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Surface Assay for Specific Detection of Soluble Amyloid Oligomers Utilizing Pronucleon Peptides Instead of Antibodies.

Immunoassays such as enzyme-linked immunosorbent assays (ELISAs) are widely used for diagnostics; however, antibodies as detection reagents may be insufficiently selective and have other shortcomings. We present a novel non-antibody-based detection method based on binding target molecules to peptides (used as recognition molecules): a surface assay for A-β oligomers employing a peptide comprising amino acid residues of the human β-amyloid protein (Pronucleon peptide) as the capture agent. For the sake of convenience, we term this the "Pronucleon peptide-linked immunosorbent assay", or PLISA. Pronucleon peptides are amino acid sequences matched to target amyloids of interest, in particular soluble Aβ-1-42 amyloid protein oligomers, which are widely considered as an early biomarker for Alzheimer's disease in body fluids. The Pronucleon peptide in a PLISA is immobilized on the surface and substitutes for the capture antibody used in an ELISA for binding the Aβ-1-42 oligomers present in the sample. We present data comparing synthetic oligomer PLISAs in spiked buffer and body fluids (such as cerebrospinal fluid, brain extracts, or whole blood) to those from an ELISA and demonstrate better selectivity of the PLISA for amyloid β-42 oligomers versus monomers and fibrils. The detection limit, calculated as the mean (blank) plus three standard deviations, was in the range of 0.35-1.5 pM (32-135 ng/L) (oligomers contained approximately 20 monomers on average).

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In Vivo and Ex Vivo Imaging of Amyloid-β Cascade Aggregates with a Pronucleon™ Peptide

Accumulation of amyloid-β (Aβ) cascade aggregates is considered a hallmark of Alzheimer's disease (AD). Current dogma holds that the appearance of Aβ oligomers and larger aggregates occur many years prior to plaque formation associated with the advanced and irreparable neurocognitive decline characteristic of AD. This premise is the impetus to identify these Aβ precursor structures prior to advanced plaque development. The Pronucleon™ technology platform is comprised of a novel series of engineered peptides that provide a unique readout when associated with beta-rich fiber and oligomeric Aβ. This technology has been applied to Ex Vivo tissue sections and In Vivo mouse models of AD to determine the potential utility of these synthetic peptides as potential imaging agents. In Ex Vivo studies, the Pronucleon™ peptide binds plaque like structures in brain sections obtained from transgenic mice overexpressing hAPP with both the human Swedish and London Aβ mutations. In Vivo, Pronucleon™ peptide administered peripherally can localize to the brain and label plaques throughout the brain in transgenic mice. Taken together, the data suggest that Pronucleon™ could provide a new imaging tool for Aβ cascade elements that precede advanced plaque and fibril formation, thereby advancing early diagnosis and treatment opportunities.

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072 Wound Healing Properties of Reconstituted Freeze-Dried Platelets

The use of fresh platelets has gained value in medicine as an essential part of wound treatments. This is not surprising since platelets contain a number of bioactive factors that contribute to the process of wound healing, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF). Fresh platelets’ short shelf life limits platelet-based therapies. If platelets can be stabilized in freeze-dried form (FDP) then long-term storage as well as pathogen inactivation methods become possible. Adlyfe and Oregon Freeze-Dry have been developing technology to stabilize freeze-dried human platelets that can be subjected to gamma irradiation and stored for a long duration. Upon reconstitution, irradiated FDP retained growth factors PDGF-AB, PDGF-BB, and TGF-B1 in quantities similar to fresh platelets as judged by capture ELISA. The rehydrated FDP promoted new DNA synthesis and cellular proliferation of primary human dermal fibroblasts and endothelial cells (HUVECs) similar to fresh platelets. The FDP also promoted remodeling of extracellular matrix by accelerating fibroblast-mediated contraction of collagen gels and stimulated HUVECs to undergo angiogenesis and form capillary structures in vitro. Therefore, we conclude that FDP and fresh platelets have comparable in vitro wound healing potential. Preclinical wound healing studies in diabetic mice are under way and further development will allow FDP to be a safe and well-suited alternative to fresh platelets for wound healing applications.

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