/E3 deleted, belong to the Ad5 serotype. Viruses were propagated in 293 human embryonic kidney cells, purified by ultracentrifugation through two cesium chloride gradients, and viral titers determined by plaque assay as previously described (Sacre (2007)).

Animals

[0309] Homozygous tenascin-C deficient mice from the original stock described by Saga (1992) on a 129/sv an inbred strain of mice with a white bellied and agouti appearance background were provided by Prof. Charles French-Constant (University of Edinburgh, UK). Age matched congenic inbred wild type 129/sv mice were obtained from Charles River (Margate, UK). All tenascin-C deficient and wild type 129/sv mice were male and between 8-10 weeks of age at the time of experimentation.

[0310] Homozygous TLR2 and TLR4 deficient mice on a C57BL/6 background (an inbred strain of mice with a black coat) were obtained from B&K Universal (Hull, UK) Hoshino (1999) and Takeuchi (1999). Homozygous MyD88 deficient mice on a C57BL/6 background were provided by the Sanger Institute (Cambridge, UK). Age matched congenic inbred wild type C57B/L6 mice were obtained from Charles River (Margate, UK). For isolation of mouse embryo fibroblasts one female aged 8-10 weeks was mated with two males aged 8-10 weeks. For isolation of bone marrow derived macrophages mice were female and between 10-12 weeks of age at the time of experimentation.

[0311] All animals were fed standard rodent chow and water ad libitum, and were housed (<6 mice/cage) in sawdust-lined cages in an air-conditioned environment with 12-hour light/dark cycles. All animal procedures were approved by the institutional ethics committee.

Statistical Methods

[0312] Mean, SD, SEM, and statistical tests were calculated using GraphPad version 3 (GraphPad Software Inc., San Diego, Calif.). Multiple group means were analyzed by one-way analysis of variance, followed by the Dunnett Multiple Comparisons test, where appropriate. Unpaired t-test was used for experiments involving only two groups.

EXAMPLE 2SYNTHESIS OF RECOMBINANT PROTEINS

[0313] Proteins corresponding to each domain of tenascin-C were synthesized (TA, EGF-L, various TNIII repeats and FBG) and purified. The recombinant proteins synthesized are depicted in FIG. 9.

Reagents

[0314] Pfu Turbo polymerase was from Stratagene (Amsterdam, Netherlands). Easy mix 50 PCR tubes were from Molecular Bioproducts (Lutterworth, UK). RNeasy kits and Ni.sup.2+-NTA-agarose columns were from Qiagen (Crawley, UK). pCR Blunt vector, pCEP4 plasmid vector, human embryonic kidney (HEK293-EBNA) cells and 4-12% Bis-Tris gradient gels were from Invitrogen (Groningen, Netherlands). pET32b vector and BL21 (DE3) Rosetta cells were from Novagen (Kent, UK). HiTrap Q columns, HiTrap S columns, Sephacryl S500 HR column and heparin sepharose columns were from Amersham (Buckinghamshire, UK).

[0315] Restriction enzymes were obtained from New England BioLabs (Hitchin, UK). DMEM, fetal bovine serum (FBS) and penicillin/streptomycin were from PAA laboratories (Yeovil, UK). FuGENE6 transfection reagent was from Roche Applied Science (Basel, Switzerland).

[0316] Anti-FLAG M2 antibody (mouse monoclonal antibody), anti-FLAG M2-agarose, FLAG peptide were from Sigma-Aldrich (Dorset, UK). Anti-tetra-his antibody (mouse monoclonal antibody) was from Qiagen (Crawley, UK). Alkaline phosphatase-conjugated goat anti-(mouse IgG) IgG and Western Blue stabilized substrate for alkaline phosphatase were from Promega (Southampton, UK). Precision Protein Standards for SDS-PAGE were from BioRad (Hemel Hempstead, UK).

Primer Design

[0317] Domain boundaries were determined using alignments published in the human tenascin-C sequence (Siri (1991) accession number P24821 (Swiss-Prot)). To clone each domain we designed PCR primers where both the forward and reverse primers contained 18-21 bases corresponding to the 5 and 3 terminal sequences of the requisite coding sequence. The forward primer contained an Nde1 restriction site, followed by an N terminal his tag, immediately before the coding sequence. The final 3 bases of the Nde1 site form the ATC methionine initiation code. The reverse primer included a TTA stop codon immediately after the coding sequence, followed by a BamH1 or a Kpn1 site to allow unidirectional cloning into pET32b expression vectors.

TABLE-US-00001 TABLE1 Protein Forwardprimer name Reverseprimer TA FW:ATA CATCATCATCATCATCATGGGGTCCTCAAG AAAGTCATCCGG[SEQIDNO:1] RV:GCC TTAGCCTGCTCCTGCAGTACATTG[SEQIDNO:2] EGF-L PCR1 FW:ACAGT ACCATGGGGGCCATGGGGGCCATGACT CAGCTGTTG[SEQIDNO:3] RV:CTTGTCATCGTCGTCCTTGTAGTCACCTTCGGTAGCGAG GGCAAG[SEQIDNO:4] PCR2 FW:GACTAGAAGGACGACGATGACAAGTGCTGTCTCCAGCC TGCCAC[SEQIDNO:5] RV:GACAGC TTAATGATGATGATGATGATGTGAGCA GTCTTCTCCGCTGTAGC[SEQIDNO:6] TN1-5 FW:ATA CATCATCATCATCATCATGAGGTGTCTCCTCC CAAAGA[SEQIDNO:7] RV:GCC TTAAGTGGATGCCTTCACACGTGC[SEQIDNO:8] TN1-3 FW:ATA CATCATCATCATCATCATGAGGTGTCTCCTC CCAAAGA[SEQIDNO:9] RV:GCC TTATGTTGTGAAGGTCTCTTTGGC[SEQIDNO:10] TN3-5 FW:ATA CATCATCATCATCATCATCGCTTGGATGCC CCCAGCCAGAT[SEQIDNO:11] RV:GCC TTAAGTGGATGCCTTCACACGTGC[SEQIDNO:12] TN5-7 FW:ATA CATCATCATCATCATCATGAGTTGGACACG CCCAAGGAC[SEQIDNO:13] RV:GCC TTATGTTGTGAACTTGGCAGTGATGGTTG[SEQ IDNO:14] TN6-8 FW:ATA CATCATCATCATCATCATGCCATGGGCTCCCC AAAGGAA[SEQIDNO:15] RV:GCC TTATGTGGTGAAGATGGTCTGGATCAT[SEQID NO:16] FBG FW:ATA CATCATCATCATCATCATATTGGACTCCTGTAC CCCTTCC[SEQIDNO:17] RV:GCC TTATGCCCGTTTGCGCCTGCCTTCAA[SEQID NO:18]

[0318] All primers above are written 5 to 3. Flag sequences are in bold, His tags (CATCATCATCATCATCAT [SEQ ID NO: 19]) are underlined, and restriction enzyme cleavage sites (CATATG=Nde1 site, GGATCC=BamH1, GGTACC=Kpn1 site) are in bold italics.

