Phosphatidylserine (PS) exposure on the cell surface is an early marker of apoptosis. To select PS binding peptides as vectors of contrast agents to image apoptosis, a phage library has been exposed to perfused mouse livers. Phages not retained on control livers during the first perfusions were used for selections on apoptotic livers in a second series of perfusions. Four selected phages were further evaluated for binding to PS-coated enzyme-linked immunosorbent assay (ELISA) plates. They presented an apparent affinity constant (Ka app ) for PS ranging from 6.08 × 10 10 M to 1.62 × 10 11 M. These phages did not bind to phosphatidylcholine, and competition with annexin V confirmed their specific interaction with PS. The phage with the highest affinity-bound PS in ELISA with a Ka app = (1.6 ± 0.2) × 10 11 M. It carried the TLVSSL peptide that was synthesized. Specific competition with annexin V and with the synthetic peptide was performed and confirms the specificity of the interaction. (Journal of Biomolecular Screening 2006:537-545)
Phage display, an extremely promising technology in the context of molecular imaging, allows for the selection of peptides interacting with virtually any target from a heterogeneous mixture of bacteriophages. In this work, we propose the concept of magnetophages, obtained by covalent coupling of ultrasmall particles of iron oxide (USPIO) to the proteins of the phage wall. To validate magnetophages as a magnetic resonance imaging contrast agent (MRI), we have used as a prototype the clone E3 because of its specific affinity for phosphatidylserine, a marker of apoptosis. Enzyme-linked immunosorbent assay showed that E3 magnetophages incubated with phosphatidylserine retained the properties of the nonmagnetically labeled phages. The usefulness of magnetophages as an MRI contrast agent was estimated by incubation with phosphatidylcholine and phosphatidylserine or with apoptotic and control cells. Under these conditions, E3 magnetophages allow the discrimination of phosphatidylserine from phosphatidylcholine and of apoptotic cells from control ones. Injected in vivo, magnetophages are rapidly cleared from the blood stream and internalized by the phagocytic cells of the liver. To abrogate this problem, USPIO were pegylated to obtain stealthy E3-PEG-magnetophages, invisible to phagocytic cells, which were successfully targeted to apoptotic liver. If this feature demonstrated for E3 magnetophages can be extrapolated to other phage display selected entities, magnetophages become an original system which allows validation of the candidate binding peptides before their synthesis is considered. The concept of the magnetophage could be extended to other imaging modalities by replacing USPIO with an adequate reporter (i.e., radiolabeled phages).
Rationale and Objectives: As a neurodegenerative pathology, Alzheimer disease (AD) is the principal cause of dementia in the elderly (before cerebral stroke and Parkinson disease) and the fourth cause of mortality in developed countries (after cardiac diseases, cancer and cerebral stroke). Its diagnosis can only be confirmed by autopsy, since the symptomatology is common to other neurodegenerative pathologies. The senile plaques, constituted essentially by the amyloid peptide Ab 1-42 (Ab 1-42 ), represent the main morpho-pathological feature of Alzheimer disease. An early and non-invasive diagnosis of this pathology would allow a more efficient treatment and may help to prolong the life expectancy of patients suffering from it. Molecular imaging is a recent and growing discipline, which could help to differentiate AD from other dementias. We propose to attempt MRI detection of the amyloid plaques by using peptides selected by phage display and subsequently grafted to adequate magnetic reporters. Methods: To select peptides with high affinity for the senile plaques, a disulfide constrained phage display heptapeptide library (New England Biolabs, The Netherlands) was incubated with Ab 1-42 (Bachem, Switzerland), which was immobilized by hydrophobic interactions on a plastic surface. After four rounds of selection, 72 candidate phage clones were arbitrarily isolated for further screening of their affinity for the target. The ELISA tests of affinity highlighted 23 phage clones with an optimal affinity for Ab 1-42 . Their peptide structure was subsequently determined by analysis of the DNA sequence of the fusion insert, and their dissociation constants (K d ) for the target were estimated with the aim of identifying the most efficient peptides. The best one was obtained by solid-phase synthesis and its K d was again evaluated after biotinylation. Results: The peptide sequence analysis of the 23 selected phage clones evidenced a more frequent representation of the amino acids Leu, Pro, His and Phe. With the exception of His, these amino acids are hydrophobic. This observation shows the importance of hydrophobic interactions with the target molecule. Concerning the amino acid representation in the peptide structures, the hydrophobic amino acids frequently occupy the first positions whereas Asn and Gln are well represented ($70%) in the seventh position of the insert. The K d of the 23 selected phage clones ranged from 2.2Â10 À10 to 2.0Â10 À9 M. Conclusion:The peptide with the most important K d was selected for further evaluation and in vivo detection of the senile plaques in AD by MRI; it will be linked to optimal paramagnetic and superparamagnetic reporters and tested with in vivo models. Rationale and Objectives: Phage display is a powerful tool in the context of molecular imaging. This technique allows one to select, from a heterogeneous mixture of bacteriophages, each displaying a different peptide on its proteic wall, a vector that is subsequently coupled to a 'contrastophore', i.e. USPIO for MRI. In a previous st...
For the first time, MQCT and MRI have been performed in parallel to investigate the same physiological problem. The existence of 2 liver groups seems to be the result of some instability of the protocol likely to be related to surgery.
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