SBIR-STTR Award

The eosinophil is a peripheral blood leukocyte containing an abundance of cytoplasmic granules, rich in cationicprotein toxins. Among these, the most abundant on a molar basis is the major basic protein-1, eMBP1. eMBP1kills helminths, bacteria, and numerous cells, such as respiratory epithelium, but also activates cells, includingbasophils and mast cells. Studies of human diseases show that eMBP1 is present in secretions from patientswith eosinophil-mediated diseases, including asthma, chronic rhinosinusitis, and gastrointestinal diseases, andit is deposited on damaged targets. These studies show that the eosinophil mediates its damage to parasitesand tissues by discharging its toxin rich granules onto microbial targets and tissues. eMBP1 is synthesized asa precursor, pro-eMBP1, composed of eMBP1 and a remarkably acidic pro-piece sequence. Developingeosinophils synthesize pro-eMBP1, and the pro-piece is removed during granule maturation. Analyses of pro-piece in different models show that it can neutralize the toxic eMBP1 effect and also the toxicity of the eosinophilcationic protein (ECP). This project proposes that neutralization of eMBP1 can be a treatment to mitigate tissuedamage in eosinophil-related diseases. It will develop a pro-piece product for treatment of eosinophil-mediateddiseases by neutralization of eMBP1 and other granule toxins. Currently, no therapies for these diseasesaddress the neutralization of granule proteins.In this project, the pro-piece will be expressed and associated glycans will be modified to identify the optimalform of the molecule for binding to and neutralizing eMBP1. These tests will determine whether glycosylation ofpro-piece importantly alters its binding to eMBP1 by analyzing whether N- or O- glycans and theglycosaminoglycan at S62 modify its activities. The project will identify the most important pro-piece regions forbinding eMBP1 and neutralizing it by creating overlapping peptides and creating mutants with increased aminoacid sequences characteristic of active regions. Evaluation of the pro-piece panel for binding to and inhibitingeMBP1 will be evaluated here in several in vitro models. The goal of this project is to identify an active form ofthe pro-piece with optimal inhibition effects on eMBP1 and other granule toxic proteins. This form will be furtherinvestigated and developed in future projects. Narrative The eosinophil, a type of immune cell in the blood circulation, presently is known to cause damage in various diseases, including asthma, chronic sinus inflammation, gastrointestinal disorders, and allergic inflammation. This proposed project is directed at developing a treatment to neutralize toxic products from eosinophils that contribute to these diseases. A successful completion of this project has the potential to treat a wide range of eosinophil-mediated diseases and improve the lives of a large population suffering from them. Amino Acid Sequence ; Primary Protein Structure ; protein sequence ; Asthma ; Bronchial Asthma ; Bacteria ; Basophils ; Basophilic Granulocyte ; Blood Basophil ; Blood Circulation ; Bloodstream ; Circulation ; Complementary DNA ; cDNA ; Cations ; Cells ; Cell Body ; Cytoplasmic Granules ; granule ; Disease ; Disorder ; Embryo ; Embryonic ; eosinophil ; Blood Eosinophil ; Eosinophilic Granulocyte ; Eosinophilic Leukocyte ; Marrow Eosinophil ; Future ; Gastrointestinal Diseases ; gastrointestinal disorder ; Glycosaminoglycans ; Mucopolysaccharides ; glycosylation ; Metabolic Glycosylation ; Goals ; Helminths ; Parasitic Worms ; Histamine Release ; Histamine Liberation ; Human ; Modern Man ; Inflammation ; Allergic inflammation ; Lead ; Pb element ; heavy metal Pb ; heavy metal lead ; Leukocytes ; Blood leukocyte ; Leukocytes Reticuloendothelial System ; Marrow leukocyte ; White Blood Cells ; White Cell ; white blood cell ; white blood corpuscle ; mast cell ; Marrow Mast Cell ; Tissue Basophils ; mastocyte ; Methods ; Molecular Weight ; Neutralization Tests ; antibody neutralization test ; Sinus ; Accessory Sinuses ; Nasal Sinuses ; Nasal cavity/Paranasal ; Nasal cavity/Paranasal sinuses ; Paranasal Sinuses ; Parasites ; Patients ; Peptides ; Polysaccharides ; Glycans ; Production ; Proteins ; Rattus ; Common Rat Strains ; Rat ; Rats Mammals ; Research ; Testing ; Tissues ; Body Tissues ; Toxin ; eosinophil peroxidase ; Measures ; Mediating ; Eosinophil Granule Proteins ; improved ; Chronic ; Phase ; Biological ; Link ; Chemicals ; Evaluation ; Structure of respiratory epithelium ; Respiratory Epithelium ; peripheral blood ; Measurement ; analog ; Inflammatory ; Deposit ; Deposition ; Immunes ; Immune ; Renal Cell ; kidney cell ; mutant ; Tumor Cell ; neoplastic cell ; success ; Surface Plasmon Resonance ; microbial ; K562 Cells ; Toxicities ; Toxic effect ; Structure ; Allergic ; Modeling ; Eosinophil-Associated Ribonuclease ; RNS3 Protein ; RNase 3 ; Serum Eosinophil Cationic Protein ; Eosinophil cationic protein ; Molecular Interaction ; Binding ; Address ; Avidity ; in vitro Model ; K-562 ; K562 ; K562 blasts ; Small Business Innovation Research Grant ; SBIR ; Small Business Innovation Research ; Characteristics ; Molecular ; Process ; Development ; developmental ; design ; designing ; Population ; human disease ; chronic rhinosinusitis ; targeted agent ; experimental study ; experiment ; experimental research ; in vivo evaluation ; in vivo testing ;

