We report the construction and the use of a phage display human antibody library (>3 × 10⁸clones) based on principles of protein design. A large repertoire of functional antibodies with similar properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto the two antibody germ line segments most frequently found in human antibodies. With this strategy we concentrated sequence diversity in regions of the antibody structure that are centrally located in the antigen binding site, while leaving residues in more peripheral positions available for further mutagenesis aimed at improving the affinity of the selected antibodies. In addition, the library was tested by selecting antibodies against six biologically relevant antigens. Using only 0.3 μg of antigen eluted from a two-dimensional gel spot, we isolated binders specific for the ED-B domain of fibronectin, a marker of angiogenesis. These antibodies recognize the native antigen with affinities in the 10⁷–10⁸ m ⁻¹ range, and perform well in immunosorbent assays, in two-dimensional Western blotting and in immunohistochemistry. The affinity of one anti-ED-B antibody was improved by 27-fold by combinatorially mutating six strategically selected residues in the heavy chain variable domain. A further 28-fold affinity improvement could be achieved by mutating residues 32 and 50 of the light chain. The resulting antibody, L19, bound to the ED-B domain of fibronectin with very high affinity (K _d = 54 pm), as determined by real-time interaction analysis with surface plasmon resonance detection, band shift analysis, and by competition experiments with electrochemiluminescent detection.

Osteosarcomas (OSs) are bone tumors most commonly found in pediatric and adolescent patients characterized by high risk of metastatic progression and recurrence after therapy. Effective therapeutic management of this disease still remains elusive as evidenced by poor patient survival rates. To achieve a more effective therapeutic management regimen, and hence patient survival, there is a need to identify more focused targeted therapies for OSs treatment in the clinical setting. The role of the OS tumor stroma microenvironment plays a significant part in the development and dissemination of this disease. Important components, and hence potential targets for treatment, are the tumor-infiltrating macrophages that are known to orchestrate many aspects of OS stromal signaling and disease progression. In particular, increased infiltration of M2-like tumor-associated macrophages (TAMs) has been associated with OS metastasis and poor patient prognosis despite currently used aggressive therapies regimens. This review aims to provide a summary update of current macrophage-centered knowledge and to discuss the possible roles that macrophages play in the process of OS metastasis development focusing on the potential influence of stromal cross-talk signaling between TAMs, cancer-stem cells and additional OSs tumoral microenvironment factors.

The available amino acid sequences of 150-kDa botulinum and tetanus neurotoxins show the presence of a closely homologous segment in the middle of the light chain (NH2-terminal 50 kDa), which is the intracellularly active portion of the toxin. This segment contains the zinc binding motif of metalloendopeptidases, HEXXH. Atomic adsorption analysis of botulinum neurotoxins (serotypes A, B, and E) made on the basis of this observation demonstrated the presence of one zinc atom/molecule of 150-kDa neurotoxin. Conditions were found for the removal of the zinc ion with chelating agents and for the restoration of the normal metal content. The conserved segment, which includes the zinc binding motif, was synthesized and shown to bind [65Zn]2+. Chemical modification experiments indicated that two histidines and no cysteines are involved in Zn2+ coordination in agreement with a probable catalytic role for the zinc ion. The present findings suggest the possibility that botulinum neurotoxins are zinc proteases.

Amyloid-β (Aβ) peptide is commonly found in human Alzheimers disease (AD) brain and is the main component of Alzheimer amyloid plaques. The predominant forms of Aβ in the human brain are Aβ(1-40) and Aβ(1-42), but Aβ(25-35) fragment, physiologically present in elderly people, is the more toxic region and has been recently found to play a relevant role in AD, due to its peculiar aggregation properties. In this work, we review the current understanding on the conformations and biological activity of Aβ(25-35) exploring aggregation, cytotoxic and neurodegenerative properties of this fundamental Aβ fragment, in order to provide an effective starting point to better approach a pathology spread and problematic as AD.