[0319] PCR

[0320] PCR amplification was carried out using 10 pmol/l of each primer, 1 g template, 5 l DMSO, and 1.25 units Pfu Turbo polymerase in a final volume of 25 l. This was added to buffer and dNTPs in Easy mix 50 tubes. The template used for all reactions was cDNA prepared from U87MG human glioma cells using RNA isolated with RNeasy kits. The reaction was cycled 40 times with denaturing, annealing and elongation temperatures of 95 C., 55-65 C. (depending on melting temperature (Tm) of primers) and 72 C. respectively.

Cloning

[0321] PCR products were ligated into pCR Blunt vectors and sequenced to ensure no errors had been introduced by PCR. Clones were selected that had no errors or silent mutations. Inserts were then ligated into pET32b using Nde1 and BamH1 restriction sites engineered into primers (TN5-7 and TN6-8). Human tenascin-C has internal BamH1 sites within the TA domain (position 494) and TNIII2 (position 2509). TA and TN1-8 were therefore cloned using the Nde1 site in the FW primer and the Kpn1 site in the cloning site of pCRBlunt. Human tenascin-C contains no internal Kpn1 sites. TN1-5, TN1-3 and TN3-5 were cloned using Nde1 and Kpn1 sites in the primers. FBG contains an internal Nde1 site (position 6439) and was therefore cloned using a two step ligation of Nde1 and BamH1 digestion, followed by Nde1 digestion. (Positions refer to sites within the full length nucleotide sequence of tenascin-C, given in FIG. 14)

Bacterial Growth, Induction and Lysis

[0322] The plasmids were transformed into BL21 (DE3) Rosetta cells, cultured in 3 L of Luria-Bertani medium containing 50 g/ml carbenicillin and induced with 1 mM isopropyl--D-thiogalactopyranoside. After 3 hours, the cells were harvested by centrifugation at 4,000 rpm for 20 min, washed twice with ice-cold wash buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM EDTA), and lysed with a French press. Inclusion bodies were collected by centrifugation at 12,000 rpm for 20 min at 4 C. With the exception of TA and FBG the proteins were located entirely in the supernatant. Recombinant TA and FBG proteins were extracted from inclusion bodies with 6 M guanidine hydrochloride, 50 mM Tris-HCl, pH 8.0, and 10 mM -mercaptoethanol at room temperature with constant stirring for 2 hours.

Purification of Bacterial Proteins

[0323] The solution containing recombinant protein was applied to a Ni.sup.2+-NTA-agarose column and washed with 50 mM Tris-HCl, pH 8.0 containing 20 mM imidazole. The column was subsequently washed with 50 mM Tris-HCl, pH 8.0 and the protein was eluted with 50 mM Tris-HCl, pH 8.0 containing 60 mM imidazole. For TA and FBG each washing and elution buffer contained 6 M guanidine hydrochloride. Following Ni chromatography TA and FBG required no subsequent purification. TN1-3 and TN6-8 were further purified by anion exchange chromatography using a HiTrap Q column, TN1-5, TN3-5 and TN5-7 by cation exchange chromatography using a HiTrap S column, and TN1-8 using a HiTrap S column followed by gel filtration using a Sephacryl S500 HR column.

Refolding of Insoluble Proteins

[0324] TA and FBG were refolded by diluting to 20 g/ml with 50 mM Tris-HCl, pH 8.0 containing 6 M guanidine hydrochloride and then treating with 20 mM cystamine with stirring for 16 hours at 4 C. The solution was then dialyzed twice against 15 volumes of 50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl, 10 mM CaCl.sub.2, 5 mM -mercaptoethanol, and 1 mM 2-hydroxyethyl disulfide for 24 hours at 4 C., twice against 20 mM Tris-HCl, pH 8.0 for 8 hours at 4 C. and then centrifuged at 12,000 rpm for 30 min at 4 C. Refolding was assessed by size shifts using SDS PAGE under reducing and non reducing conditions. Protein activity was confirmed by TA domain polymerization and FBG binding to heparin sepharose columns.

Synthesis of EGF-L Domain Using Mammalian Cells

[0325] Initial attempts to express and purify the EGF-L repeats region using an E. coli expression system were unsuccessful. This is most likely to be attributable to difficulty in achieving protein folding due to a total of 91 cysteines in this region. Therefore, the EGF-like domains of TN-C were expressed using HEK293 cells.

[0326] Two PCR reactions were carried out. The first PCR product consisted of a restriction enzyme KpnI site, a Kozak sequence followed by the TN-C signal sequence. The second PCR product consisted of a FLAG peptide, the EGF-like domain sequence, followed by a histidine tag and a BamH1 restriction enzyme sequence.

[0327] The two PCR products were ligated together as described by Ho (1989). PCR reactions were carried out as described above. The entire construct was cloned into the PCR blunt vector and sequenced. It was then subcloned into the pCEP4 vector. The DNA was transfected into HEK293 cells using Fugene and cells were selected for hygromycin resistance (200 g/ml) in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum, penicillin (100 units/ml) and streptomycin (100 units/ml). 2 litres conditioned medium (collected after cells have been cultured in medium) from stably transfected cells was collected and pooled. The pooled conditioned medium (2 litres) was centrifuged at 3000 rpm to separate cell debris from the medium.

[0328] The medium was then applied to an anti-FLAG column. Material was collected in 50 ml fractions for the flow-through. The column was washed with 10 column volumes of 1M NaCl, 50 mM Tris-HCl, pH 7.5 and then washed with 10 column volumes of 60% isopropanol to ensure removal of LPS. The column was then washed with 50 mM Tris-HCl buffer, pH 7.5 and finally the protein was eluted using 200 g/ml FLAG peptide in 50 mM Tris-HCl buffer, pH 7.5.

Analysis of Protein Purity

[0329] Each protein was dialysed against 1000 volumes of 150 mM NaCl and 50 mM Tris pH 7.5. Protein purity was analyzed by SDSPAGE under reducing conditions. To do this 1 g of each purified recombinant protein was run on a 4-12% Bis-Tris gradient gel and the gel was subsequently silver stained to demonstrate a single band (FIG. 10). Western blotting analyses were also carried out. Proteins separated by SDS-PAGE were electrotransferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA in Tris-buffered saline and then incubated with primary antibodies recognizing FLAG M2 (1:2000 dilution)(EGF-L) or tetra-his antibodies (1:2000)(all other proteins). The blot was then incubated with secondary antibody conjugated to alkaline phosphatase and the protein bands visualized using Western Blue stabilized substrate whereby the gels show a single specific band recognised by each antibody at the expected Mw (not shown)

EXAMPLE 3ANIMAL MODELS

Zymosan-Induced Arthritis

[0330] Zymosan-induced arthritis (ZIA) was induced in tenascin-C deficient and wild type mice by injection of zymosan (Saccharomyces cerevisiae), as described in Keystone (1977). Zymosan was prepared by dissolving 15 mg of zymosan in 1 ml of sterile PBS. The solution was boiled twice and sonicated. Mice were anesthetized by intraperitoneal injection of 150 l of Hypnorm diluted 1:10 in sterile water, then injected with zymosan (10 l) into the right footpad (d=0).

[0331] Control mice received an injection of 10 l PBS alone or were not injected. For macroscopic assessment of arthritis, the thickness of each hind paw was measured daily with microcalipers (Kroeplin, Schluchlem, Germany) and the diameter expressed as an average for each inflamed hind paw per mouse.

[0332] Following completion of the experiment (day=4), mice were euthanized and hind paws fixed in 10% (v/v) buffered formalin, decalcified with 10% EDTA and processed to paraffin.

Antigen-Induced Arthritis

[0333] Antigen-induced arthritis (AIA) was induced in tenascin-C-deficient and wild-type mice as described previously by Brackertz (1977). Briefly, at day 0 mice were anesthetized by intraperitoneal injection of 150 l of Hypnorm diluted 1:10 in sterile water, then immunized with 200 g of methylated BSA. mBSA was emulsified in 0.2 ml of Freund's complete adjuvant and injected intra-dermally at the base of the tail.

[0334] At day 7, arthritis was induced by intra-articular injection of mBSA (100 g in 10 l of sterile PBS) into the right knee joint using a sterile 33-gauge microcannula. Control mice received an injection of 10 I PBS alone or were not injected.

[0335] On day 14, mice were euthanized, the knee joints were excised and fixed in 10% (volume/volume) buffered formalin, decalcified, with 10% EDTA and processed to paraffin.

Injection of FBG

[0336] Wild type mice were anesthetized by intraperitoneal injection of 150 l of Hypnorm diluted 1:10 in sterile water and then injected with 100 ng, 1 or 3 g FBG in 10 l of sterile PBS into the right knee joint using a sterile 33-gauge microcannula. Control mice received an injection of 10 l PBS alone or were not injected.

[0337] On days 3 and 7, mice were euthanized, the knee joints were excised and fixed in 10% (volume/volume) buffered formalin, decalcified, with 10% EDTA and processed to paraffin.

Histology of Knee Joints

[0338] Coronal tissue sections (4 m) were cut at 7 depths throughout the joint; 80 m apart and stained with hematoxylin and eosin or Safranin-O to assess joint pathology. Histopathologic changes were scored using the following parameters as described in Van Lent (2006).

[0339] Inflammation (the influx of inflammatory cells into synovium (infiltrate) and the joint cavity (exudates), was graded using an arbitrary scale from 0 (no inflammation) to 3 (severe inflammation). Chondrocyte death was determined as the percentage of cartilage area containing empty lacunae in relation to the total area. Cartilage surface erosion was determined as the amount of cartilage lost in relation to the total cartilage area. Bone destruction was determined in 10 different areas of the total knee joint section. Destruction was graded on a scale of 0 (no damage) to 3 (complete loss of bone structure). Histological analysis was performed by an investigator who was blinded to the experimental groups. The mean score for each animal in an experimental group was calculated by averaging the histopathologic scores in at least 5 section depths per joint.

Results

Zymosan Induced Joint Inflammation is not Sustained in Tenascin-C Deficient Mice

[0340] Zymosan injection into the footpad was used to induce acute synovitis in mice. Wild type mice exhibited rapid paw swelling reaching maximal paw diameter by 24 hours (2.56 mm, an increase of 62% of the starting paw diameter). This was maintained for a further 24 hours. After 2 days paw diameter decreased but paws remained swollen by 4 days (2.08 mm, an increase of 32%) (FIG. 1a). tenascin-C deficient mice exhibited a similar degree of paw swelling to wild type mice 24 hours post injection (2.41 mm, an increase of 57% of starting paw diameter). However, swelling in the tenascin-C null mice subsided faster than in the wild type mice; paw diameter was significantly reduced at 2 days and had declined to 1.7 mm (an increase of only 11%) by 4 days (FIG. 1a). By day 4 post injection the paws of wild type mice were still visibly swollen and red, whereas the paws of tenascin-C null mice were not visibly swollen or red and resembled non-injected paws (FIG. 9).

[0341] This difference was reflected histologically at 4 days. The synovia of wild type mice were significantly inflamed and exhibited cellular infiltration and cartilage proteoglycan loss was observed (FIG. 1b, c). In contrast, the synovium of tenascin-C deficient mice exhibited no synovitis, cellular infiltrate or cartilage proteoglycan loss (FIG. 1d, e) and resembled the joints of sham injected and non injected mice (not shown). Quantification of joint inflammation revealed whilst there was little exudate (cellular mass in the joint cavity) in either wild type or tenascin-C null mice, levels of infiltrate (cellular mass in the synovial layer) were significantly reduced in tenascin-C null mice (FIG. 1f). No erosion of cartilage or bone occurred in mice of either genotype (not shown), however a low level of chondrocyte death occurred in wild type mice, that was not observed in tenascin-C null mice (FIG. 1g). Thus tenascin-C expression appears to promote the maintenance of acute inflammation.

Tenascin-C Null Mice are Protected from Persistent Inflammation and Structural Damage During Antigen Induced Arthritis

[0342] To determine whether tenascin-C also contributes to more destructive inflammatory joint disease, erosive arthritis was induced by intra-articular injection of mBSA into the knee joint following immunization with mBSA. This model involves both cellular and humoral immune responses and induces pathological changes similar to human RA (Brackertz (1977)). Injection of mBSA induced a similar inflammatory response in both tenascin-C null and wild type mice. Cell infiltration and synovial thickening is apparent by 24 hours in mice of both genotypes (FIG. 2c-f, h,i) compared to sham injected (FIG. 2a, b, g) or non injected (not shown) mice.

[0343] However, this does not persist in tenascin-C null mice as it does the wild type mice. By 3 days post injection wild type mice exhibit increased inflammation of the meniscus and capsule, synovial hyperplasia, cells and fibrin deposits in the joint space, pannus formation and localized cartilage proteoglycan loss (FIG. 3a, b, f). In contrast, by 3 days in tenascin-C null mice inflammation is limited to the capsule, synovial inflammation has subsided and there are no fibrin/cell aggregates present in the joint space, no pannus formation and no cartilage proteoglycan loss (FIG. 3c, d, e).

[0344] By 7 days wild type mice exhibited persistent inflammatory cell infiltration and joint space exudate, extensive synovitis and pannus formation and destruction of articular cartilage and bone, erosion (FIG. 4a, b). Sham injected knees and knees from mice that had undergone no injection were healthy and exhibited no inflammation or joint destruction (not shown). tenascin-C deficient mice also had healthy joints that exhibited only mild inflammatory cell infiltration, with no joint space exudate, synovitis, pannus formation, destruction of articular cartilage or bone erosion (FIG. 4c, d). Joints from tenascin-C deficient mice that had been sham injected and or that had undergone no injection were also healthy (not shown).

[0345] These histological data are reflected upon scoring of joint disease as described in materials and methods. Levels of cellular infiltrate and exudate observed in both wild type mice and tenascin-C null mice 24 hours post injection were not significantly different. However, whilst cellular mass continued to increase in wild type mice over time, this response was attenuated in tenascin-C null mice and cell numbers in the joint decreased over time (FIG. 4e). Increasingly high levels of chondrocyte death occurred in the cartilage of wild type mice over time, but no significant death was observed in tenascin-C null mice (FIG. 4f). No cartilage surface erosion and bone erosion was evident in wild type mice at 24 hours or 3 days (not shown) but significant tissue destruction had occurred by 7 days. In contrast tenascin-C null mice exhibited no tissue destruction at 24 hours, 3 days (not shown) or 7 days (FIG. 4f). These data indicate that whilst the initiation of joint inflammation (cell influx into the synovium and joint space) is unaffected in tenascin-C null mice, unlike in wild type mice disease does not progress to tissue destruction and cell death. These results demonstrate that expression of tenascin-C is required for persistent synovial inflammation and joint destruction in this model.

EXAMPLE 4CELL CULTURE

Patient Specimens

[0346] Human monocytes were isolated from peripheral blood (London Blood Bank) and macrophages were derived from monocytes after differentiation for 4 days with 100 ng/ml of M-CSF as previously described (Foxwell (1998)).

[0347] RA membrane cells (representing a mixed population of all synovial cell types) were isolated from synovial membranes obtained from patients undergoing joint replacement surgery as previously described (Brennan(1989)). RA synovial fibroblasts were isolated from the mixed population of RA membrane cells as previously described (Brennan(1989)). The study was approved by the local Trust ethics committee (Riverside NHS Research Committee), and waste tissue (synovium after joint replacement surgery) was obtained only after receiving signed informed consent from the patient and anonymyzing the tissue to protect patient identity.

[0348] Immediately after isolation, RA membrane cells and macrophages were cultured at 110.sup.5 cells/well in RPMI 1640 containing 10% (v/v) FBS and 100 U/ml (Units/nil) penicillin/streptomycin in 96-well tissue culture plates for 24 hours before stimulation. Synovial fibroblasts (used only at either passage number 2 or 3) were cultured at 110.sup.4 cells/well in DMEM containing 10% (v/v) FBS and 100 U/ml penicillin/streptomycin in 96-well tissue culture plates for 24 hours before stimulation.

Mouse Embryonic Fibroblasts (MEFs) and Bone Marrow Derived Macrophages (BMDMs)

[0349] MEFs express high levels of mRNA of all 9 murine TLRs and are specifically and highly responsive to TLR ligand activation. MEFs from mice with targeted deletions of TLR2, TLR4 and MyD88 demonstrate profound defects in their IL-6 response to specific ligands (Kurt-Jones (2004)). MEFs were isolated from d13 embryos harvested from age-matched, pregnant female wild type, TLR2, TLR4 and null mice (as described in Todaro (1963)). Fibroblasts were cultured at 210.sup.4 cells/well in DMEM containing 10% (v/v) FBS and 100 U/ml penicillin/streptomycin in 96-well tissue culture plates for 24 hours before stimulation.

[0350] BMDMs were derived by aspirating the femurs of age matched female wild type, TLR2 and TLR4 null mice as described in Butler (1999)) and culturing the cells for 7 days in DMEM, 20% (v/v) FBS, 10 ml/L (v/v) antibiotic-antimycotic solution PSA, 50 M Mercaptoethanol and 10 ng/ml M-CSF. Macrophages were then cultured at 110.sup.5 cells/well in DMEM, 20% (v/v) FBS, 10 ml/L (v/v) antibiotic-antimycotic solution PSA, 50 M -Mercaptoethanol in 96-well tissue culture plates for 24 hours before stimulation.

HEK293 Cell Lines

[0351] HEK293 cell lines expressing TLR2 and TLR4/CD14/MD-2 were cultured at 110.sup.4 cells/well in DMEM containing 10% (v/v) FBS and 10 g/ml biasticidin in 96-well tissue culture plates for 24 hours before stimulation.

Cell Stimulation and Assessment of Cytokine Synthesis

[0352] Cells were incubated for 24 hours at 37 C. with the indicated doses of tenascin-C and recombinant tenascin-C fragments (1.0 M-1.0 nM). Cells were also stimulated where indicated with LPS (1 ng/ml for human macrophages, 10 ng/ml for human fibroblasts, RA membrane cells and HEKs, 100 ng/ml for MEFS and BMDMs and 10 ng/ml for HEKS), PAM3 (10 ng/ml for human macrophages, human fibroblasts, and HEKs, 100 ng/ml for MEFs and BMDMs), murine IL-1 (5 ng/ml for MEFS) and murine TNF- (100 ng/ml for MEFS). Unless specifically stated otherwise rough LPS was used for in vitro studies.

[0353] For adenoviral gene transfer experiments, human RA synovial fibroblasts were incubated with adenoviral vectors at a multiplicity of infection of 100, washed after 2 hours, cultured in complete medium for 24 hours, then stimulated for 24 hours, after which time supernatants were collected.

[0354] Where stated, cells were pre-incubated with 10 g/ml anti-CD14 antibody, 10 g/ml receptor antagonist, 10 g/ml anti-TLR2 antibody, 25 g/ml anti-TLR4 antibody, 10 or 25 g/ml isotype control antibody, 25 g/ml polymyxin B, or 1 g/ml msbB LPS, for 30 minutes at 37 C. before stimulation. Where stated, recombinant tenascin-C and FBG, and LPS were boiled for 15 minutes before addition to cells

[0355] In all cases, viability of the cells was not significantly affected throughout the experimental time period when examined by the MTT cell viability assay (Sigma, Poole, UK).

[0356] Supernatants were subsequently examined for the presence of the cytokines TNF-, IL-6, and IL-8 by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic, Vantaa, Finland) and analyzed using the Ascent software program (Thermo Labsystems, Altrincham, UK).

Results

Tenascin-C Induces TNF-, IL-6 and IL-8 Synthesis in Primary Human RA Synovial Fibroblasts and Macrophages

[0357] We next investigated whether tenascin-C might activate the innate immune response. tenascin-C was used to stimulate primary human macrophages and RA synovial fibroblasts and the production of the pro-inflammatory cytokines TNF-, IL-6 and IL-8 examined. The bacterial cell wall component LPS was used as a positive control. tenascin-C induced a cell type specific cytokine profile which was significantly different from LPS. It dose dependently stimulated the production of TNF-, IL-6 and IL-8 in human macrophages (FIG. 5a). However, tenascin-C only induced IL-6 synthesis in synovial fibroblasts, whereas LPS induced both IL-6 and IL-8 (FIG. 5b). Neither LPS nor tenascin-C induced TNF- synthesis in fibroblasts (data not shown). tenascin-C stimulation of IL-6 (FIG. 5c), IL-8 and TNF- by human macrophages and IL-6 by synovial fibroblasts (not shown) was heat sensitive and unaffected by the LPS inhibitor, polymyxin B. Together these results provide strong evidence that cytokine induction by tenascin-C is not due to LPS contamination.

The Fibrinogen-Like Globe (FBG) Mediates Tenascin-C Activation of Cells.