The eosinophil is a peripheral blood leukocyte containing an abundance of cytoplasmic granules, rich in cationicprotein toxins. Among these, the most abundant on a molar basis is the major basic protein-1, eMBP1. eMBP1kills helminths, bacteria, and numerous cells, such as respiratory epithelium, but also activates cells, includingbasophils and mast cells. Studies of human diseases show that eMBP1 is present in secretions from patientswith eosinophil-mediated diseases, including asthma, chronic rhinosinusitis, and gastrointestinal diseases, andit is deposited on damaged targets. These studies show that the eosinophil mediates its damage to parasitesand tissues by discharging its toxin rich granules onto microbial targets and tissues. eMBP1 is synthesized asa precursor, pro-eMBP1, composed of eMBP1 and a remarkably acidic pro-piece sequence. Developingeosinophils synthesize pro-eMBP1, and the pro-piece is removed during granule maturation. Analyses of pro-piece in different models show that it can neutralize the toxic eMBP1 effect and also the toxicity of the eosinophilcationic protein (ECP). This project proposes that neutralization of eMBP1 can be a treatment to mitigate tissuedamage in eosinophil-related diseases. It will develop a pro-piece product for treatment of eosinophil-mediateddiseases by neutralization of eMBP1 and other granule toxins. Currently, no therapies for these diseasesaddress the neutralization of granule proteins.In this project, the pro-piece will be expressed and associated glycans will be modified to identify the optimalform of the molecule for binding to and neutralizing eMBP1. These tests will determine whether glycosylation ofpro-piece importantly alters its binding to eMBP1 by analyzing whether N- or O- glycans and theglycosaminoglycan at S62 modify its activities. The project will identify the most important pro-piece regions forbinding eMBP1 and neutralizing it by creating overlapping peptides and creating mutants with increased aminoacid sequences characteristic of active regions. Evaluation of the pro-piece panel for binding to and inhibitingeMBP1 will be evaluated here in several in vitro models. The goal of this project is to identify an active form ofthe pro-piece with optimal inhibition effects on eMBP1 and other granule toxic proteins. This form will be furtherinvestigated and developed in future projects.

Public Health Relevance Statement:
Narrative The eosinophil, a type of immune cell in the blood circulation, presently is known to cause damage in various diseases, including asthma, chronic sinus inflammation, gastrointestinal disorders, and allergic inflammation. This proposed project is directed at developing a treatment to neutralize toxic products from eosinophils that contribute to these diseases. A successful completion of this project has the potential to treat a wide range of eosinophil-mediated diseases and improve the lives of a large population suffering from them.

Project Terms: