Use of recombinant human uteroglobin in treatment of inflammatory and fibrotic conditions
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example 1
Production and Characterization of rhUG
[0054] Recombinant human UG is produced by the following procedures, and possesses a level of purity exceeding 99% such that it may be used according to the present invention to inhibit PLA2 activity and reduce fibronectin deposition in vitro and in vivo.
[0055] Construction of Bacterial Gene for rhUG
[0056] A synthetic gene for human UG is assembled from oligonucleotides and cloned into pBR322 for DNA sequencing to confirm the sequence. Because native UG has a glutamic acid residue at its N-terminus, an initiator methionine will be added. Mantile et al (1993; supra) have previously shown that addition of Met-Ala-Ala (SEQ ID NO:5) at the N-terminus of recombinant hUG does not alter its activity as an inhibitor of PLA.sub.2. Nor does a Met residue at the N-terminus (which allows bacterial translational initiation) effect is activity as a PCB-binding protein (Hard et al, 1996; supra). Bam Hi sticky ends are located at both ends of the annealed g...
example 2
In Vivo Experiments
[0084] One male and one female of the species P. cyanocephalus, weighing approximately 400 grams each were delivered by C-section at 142 days of gestation. This is an established model of RDS (Coalson, J. J., et al. Baboon Model of BPD. II: Pathologic features. Exp. Mol. Pathol. 37: 355-350 (1982)).
[0085] After delivery, the infants were anesthetized with ketamine (10 mg / kg) and intubated with a 2.5 mm diameter endotracheal tube. Blood gases and pressure were monitored via an arterial line placed by percutaneous injection into the radial artery. A deep venous line was placed percutaneously into this saphenous vein through which fluids and drugs were administered. Animals were maintained on servo-controlled infrared warmers and ventilated with a standard time-cycled, pressure-regulated ventilator with humidifiers maintained at 36-37° C. Initial settings were FiO21.0, rate 40 / min., I / E ratio 1:1.5, positive end expiratory pressure (PHEP) at 4 cm H2O, and peak insp...
example 3
Inhibition of Hydrolysis of Artificial Surfactant by Soluble PLA2 s in vitro
[0088] RhUG inhibits hydrolysis of artificial surfactant by soluble PLA2s in vitro. Survanta is an artificial surfactant derived from bovine lung and is used to treat pre-term neonates with RDS and adults with RDS (ARDS). Hydrolysis of Survanta by a Group I soluble PLA2, i.e., porcine, pancreatic PLA2 (Sigma) is characterized by its ability to compete as a substrate with a fluorescent phosphatidylcholine substrate (Cayman Chemicals), generating arachidonic acid as a product.
[0089] Survanta is a substrate for in vitro degradation by Group I soluble PLA2s. Survanta is rapidly degraded in vitro by PLA2s found in the extracellular fluids of a human lung. RhUG inhibits degradation of Survanta in vitro.
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