Biological materials and therapeutic uses thereof

A use, biologically active technology in the field of biological materials and their therapeutic uses

Inactive Publication Date: 2016-10-12
IMPERIAL INNOVATIONS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, acute joint inflammation induced by zymosan was not as protracted as in tenascin-C-deficient mice

Method used

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  • Biological materials and therapeutic uses thereof
  • Biological materials and therapeutic uses thereof
  • Biological materials and therapeutic uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0302] Example 1: General method

[0303] Reagent

[0304] Zymosan, methylated BSA and Freund's complete adjuvant (Freund's complete adjuvant), anti-FLAG M2 antibody (mouse monoclonal antibody), blasticidin, and isotype control antibody (mouse IgG2a , IgG1) were from Sigma-Aldrich (Dorset, UK). Hypnorm (fentanyl-fluanone) was from VetaPharma Ltd (Leeds, UK). Limulus amebocyte 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-1β were from PeproTech (Neuilly-Sur-Seine, France). DMEM, RPMI 1640, fetal bovine serum (FBS), penicillin / streptomycin, antibiotic-antimycotic solution PSA, and β-mercaptoethanol were from PAA Laboratories (Yeovil, UK) . HEK293 cell lines stably expressing human TLR2 and TLR4 / CD14 / MD-2, polymyxin B, msbB LPS, and functionally blocked TLR2 (clone: ​​TL2.1; isotype: mouse IgG2a) and TLR4 antibody (clone : HTA125; isotype:...

Embodiment 2

[0319] Embodiment 2: the synthesis of recombinant protein

[0320] Proteins corresponding to each domain of tenascin-C (TA, EGF-L, various TNIII repeats, and FBG) were synthesized and purified. The recombinant protein synthesized is depicted in Figure 9 middle.

[0321] Reagent

[0322] Pfu Turbo polymerase was from Stratagene (Amsterdam, Netherlands). Easy mix 50 PCR tubes were from Molecular Bioproducts (Lutterworth, UK). RNeasy kit and Ni 2+ -NTA-agarose columns were from Qiagen (Crawley, UK). The pCR Blunt vector, pCEP4 plasmid vector, human embryonic kidney (HEK293-EBNA) cells and 4%-12% Bis-Tris gradient gel were from Invitrogen (Groningen, Netherlands). The pET32b vector and BL21(DE3) Rosetta cells were from Novagen (Kent, UK). HiTrapQ column, HiTrap S column, Sephacryl S500 HR column and heparin sepharose column were from Amersham (Buckinghamshire, UK).

[0323] Restriction enzymes were obtained from New England BioLabs (Hitchin, UK). DMEM, fetal bovine serum...

Embodiment 3

[0348] Embodiment 3: animal model

[0349] Zymosan-induced arthritis

[0350] 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). Prepare zymosan 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 μl of Hypnorm (diluted 1:10 in sterile water), followed by injection of zymosan (10 μl) into the right footpad (d=0).

[0351] Control mice received an injection of 10 μl of PBS alone or no injection. For the gross assessment of arthritis, the thickness of each hind paw was measured daily with a micrometer (Kroeplin, Schluchlem, Germany) and the diameter was expressed as the average of each inflamed hind paw per mouse. value.

[0352] After completion of the experiment (days = 4), mice were euthanized and hind paws were fixed in 10% (v / v) buffered formalin...

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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.

Description

technical field [0001] The present invention relates to citrullinated tenascin-C and its activity in chronic inflammation. Also provided are citrullinated tenascin-C and modulators of its biological activity and additional uses of citrullinated tenascin-C to identify agents that up-regulate or down-regulate chronic inflammation. Background technique [0002] Inflammation is a complex biological response of tissues to noxious stimuli, such as pathogens, tissue damage, or irritants. It is the tissue's protective attempt to remove harmful stimuli and initiate the tissue's healing process. Abnormalities associated with inflammation encompass a large group of unrelated disorders (inflammatory disorders) that underlie a variety of human diseases. Examples of diseases with an aspect of inflammation include, but are not limited to, asthma, autoimmune disease, glomerulonephritis, allergies (hypersensitivity), inflammatory bowel disease, reperfusion injury, rheumatoid arthritis, and...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K45/06A61P29/00C12Q1/02A61P19/02A61P37/02A61P1/00A61P17/02A61P35/00A61P9/10A61P3/10A61P11/06A61P11/00A61P1/16A61P39/06A61P17/06A61P19/08A61P9/00
CPCA61K45/00A61K45/06G01N33/5008G01N2500/10C12N2503/02C12N2310/14C07K16/44C07K2317/34G01N2333/78G01N2440/18G01N2800/7095C12N15/113A61P1/00A61P1/04A61P1/16A61P11/00A61P11/06A61P17/02A61P17/06A61P17/16A61P19/02A61P19/04A61P19/08A61P25/08A61P25/28A61P29/00A61P31/04A61P35/00A61P37/02A61P37/06A61P39/06A61P43/00A61P5/44A61P9/00A61P9/10A61P3/10A61K39/3955G01N33/68C07K16/18C07K2317/24C07K2317/76C12N2320/30
Inventor K·S·米德伍德P·J·维纳布尔斯
Owner IMPERIAL INNOVATIONS LTD
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