[0358] Tenascin-C is a large hexameric molecule, each domain of which binds to different cell surface receptors (reviewed in Orend (2005)). Understanding the mechanism of action of tenascin-C will require identification of which domain(s) are critical for promoting cytokine production. We synthesized recombinant proteins comprising different domains of the molecule (FIG. 10). Each domain was made in E. coli, purified (FIG. 11), and found to contain <10 pg/ml LPS by subjecting neat protein to the Limulus amaebocyte lysate assay. Only one domain of tenascin-C was active. The fibrinogen-like globe (FBG) stimulated TNF- synthesis in human macrophages (FIG. 6a), IL-6 and IL-8 synthesis in human macrophages (not shown) and IL-6 in RA synovial fibroblasts (not shown) to an equal extent to full-length tenascin-C. Like full-length tenascin-C, FBG did not induce IL-8 synthesis in RA synovial fibroblasts where LPS did (data not shown). FBG induced cytokine synthesis was also heat sensitive and unaffected by polymyxin B (data not shown).

The FBG Domain of Tenascin-C Induces Cytokine Production in Human RA Synovium and Joint Inflammation in Mice.

[0359] We investigated whether FBG could promote expression of inflammatory cytokines in synovial membranes from RA patients. This tissue model of RA (comprising a mixed population of all synovial cell types) spontaneously produces high levels of IL-6, IL-8 and TNF- (Brennan (1989)) (FIG. 6b). FBG further enhanced synthesis of all these cytokines (FIG. 6b). To determine whether FBG could induce inflammation in vivo, wild type mice were injected intra-articularly with FBG. We observed a transient and dose dependent stimulation of joint inflammation. No inflammation or proteoglycan loss occurred in non-injected mice or in mice injected with PBS (FIG. 6c-e) or Wong FBG (data not shown). In mice injected with 1 g FBG inflammatory cell infiltration (FIG. 60, mild synovitis, pannus formation (FIG. 6g) and proteoglycan loss (FIG. 6h) was observed. A similar response was seen in mice injected with 3 g FBG (data not shown). Upon histological quantification, high levels of cellular infiltrate and exudate and chondrocyte death were observed in mice injected with FBG, together with a modest amount of cartilage surface erosion and bone damage (FIG. 6i).

FBG Mediated Cytokine Synthesis is Dependent on Myd88

[0360] Many DAMPs, including fibrinogen (Smiley (2001)), have been shown to stimulate the innate immune response by activation of TLRs. Therefore, we investigated whether TLRs might also mediate tenascin-C induced cytokine production. Myeloid differentiation factor 88 (MyD88) is required for signalling by all TLRs, except TLR3 (O'Neill (2008)). Infection of synovial fibroblasts with adenovirus expressing dominant negative MyD88, but not GFP control virus, abolished FBG induction of IL-6 (FIG. 7a). These data suggest that FBG induced inflammation is dependent on functional MyD88. This effect of FBG did not appear to be mediated by IL-1 as addition of IL-1 receptor antagonist did not inhibit induction of cytokines (data not shown). To confirm that FBG action is MyD88 dependent we demonstrated that FBG does not stimulate cytokine synthesis in embryonic fibroblasts isolated from mice with targeted deletions in the MyD88 gene. The TLR2 ligand PAM3, TLR4 ligand LPS and IL-1 all signal via MyD88. Stimulation with these was also abolished in MEFs from deficient mice. However, TNF-, which does not signal via MyD88, was unaffected (FIG. 7b). Re-transfection of wild type MyD88 restored the responsiveness of these cells to FBG, PAM3, LPS and IL-1 (data not shown).

FBG Signals Via TLR4

[0361] TLRs exhibit specificity for endogenous ligands; proteins are recognised by one or both of TLR2 and 4 (reviewed in O'Neill (2008)). Neutralising antibodies to TLR4 inhibited both FBG and LPS induced IL-6, IL-8 and TNF- synthesis in human macrophages and IL-6 synthesis in RA synovial fibroblasts but had no effect on the function of the TLR2 ligand, PAM3. Antibodies to TLR2 inhibited PAM3 mediated cytokine synthesis but had no effect on LPS or FBG induced cytokine synthesis. Isotype matched controls had no effect on cytokine synthesis induced by any ligand (TNF- synthesis by human macrophages is shown in FIG. 8a). To confirm that FBG action is TLR4 dependent we demonstrated that FBG does not stimulate cytokine synthesis in embryonic fibroblasts or macrophages isolated from mice with targeted deletions in the TLR4 gene. FBG mediated cytokine synthesis was unaffected in embryonic fibroblasts or macrophages isolated from mice with targeted deletions in the TLR2 gene. Cells isolated from TLR2 deficient mice were unresponsive to PAM3 but responsive to LPS and IL-1. Cells isolated from TLR4 deficient mice were unresponsive to LPS but did respond to PAM3 and IL-1 (FIG. 8b, c). In addition, expression of TLR4 was required for the arthritogenic action of FBG in vivo; FBG was able to induce joint inflammation in TLR2 null mice but not in TLR4 null mice (FIG. 12).

Different Co-Receptor Requirements for FBG and LPS

[0362] LPS signalling via TLR4 is mediated by a receptor complex including the soluble protein MD-2 and GPI-linked cell surface or soluble CD14 (reviewed in Fitzgerald (2004)). We next examined whether CD14 and MD-2 are required for FBG activation of TLR4. As a positive control here we examined the activity of smooth glycosylated LPS which requires both MD-2 and CD14 (Jiang (2005)). LPS mediated IL-6, IL-8 and TNF- synthesis by human macrophages and IL-6 synthesis by RA synovial fibroblasts was inhibited by anti-CD14 antibodies and an antagonistic LPS derived from the msbB mutant E. coli which competes for LPS binding to MD-2 (Coats(2007)). Conversely, both PAM3, which does not require these co-receptors for activation of TLR2, and FBG-mediated cytokine synthesis was unaffected by anti CD14 antibodies or msbB mutant LPS (FIG. 8d shows TNF- synthesis by human macrophages). These data suggest that neither CD14 nor MD-2 is required for FBG mediated cytokine synthesis. Therefore, whilst LPS and FBG both signal via activation of TLR4, they may have different co-receptor requirements.

EXAMPLE 5INHIBITION OF TENASCIN-C ACTION AND SYNTHESIS IN HUMAN TISSUE

[0363] This example studies the effect of (1) prevention of the pro-inflammatory action of tenascin-C and (2) inhibition of tenascin-C expression in the human RA synovium.

Methods

Peptide Synthesis

[0364] Nine overlapping peptides comprising the entire FBG domain (table 2) were synthesized by Biogenes, Germany. Peptides were cleaved at room temperature (cleavage mixture: 90% trifluoroacetate, 5% thioanisol, 3% ethanedithiol, 2% anisole), purified by reverse phase high performance liquid chromatography, and characterized by MALDI TOF mass spectral analysis. The purity of the peptides was >85% as determined high performance liquid chromatography.

[0365] The facility was unable to synthesize peptide 7, presumably due to the formation of secondary structure that prevented elongation of the peptide chain (as previously reported (LaFleur (1997)).

