Pleiotrophin (PTN), a neurotrophic factor with important roles in survival and differentiation of dopaminergic neurons, is up-regulated in the nucleus accumbens after amphetamine administration suggesting that PTN could modulate amphetamine-induced pharmacological or neuroadaptative effects. To test this hypothesis, we have studied the effects of amphetamine administration in PTN genetically deficient (PTN -/-) and wild type (WT, +/+) mice. In conditioning studies, we found that amphetamine induces conditioned place preference in both PTN -/- and WT (+/+) mice. When these mice were re-evaluated after a 5-day period without amphetamine administration, we found that WT (+/+) mice did not exhibit amphetamine-seeking behaviour, whereas, PTN -/- mice still showed a robust drug-seeking behaviour. In immunohystochemistry studies, we found that amphetamine (10 mg/kg, four times, every 2 hours) causes a significant increase of glial fibrillary acidic protein positive cells in the striatum of amphetamine-treated PTN -/- mice compared with WT mice 4 days after last administration of the drug, suggesting an enhanced amphetamine-induced astrocytosis in the absence of endogenous PTN. Interestingly, we found in concomitant in vitro studies that PTN (3 µM) limits amphetamine (1 mM)-induced loss of viability of PC12 cell cultures, effect that could be related to the ability of PTN to induce the phosphorylation of Akt and ERK1/2. To test this possibility, we used specific Akt and ERK1/2 inhibitors uncovering for the first time that PTN-induced protective effects against amphetamine-induced toxicity in PC12 cells are mediated by the ERK1/2 signalling pathway. The data suggest an important role of PTN to limit amphetamine-induced neurotoxic and rewarding effects.

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.

No abstract

No abstract