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International audience; Prions are infectious proteins responsible for a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs), or prion diseases. In mammals, prions reproduce themselves by recruiting the normal cellular protein (PrPC) and inducing its conversion to the disease-causing isoform denominated PrPSc. Recently, anti-prion antibodies have been shown to permanently cure prion-infected cells. However, the inability of full-lenght antibodies and proteins to cross the blood-brain barrier (BBB) hampers their use in the therapy of TSEs in vivo. Alternatively, brain delivery of prion-specific scFv by adeno-associated virus (AAV) transfer delays the onset of the disease in infected mice, although protection is not complete. We investigated the anti-prion effects of a recombinant anti-PrP (D18) single-chain variable fragment (scFv) by direct addition to scrapie infected cell cultures or by infection with both lentivirus and adeno-associated virus (AAV) transducing vectors. We show that recombinant anti-PrP scFv is able to reduce proteinase K-resistant PrP content in infected cells. In addition, we demonstrate that lentiviruses are more efficient than AAV in gene transferring of the anti-PrP scFv gene and in reducing PrPSc content in infected neuronal cell lines. Finally, we have used a bioinformatic approach to construct a structural model of D18scFv-PrPC complex. Interestingly, according to the docking results, ArgPrP151 (Arg151 from prion protein) is the key residue for the interactions with D18scFv, anchoring the PrPC to the cavity of the antibody. Taken together, these results indicate that combined passive and active immunotherapy targeting PrP might be promising strategies for therapeutic intervention in prion diseases.

The most relevant cause of morbidity and mortality in cystic fibrosis (CF) patients is the lung pathology characterized by chronic infection and inflammation sustained mainly by Pseudomonas aeruginosa (P. aeruginosa). Innovative pharmacological approaches to control the excessive inflammatory process in the lung of CF patients are thought to be beneficial to reduce the extensive airway tissue damage. Medicinal plants from the so-called traditional Asian medicine are attracting a growing interest because of their potential efficacy and safety. Due to the presence of different active compounds in each plant extract, understanding the effect of each component is important to pursue selective and reproducible applications. Extracts from Emblica officinalis (EO) were tested in IB3-1 CF bronchial epithelial cells exposed to the P. aeruginosa laboratory strain PAO1. EO strongly inhibited the PAO1-dependent expression of the neutrophil chemokines IL-8, GRO-alpha, GRO-gamma, of the adhesion molecule ICAM-1 and of the pro-inflammatory cytokine IL-6. Pyrogallol, one of the compounds extracted from EO, inhibited the P. aeruginosa-dependent expression of these pro-inflammatory genes similarly to the whole EO extract, whereas a second compound purified from EO, namely 5-hydroxy-isoquinoline, had no effect. These results identify Pyrogallol as an active compound responsible for the anti-inflammatory effect of EO and suggest to extend the investigation in pre-clinical studies in airway animal models in vivo, to test the efficacy and safety of this molecule in CF chronic lung inflammatory disease.

Human NK cells cultured in the presence of IL-12 or IL-4 differentiate into cell populations with distinct patterns of cytokine secretion similar to Th1 and Th2 cells. NK cells grown in IL-12 (NK1) produce IL-10 and IFN-gamma, whereas NK cells grown in IL-4 (NK2) produce IL-5 and IL-13. Although these NK cell subsets do not differ in cytotoxic activity, NK1 cells express higher levels of cell surface CD95 (Fas) Ag than NK2 cells and are more sensitive to Ab or chemically induced apoptosis. Like Th1 cells, NK1 cells accumulate much higher levels of the IL-12Rbeta2-chain mRNA and are significantly more responsive to IL-12 than NK2 cells at the level of activation of STAT4 transcription factor. The identification of NK cell subsets that are analogous to T cell subsets suggests a new role for NK cells in innate inflammatory responses and in their effect on adaptive immunity.

We have studied the expression of the intermediate filament (IF) proteins, vimentin and glial fibrillary acidic protein (GFAP), in cultured human Schwann cells (SC) from patients with different neuropathies and normal control cases. SC cultures from sural nerve biopsies of 8 subjects with axonal neuropathies, 8 with demyelinating neuropathies and 3 normal controls were included in this study and processed with double immunofluorescence technique, using anti-vimentin and anti-GFAP antibodies, during the 2nd, 4th and 6th week of culture. Five cultures incubated with anti-GFAP antibodies were also processed for immunoelectron microscopy. Specificity tests of the used antibodies were performed. We have found that: (1) cultured human SC constantly express vimentin; (2) SC from normal controls are GFAP-negative in the first period of culture; (3) SC from pathologic nerves can contain GFAP-immunoreactive IF and the percentage of GFAP-positive SC is higher in axonal than in demyelinating neuropathies; (4) during the permanence in culture human SC from both normal and pathologic cases acquire the ability to synthesize GFAP. The obtained data suggest that the removal from axonal contact and the resulting loss of myelinating function induce a cytoskeletal cellular response in human SC characterized by the cytoplasmic accumulation of GFAP-immunoreactive IF.

In order to better characterize the mechanisms which regulate the immune response at the pulmonary level, the effects of beta-interferon (IFN-beta) and lymphokines (LK) on prostaglandin E (PGE) release and the suppressive capacity of mouse resident alveolar (AM phi) and pleural macrophages (PlM phi) were investigated in comparison with peritoneal macrophages (PM phi). After in vitro exposure to IFN-beta, PlM phi and PM phi showed a significant decrease of suppressive capacity and PGE release, whereas LK treatment did not affect such activities. In contrast, pre-treatment of AM phi with LK caused a strong impairment of their suppressive capacity. This effect was optimal after an incubation time of 20 h, was evident also at very low doses of LK and was not paralleled by any change of PGE release. Again in contrast with PlM phi and PM phi, suppressive capacity of AM phi was decreased only by very high doses of IFN-beta, whereas lower doses caused either an increase or no change of this activity. Furthermore, PGE release by AM phi was markedly increased after treatment with IFN-beta. Thus, suppressive capacity of AM phi appears to be controlled by different mechanisms from those of PlM phi and PM phi. In addition, a dissociation is evident between suppressive capacity and PGE release by AM phi.