TABLE-US-00002 TABLE2 OverlappingpeptidesthatspantheentireFBG domainofhumantenascin-C Peptide# Aminoacidsequence 1 TIGLLYPFPKDCSQAMLNGDTTSGLYTIYL[SEQ IDNO:20] 2 YTIYLNGDKAEALEVFCDMTSDGGGWIVFL [SEQIDNO:21] 3 WIVFLRRKNGRENFYQNWKAYAAGFGDRRE [SEQIDNO:22] 4 GDRREEFWLGLDNLNKITAQGQYELRVD[SEQ IDNO:23] 5 ELRVDLRDHGETAFAVYDKFSVGDAKTRYK [SEQIDNO:24] 6 KTRYKLKVEGYSGTAGDSMAYHNGRSFST [SEQIDNO:25] 7 RSFSTFDKDTDSAITNCALSYKGAFWYRN[SEQ IDNO:26] 8 WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG [SEQIDNO:27] 9 FHWKGHEHSIQFAEMKLRPSNFRNLEGRRKRA [SEQIDNO:28]

Patient Specimens and Cell Culture

[0366] RA membrane cells (representing a mixed population of all synovial cell types) were isolated from synovial membranes obtained from patients undergoing joint replacement surgery (Brennan (1989)). Synovial membrane tissue was digested in RPMI 1640 (GIBCO) containing 5% fetal calf serum (FCS) (GIBCO), 5 mg/ml collagenase type IV (Sigma) and 0 15 mg/ml DNAse type I (Sigma) and incubated at 37 C. for 2 h.

[0367] After incubation the tissue was pipetted through a nylon mesh into a sterile beaker. The cells were then washed three times in complete medium (RPMI 1640 supplemented with 10% FCS). RA synovial fibroblasts were isolated from the mixed population of RA membrane cells by selection in DMEM (Bio-Whittaker) supplemented with 10% FBS, 1 M glutamine, 100 U/ml penicillin, and streptomycin. Human monocytes were isolated from peripheral blood (London Blood Bank) and macrophages were derived from monocytes after differentiation for 4 days with 100 ng/ml of M-CSF.

[0368] The study was approved by the local Trust ethics committee, and waste tissue (synovium after joint replacement surgery) was obtained only after receiving signed informed consent from the patient and anonymising the tissue to protect patient identity.

Cell Stimulation and Assessment of Cytokine Synthesis

[0369] Immediately after isolation, RA membrane cells were cultured at 110.sup.5 cells/well in RPMI 1640 containing 10% (v/v) FBS and 100 U/ml penicillin/streptomycin in 96-well tissue culture plates. Cells were incubated for 24 h at 37 C. with no addition, buffer control (PBS, 1% BSA, 0.01% NaN.sub.3), or with 25 m, 100 M or 250 M of each FBG spanning peptide.

[0370] Synovial fibroblasts (used only at either passage number 2 or 3) were seeded at a concentration of 510.sup.4 cells in a 3.5-cm dish. siRNA was transfected at a final concentration of 10 nM using Lipofectamine 2000 (Invitrogen) for 4 h in serum-free OptiMEM I. Two different siRNAs against human tenascin-C were used (s7069 and s229491) (Applied Biosystems).

[0371] siRNA sequences of s7069 are: (sense 5 CGCGAGAACUUCUACCAAAtt 3 [SEQ ID NO: 29], antisense 5 UUUGGUAGAAGUUCUCGCGtc 3 [SEQ ID NO: 30]) and of s229491 are (5 GGAAUAUGAAUAAAGAAGAtt 3 [SEQ ID NO: 31], antisense 5 UCUUCUUUAUUCAUAUUCCgg 3 [SEQ ID NO: 32]). siRNA against luciferase (Dharmacon) was transfected as a non-targeting control.

[0372] Four hours after transfection, medium was changed with pre-equilibrated Dulbecco's modified Eagle's medium containing 10% FBS (v/v) and cells were incubated for a further 48 h and 72 h. Cells were then stimulated with 10 ng/ml LPS for 24 h at 37 C. Tenascin-C mRNA and protein levels were quantitated by PCR and western blotting respectively. Total RNA was extracted from cells using a QiaAmp RNA Blood mini kit (Qiagen, Germany). cDNA was synthesised from equivalent amounts of total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and 18-mer oligo dTs (Eurofins MWG Operon).

[0373] Gene expression was analysed by delta-delta ct methods based on quantitative real-time PCR with TaqMan primer set human tenascin-C(Hs01115663-ml) and human ribosomal protein endogenous control (RPLPO) (4310879E) (Applied Biosystems) in a Corbett Rotor-gene 6000 machine (Corbett Research Ltd). Tenascin-C protein was detected in cell supernatants and cell lysates by by SDS PAGE and western blotting using antibody MAB1908 (Millipore).

[0374] Macrophages were cultured at 110.sup.5 cells/well in RPMI 1640 containing 5% (v/v) FBS and 100 U/ml penicillin/streptomycin in 96-well tissue culture plates for 24 h before stimulation. Cells were incubated for 24 h at 37 C. with no addition, 1.0 M FBG, 1 ng/ml LPS or 1 or 20 M FBG peptide. Where stated, cells were pre-incubated with 20 M FBG peptides for 15 min.

[0375] The viability of the cells was not significantly affected throughout the experimental time period when examined by the MTT cell viability assay (Sigma, Poole, UK). Supernatants were examined for the presence of the cytokines TNF-, IL-6, and IL-8 by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&D systems). Absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic, Vantaa, Finland) and analyzed using the Ascent software program (Thermo Labsystems, Altrincham, UK).

Statistical Methods

[0376] Mean, SD and SEM were calculated using GraphPad (GraphPad Software Inc., San Diego, Calif.).

Results

Blockade of Cytokine Synthesis in RA Membrane Cultures by Specific FBG Peptides

[0377] The approach of peptide inhibition has been used successfully to pinpoint the v3 integrin binding site in the FBG domain of tenascin-C and to prevent cell adhesion in response to this domain of tenascin-C (Lafleur (1997) and Yokoyama (2000)).

[0378] We synthesized a series of 8 overlapping peptides of 30 amino acids that span the entire sequence of FBG (Table 2). Peptides were tested for the ability to block spontaneous cytokine synthesis in RA synovial membrane cultures. TNF and IL8 synthesis was inhibited by peptides 3 and 8, but not by any other peptide (TNF shown in FIG. 15). Peptides 3 and 8 dose dependently inhibited cytokine synthesis with the highest concentrations achieving 95% and 56% inhibition respectively (FIG. 16). Whilst peptide 5 had no effect on TNF synthesis, it dose dependently blocked IL8 synthesis in RA membrane cells with a maximal inhibition of 81% (FIG. 16).

[0379] To map the active domain within FBG responsible for inducing cytokine production we stimulated primary human macrophages with each FBG peptide. Peptides 1, 5 and 6 all induced cytokine synthesis in a dose dependent manner (FIG. 17).

[0380] To determine if any peptide could block FBG induced cytokine synthesis in human macrophages, cells were pre-incubated with each FBG peptide before stimulation with either whole FBG or LPS. Peptide 5 specifically blocked FBG mediated cytokine synthesis, whilst peptide 8 blocked cytokine synthesis in response to both LPS and FBG (FIG. 18).

