Amelioration of inflammatory arthritis by targeting the pre-ligand assembly domain (PLAD) of tumor necrosis factor receptors
A PLAD, receptor technology, applied in cytokine/lymphokine/interferon receptors, receptors/cell surface antigens/cell surface determinants, animal/human proteins, etc.
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Embodiment 1
[0195] H9 lymphoma cells were washed and resuspended in PBS. Cells were then incubated with 100 ng / ml human recombinant TNFα (R&D Systems) under rotation for 1 hour at 4°C. Cells were then treated with 2 mM crosslinker DTSSP (Pierce) for 30 minutes and the reaction was stopped with 20 mM Tris.Cl [pH 7.5] for 15 minutes on ice. Cells were lysed with 150 mM NaCl, 20 mM Tris.Cl [pH 7.5], 1 mM EDTA, 30 mM NaF, 2 mM b-glycerol phosphate and 1 mM sodium orthovanadate supplemented with protease inhibitors (Boehringer Mannheim). Equal amounts of lysates were electrophoresed under non-reducing (without β-mercaptoethanol) or reducing (with 280 mM β-mercaptoethanol) conditions and analyzed for p60 and p80 complexes with specific antibodies (19). Densitometric analysis was performed with a Kodak Image Station 440.
[0196] The p80 complex was found to show a molecular size approximately three times larger than the unit size, consistent with glycosylated and unglycosylated trimers ( Fig...
Embodiment II
[0213] In human autoimmune lymphoproliferative syndrome (ALPS), heterozygous mutations encoding an abnormal form of Fas (CD95 / APO-1) interfere dominantly with Fas-induced lymphocyte apoptosis. The present invention demonstrates that this arises from pre-association of wild-type and mutant Fas receptors utilizing the extracellular domain, independent of ligand-induced receptor oligomerization. The pre-associated Fas receptor complex was found to be required for signal transduction and confirmed in living cells by the novel application of FRET between green fluorescent protein (GFP) variants body. These results provide a new molecular mechanism for Fas signaling and dominant interference in human disease.
[0214] Fas(APO-1 / CD95) is a cell surface receptor that transduces apoptotic signals critical for immune homeostasis and tolerance (19-21). Fas is a 317 amino acid type 1 membrane glycoprotein with three cysteine-rich extracellular domains (CRDs) that are characteristic of t...
Embodiment III
[0229] method
[0230] Mice and Reagents
[0231] BALB / c, C57BL / 6, DBA / 1J, C3H / HeJ, C3H / HeN, TNFR1 and TNFR2 knockout mice were purchased from Jackson Laboratory. TNF-α transgenic mice were purchased from Taconic. Male mice aged 6-8 weeks were used in all experiments and housed in the animal room of the Immunology Laboratory (NIAID, NIH). CpG DNA was synthesized using CBER. The CpG DNA 1668 sequence is 5'-TCCATGACGTTCCTGATGCT-3' (SEQ ID NO: 61) (50). Anti-P60(1H11) or anti-P80PLAD(3H11) mouse monoclonal antibody (MAb) was prepared by using P60 PLAD and P80PLAD.
[0232] Purification of PLAD-GST fusion protein
[0233]Cloning and purification of the PLAD-GST fusion protein was performed as described in the literature (78). Purified PLAD-GST fusion proteins were verified by 4-20% Tris-glycine gels and mass spectrometry and stored at -80°C. LPS was removed with Detoxi-Gel AffinityPak columns (Pierce).
[0234] In vitro testing of P60 and P80 PLAD proteins
[0235] L929 c...
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