Neutrophils are a major contributor to the cellular damage that accompanies reperfusion of ischemic tissues. It is planned to clone and produce through recombinant DNA methods a human protein that blocks attachment of neutrophils to endothelial cells in vivo and thereby prevents the entry of neutrophils into reperfused tissues and the consequent organ damage. This research will also lead to the development of a new technology for the treatment of a large class of human inflammatory diseases, such as adult respiratory distress syndrome, irritable bowel disease, and idiopathic pulmonary fibrosis, in which neutrophils appear to play a destructive role.In Phase I, Synergen, Inc., will immunize mice and select for monoclonal antibodies that react with the protein, using an in vitro functional assay. These monoclonals will be used to identify the protein on Western blots and in immunoprecipitation experiments. In Phase II, the monoclonal antibodies will be used to purify enough of the protein to determine a partial amino acid sequence. Oligonucleotides corresponding to the determined amino acid sequence and/or the monoclonal antibodies will be used to isolate a CDNA encoding the protein from a CDNA expression library. The protein will be produced in large quantities through recombinant DNA methods to evaluate its effectiveness in vitro and in vivo for preventing attachment of the neutrophils to endothelial cells and consequent damage to reperfused tissues.
Anticipated Results:The human receptor protein developed from this research will aid in preventing death and organ damage that accompanies reperfusion of ischemic tissues. Human diseases for which the protein will have therapeutic benefits include heart attacks, strokes, and organ transplants. Once cloned, the receptor protein will permit the rapid screening, identification, and development of new or second-generation drugs that can be administered continuously to patients suffering from chronic inflammatory diseases.National Heart, Lung, And Blood Institute (NHLBI)