[0381] Peptide 8 therefore non-specifically blocks cytokine production induced by any stimuli. This domain is the integrin binding domain of FBG that mediates cell adhesion and thus may be acting to prevent cell attachment to tissue culture plates. Peptide 5 specifically blocks FBG-induced cytokine synthesis suggesting that targeting this domain may be useful in preventing tenascin-C induced inflammation.

Silencing Tenascin-C Gene Expression Inhibits Cytokine Synthesis in RA Synovial Fibroblasts

[0382] Examination of the effect of inhibiting tenascin-C expression in the human RA synovium has identified synovial fibroblasts as the major source of tenascin-C in RA (FIG. 1C) (in Goh 2010).

[0383] siRNA mediated knockdown of tenascin-C expression in these cells has been shown with a maximal efficiency between 94-96% (FIG. 19). In cells transfected with tenascin-C siRNA, both the basal level of cytokine synthesis and LPS induced cytokine production was inhibited by 38% and 44% respectively compared to control cells (FIG. 19)

[0384] These data reveal that silencing tenascin-C in RA synovial fibroblasts reduces the synthesis of pro-inflammatory cytokines and suggest that ablation of tenascin-C expression is a viable strategy to inhibit inflammation in the synovium.

[0385] This work has established that blocking tenascin-C activity (with peptides) and tenascin-C expression (with siRNA) reduces inflammatory cytokine synthesis in human RA synovia. These data shows that tenascin-C blockade is of potential clinical benefit in treating RA and other inflammatory diseases.

EXAMPLE 6IN VITRO CITRULLINATION

[0386] Equal volumes of protein and 2 citrullination buffer (200 mM Tris HCl, pH 7.4, 20 mM CaCl.sub.2), 10 mM DTT) were mixed. 8.75 U rabbit skeletal PAD (product number P1584 from Sigma) per mg substrate protein was added and incubated for 3 hours at 37 C. or 2 hours at 50 C. Citrullination was confirmed by size shift visualized by coomassie blue staining of SDS PAGE or by AMC detection.

[0387] The results shown in FIG. 20 demonstrate that purified fibrinogen and FBG can be citrullinated in vitro.

[0388] In vitro citrullination of purified full length tenascin-C is shown by the results in FIG. 21.

EXAMPLE 7AMC (ANTI-MODIFIED CITRULLINE) DETECTION OF CITRULLINATED PROTEINS

[0389] Citrullination was detected using a protocol from an anti-citrulline (Modified) Detection Kit (Millipore (catalogue: 17-347)).

A. Nitrocellulose Blot Preparation

[0390] 1. Run SDS-PAGE and transfer to nitrocellulose. Wash blot with water 25 min. [0391] 2. Incubate blot in 10 ml 0.1% ovalbumin in TBS 15 min RT [0392] 3. Wash with water 210 min

[0393] B. Modification of Citrulline Residues [0394] 1. Mix 3 ml Reagent A and 3 ml Reagent B. Prepare just before use. [0395] 2. Add modification buffer to blot in light-proof container, incubate at 37 C. 5-7 hrs. [0396] 3. Rinse blot 4-5 times in water.
C. Detection of modified Citrulline Residues [0397] 1. Block blot in freshly prepared 3% non-fat dried milk in TBS for 30 min-1 hr. [0398] 2. Incubate blot with 5-8 ml of 1:1000 dil of anti-modified citrulline antibody diluted in TBS-MLK overnight with agitation at 4 C. (Seems to work with a 2-3 hr incubation RT). [0399] 3. Rinse blot 3 with water, then wash 115 min, then 35 min. [0400] 4. Incubate the blot with 5-8 ml of 1:5000 dil of goat anti-rabbit HRP-conjugated IgG in TBS-MLK for 1 hr at RT with agitation. [0401] 5. Wash blot as in step 3. [0402] 6. Wash blot in TBS-0.05% Tween 20 for 3-5 min. [0403] 7. Rinse blot in 4-5 changes of water. [0404] 8. Use ECL-plus for detection.

[0405] FIG. 22 shows confirmation of the citrullination of FBG and tenascin-C by western blot.

EXAMPLE 8FBG IS CITRULLINATED BY PAD IN VITRO

[0406] FIG. 23 shows FBG is citrullinated by PAD in vitro. In particular, an increased molecular weight of FBG in Coomassie-blue stained SDS PAGE and Western Blotting with an anti-modified citrulline specific antibody demonstrates that FBG is citrullinated by rPAD2, hPAD2 and hPAD4 in a dose- and time dependent manner.

EXAMPLE 9DEMONSTRATION THAT CITRULLINATION MODULATES THE PRO-INFLAMMATORY ACTIVITY OF TENASCIN-C

[0407] Primary human macrophages were isolated as described above and cultured at 110.sup.5 cells/well in RPMI 1640 containing 5% (v/v) FBS and 100 U/ml penicillin/streptomycin in 96-well tissue culture plates for 24 h before stimulation. Cells were incubated for 24 h at 37 C. with no addition (UN), different concentrations (uM) of native FBG (nFBG) or cit FBG (cFBG) or citrullination buffer alone (CIT) or cit buffer plus PAD enzyme (CIT+PAD).

[0408] The viability of the cells was not significantly affected throughout the experimental time period when examined by the MTT cell viability assay (Sigma, Poole, UK). Supernatants were examined for the presence of the cytokines TNF- by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&D systems). Absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic, Vantaa, Finland) and analyzed using the Ascent software program (Thermo Labsystems, Altrincham, UK).

[0409] FIG. 24 shows that citrullination enhances cytokine production stimulated by FBG.

EXAMPLE 10SERUM FROM RA PATIENTS REACT WITH CITRULLINATED TENASCIN-C AND CITRULLINATED FBG UNLIKE HEALTHY CONTROLS

SDS PAGE of Citrullinated Proteins

[0410] 33.33 ul of citrullination reaction (cit-TNC alone or cit-TNC combined with cit-FBG) was loaded into NuPAGE Novex 4-12% Bis-tris gel using a 1.0 mm 2D single lane well.

Western Blot Detection with RA Serum

[0411] After electrophoresis proteins were transferred to nitrocellulose and the membrane blocked and then cut into strips (up to 8 strips0.7 cm wide). Strips were incubated with serum from RA patients or normal healthy individuals diluted 1:100 in 5% milk TBS-tween. Strips were incubated in 15 ml falcon tubes on rollers 1 h room temperature, then washed 35 min, 115 min in tubes with TBS-Tween. Strips were then incubated with seondary mouse anti-human Ig 1:5000 1 h in room temperature, then washed 35 min, 115 min in the dish with TBS-tween. Strips were finally incubated with ECL reagents and exposed to film.

[0412] FIG. 25A shows a coomassie stained gel of native (nTNC) and citrullinated (cTNC) purified human recombinant tenascin-C (top panel) and citrullination of tenascin-C demonstated by western blot with the AMC (Anti-modified citrulline) kit (bottom panel).

[0413] FIG. 25B shows a representative western blot of cTNC probed with serum from RA patients (RA) or normal healthy controls (NH) (n=50). No reactivity was observed with any sera in blots of nTNC (not shown).

