Patients were informed by the clinicians; if they did not express their opposition to research, the de-identified samples were immediately processed and embedded in paraffin. Paraffin-embedded sections of arterial tissue from mouse, rabbit or human were used in IHC experiments. vector (pPICZA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in (30 mg/L culture). The advantage of as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. Introduction Atherosclerosis is an inflammatory disease associated with the formation of unstable thrombosis-prone atheroma plaques made of large lipid cores, thin fibrous cap and inflammatory cell infiltrates within the walls of arteries.[1] Atherosclerotic plaque rupture is the mechanistic cause of about 75% of all sudden and often fatal heart attacks.[2] As the risk of plaque rupture is more related to the plaque contents than to the plaque size, Neostigmine bromide (Prostigmin) molecular imaging modalities have risen as a new F2 imperative. Current studies tend towards the development of non-invasive targeted methods to assess the cellular components that underlie the risk of rupture.[3,4] Molecular imaging requires highly sensitive and specific probes made of a signal detection compound and an affinity ligand for targeting. The affinity ligand should be able to recognize molecules and cells over-expressed during the course of atherogenesis. Inflammation is a well-recognized pathophysiological process involving both innate and adaptive immune cells.[5] Recruitment of monocytes in the vascular wall and macrophage differentiation and proliferation represent a hallmark in the pathology of atherosclerotic lesions.[6] They contribute to the processes that underlie atherogenesis such as lipid accumulation, secretion of pro-inflammatory cytokines, extracellular matrix remodelling. Moreover, the observation of activation and oligoclonal expansion of T cells has suggested the presence of inciting antigens (Ags) that sustain T cell recruitment within coronary lesions.[7] B cells also play a pro or anti-atherogenic role depending on the subtypes (B1(a) or B2), and in atherosclerosis they accumulate both in the atherosclerotic intima and associated adventitia.[8C10] More recently, platelets have come to the forefront as partners of macrophages, T cells and B cells in inflammation and immune responses. They are now recognized as key players in innate and adaptive immune responses [11, 12] and notably shown to modulate the T-effector/T-regulator balance via the CD40 ligand.[13,14] Platelet-derived CD40 ligand has also been reported to support B-cell differentiation and immunoglobulin class switching in mice.[15] Several cytokines released by activated platelets have been demonstrated to modulate monocyte and macrophage function.[16] Moreover plateletleukocyte interactions largely contribute to OxLDL uptake and foam cell formation.[17] A recent study has underlined the presence of platelets not only in thrombi and intraplaque hemorrhage but also in atheroma burden, around necrotic areas and neovessels, shedding light on the rationale for targeting platelets within atherosclerotic lesions.[18] Today, antibodies are used for several applications in research, diagnostics, and therapy.[19] Technology improvements are focused on several approaches to manufacturing recombinant human antibodies.[20] Moreover, selection technologies such as antibody phage display or ribosomal display have accelerated the generation of these recombinant human antibodies.[21C23] To develop a novel non-invasive targeting approach for atheroma, our team previously selected, using phage display biotechnology on activated platelets, a phage-scFv fully human antibody (HuAb) specific to the IIb3 integrin, which is an integrin only expressed on platelets and not on other immune cells.[24] This human antibody was further processed as a whole human IgG4 molecule in baculovirus system. [18] We proved the maintenance of the bioactivity after grafting onto superparamagnetic nanoparticles dedicated to MRI imaging. However, the chemical functionalization was hard to proceed, time-consuming and we did not succeed in grafting more than one HuAb Neostigmine bromide (Prostigmin) Neostigmine bromide (Prostigmin) onto each nanoparticle [18]. To overcome these drawbacks and obtain a better conjugation ratio, another type of protein engineering has been applied to reduce the probe size. A scFv protein composed of the heavy (VH) and light (VL) chains of an antibody linked with a flexible peptide, has been constructed by recombinant DNA technology. The diameter of scFv fragments (5 nm), one-fifth the size.