[0414] FIGS. 26 and 27 shows that serum from a subset of RA patients exhibits reactivity with citTNC (see RA patient samples in lane 4 on each gel).

[0415] FIG. 28 shows that serum from normal healthy controls exhibit no reactivity with citTNC.

[0416] FIG. 29 shows RA serum reactivity against citrullinated TNC and citrullinated FBG. A western blot of cTNC plus cFBG was run together in the same well, probed with serum from 7 different RA patients. Patient subsets were observed that reacted with full length TNC (320 kD) but not FBG (lanes 4, 5) or patients that reacted with citrullinated FBG (27 kd) but not full length TNC (lane 6).

[0417] Gels run with full length citrullinated TNC and with citrullinated FBG in the same lane show some patients react with full length TNC. Of those that do react with TNC only a subset also react with FBG thus they must by definition recognize a different domain.

EXAMPLE 11DEFINING THE SITES OF CITRULLINATION BY LC-MS/MS

[0418] With reference to FIG. 30 and table 3, citrullinated arginine residues were identified by LC-MS/MS. From 17 Arginines, 5 are not citrullinated, 3 arginines are not covered by this LC-MS/MS analysis, and 9 arginine residues are modified to citrulline residues, independent of the enzyme used for citrullination.

TABLE-US-00003 TABLE 3 Position R Sequence m/z Charge 55 NGR(+.98)ENFYQNWK 728.8331 2+ [SEQ ID NO: 33] 72 AYAAGFGDR(+.98)R 542.7619 2+ [SEQ ID NO: 34] 120 TR(+.98)YKLK 405.2478 2+ [SEQ ID NO: 35] 169 GAFWYR(+.98)RNC(+57.02)HR 684.3063 2+ [SEQ ID NO: 36] 173 NC(+57.02)HR(+.98)VNLM(+15.99)GR 637.2995 2+ [SEQ ID NO: 37] 173 NC(+57.02)HR(+.98)VNLMGR 629.3023 2+ [SEQ ID NO: 38] 173 NC(+57.02)HR(+.98)VNLM(+15.99)GR 425.2011 3+ [SEQ ID NO: 39] 173 N(+27.99)C(+57.02)HR(+.98)VNLMGR 643.2988 2+ [SEQ ID NO: 40] 209 L(+57.02)R(+.98)PSNFR 474.2563 2+ [SEQ ID NO: 41] 214 L(+27.99)RPSNFR(+.98)NLEGR 744.3896 2+ [SEQ ID NO: 42] 214 LRPSNFR(+.98)NLEGR 730.3915 2+ [SEQ ID NO: 43] 214 LRPSN(+.98)FR(+.98)NLEGR 487.5948 3+ [SEQ ID NO: 44] 214 L(+27.99)RPSNFR(+.98)NLEGR 496.5952 3+ [SEQ ID NO: 45] 214 LRPSNFR(+.98)NLEGRR 404.7247 4+ [SEQ ID NO: 46] 214 LRPSNFR(+.98)NLEGR 487.2641 3+ [SEQ ID NO: 47] 219 N(+27.99)LEGR(+.98)RK 451.2462 2+ [SEQ ID NO: 48] 219 L(+27.99)RPSNFRNLEGR(+.98)R 548.6292 3+ [SEQ ID NO: 49] 214/219 LRPSN(+.98)FR(+.98)NLEGR(+.98)R 539.9565 3+ [SEQ ID NO: 50] 214/219 LRPSNFR(+.98)NLEGR(+.98) 730.8856 2+ [SEQ ID NO: 51] 214/219 LRPSNFR(+.98)NLEGR(+.98)R 808.9345 2+ [SEQ ID NO: 52] 214/219 LRPSNFR(+.98)NLEGR(+.98)R 539.6262 3+ [SEQ ID NO: 53]

EXAMPLE 12IDENTIFYING THE CITRULLINATED ANTIBODY EPITOPE

[0419] With reference to FIG. 31, the antibody response in 20 RA and 20 control sera to 6 cyclic FBG peptides, encompassing citrulline residues was evaluated.

[0420] The peptides evaluated where as follows:

TABLE-US-00004 R55: [SEQIDNO:54] CVFLRRKNG-R-ENFYQNWC CIT55: [SEQIDNO:55] CVFLRRKNG-Cit-ENFYQNWC R72: [SEQIDNO:56] CAYAAGFGD-R-REEFWLGLC CIT72: [SEQIDNO:57] CAYAAGFGD-Cit-REEFWLGLC R120: [SEQIDNO:58] CFSVGDAKT-R-YKLKVEGYC CIT120: [SEQIDNO:59] CFSVGDAKT-Cit-YKLKVEGYC R169/173: [SEQIDNO:60] CKGAFWY-R-NCH-R-VNLMGRC CIT169/173: [SEQIDNO:61] CKGAFWY-Cit-NCH-Cit-VNLMGRC R209/214/219/220: [SEQIDNO:62] CEMKL-R-PSNF-R-NLEG-R-R-KRC CIT209/214: [SEQIDNO:63] CEMKL-Cit-PSNF-Cit-NLEGRRKRC CIT219/220: [SEQIDNO:64] CEMKLRPSNFRNLEG-Cit-Cit-KRC

[0421] A strong antibody response towards citrulline containing peptides CIT55, CIT209/214 and CIT219/220 was detected in RA sera but not control sera, and no response was observed against arginine containing control peptides. Data from this small cohort shows that about 35% of RA patients will be positive for the CIT55 epitope, 20% for the CIT 209/214 and 30% for the CIT219/220 whilst none of the healthy controls exhibited positivity, defined using the 95th percentile of the normal group. The same calculations may be used to diagnose RA compared to a group of healthy controls and a similar percentage in these groups may be expected.

[0422] These data will enable the stratification of patients based on citFBG antibody positivity or on citFBG positivity. Those patients that were positive would be candidates for treatment with any agents designed to target the activity of citrullinated FBG. The presence of different epitopes also informs how to target citFBGe.g. if CIT55 was present treatment may comprise blocking the immunogenic activity of FBG, but if the integrin binding site was citrullinated treatment may comprise blocking the action of this domain.

EXAMPLE 13CITRULLINATION OF FBG REDUCES CELL ADHESION OF HDF AND RAF

[0423] With reference to FIG. 32, FBG is proadhesive for human dermal fibroblasts (HDF) and synovial fibroblasts from RA joints (RAF). Citrullination of FBG reduces adhesion of HDF and RAF. Two arginines within the sequence known to bind integrin v3 are citrullinated, therefore reduced adhesion may be due to reduced binding of cFBG to integrin v3. Yokoyama et al. determined that this peptide sequence within FBG mediates cell adhesion via binding to cell surface v3 integrins. Here it is shown 1) citrullination occurs in vitro in the integrin binding site, 2) this reduces cell adhesion, and 3) this happens in RASF meaning that, in an RA joint, citrullination of FBG at this site may reduce the adhesion of RASF. This can have many implications for disease progression, for example, it may promote cell migration (e.g. invasion into and degradation of healthy joint tissue), and it may facilitate RASF proliferation (thus perpetuating synovial hyperplasia).

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