Novel anti-ErbB3 monoclonal antibodies show therapeutic efficacy in xenografted and spontaneous mouse tumors

The role of the ErbB3 receptor in signal transduction is to augment the signaling repertoire of active heterodimeric ErbB receptor complexes through activating the PI3K/AKT pathway, which in turn promotes survival and proliferation. ErbB3 has recently been proposed to be involved in acquired resistance to tyrosine kinase inhibitors (TKIs), and is therefore a promising new drug cancer target. Since ErbB3 is a kinase defective receptor, it cannot be targeted by small molecule inhibitors, whereas monoclonal antibodies may offer a viable strategy for pharmacological intervention. In this study, we have utilized DNA electroporation (DNA-EP) to generate a set of novel hybridomas directed against human ErbB3, which have been characterized for their biochemical and functional properties and selected for their ability to negatively regulate the ErbB3-mediated signaling pathway. In vitro, the anti-ErbB3 antibodies modulate the growth rate of cancer cells of different origins. In vivo they show antitumoral properties in a xenograft model of human pancreatic tumor and in the ErbB2-driven carcinogenesis genetically engineered mouse model (GEMM) for mammary tumor, the BALB/neuT. Our data confirm that downregulating the ErbB3-mediated signals with the use of anti-ErbB3 monoclonal antibodies is both feasible and relevant for therapeutic purposes and provides new opportunities for novel anti-ErbB3 combinatory strategies for cancer treatment.     

Applications of microbial fermentations for production of gluten-free products and perspectives

A gluten-free (GF) diet is recognised as being the only accepted treatment for celiac disease—a permanent autoimmune enteropathy triggered by the ingestion of gluten-containing cereals. The bakery products available in today’s gluten-free market are characterised by lower palatability than their conventional counterparts and may lead to nutritional deficiencies of vitamins, minerals and fibre. Thus, the production of high-quality gluten-free products has become a very important socioeconomical issue. Microbial fermentation by means of lactic acid bacteria and yeast is one of the most ecological/economical methods of producing and preserving food. In this review, the role of a fermentation process for improving the quality of GF products and for developing a new concept of GF products with nutraceutical and health-promoting characteristics will be examined.     

Decreased subunit stability as a novel mechanism for potassium current impairment by a KCNQ2 C terminus mutation causing benign familial neonatal convulsions

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 microm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates.     

Human chondrocytes and mesenchymal stem cells grown onto engineered scaffold

The development of improved methods for treatment of chondral defects using autologous cells in combination with biomaterials leads to a new generation of implantable devices. Their association gives rise to a hybrid construct combining biological and material components that can be specifically committed. The comprehension of cellular and molecular mechanisms of cartilage repair and the use of biomaterials in combination with chondrocytes or mesenchymal stem cells in the treatment of cartilage defects has opened a new era of therapeutical strategies. Recently, their applicability in the treatment of early lesions in osteoarthritis is under investigation. To obtain new information on the behaviour of chondrocytes and mesenchymal stem cells grown on a hyaluronan derivative scaffold (Hyaff-11) already used in cartilage repair, we analysed a series of molecules expressed by these cells by Real-Time RT-PCR and immunohistochemical analyses. The data obtained with this work showed that this biomaterial is able to reduce the expression of some catabolic molecules by human chondrocytes and provide a good environment to support the differentiation of mesenchymal stem cells in chondrogenic sense. These observations confirm Hyaff-11 as a suitable scaffold both for chondrocytes and mesenchymal stem cells for the treatment of articular cartilage defects.     

Vav1 controls DAP10-mediated natural cytotoxicity by regulating actin and microtubule dynamics

The NK cell-activating receptor NKG2D recognizes several MHC class I-related molecules expressed on virally infected and tumor cells. Human NKG2D transduces activation signals exclusively via an associated DAP10 adaptor containing a YxNM motif, whereas murine NKG2D can signal through either DAP10 or the DAP12 adaptor, which contains an ITAM sequence. DAP10 signaling is thought to be mediated, at least in part, by PI3K and is independent of Syk/Zap-70 kinases; however, the exact mechanism by which DAP10 induces natural cytotoxicity is incompletely understood. Herein, we identify Vav1, a Rho GTPase guanine nucleotide exchange factor, as a critical signaling mediator downstream of DAP10 in NK cells. Specifically, using mice deficient in Vav1 and DAP12, we demonstrate an essential role for Vav1 in DAP10-induced NK cell cytoskeletal polarization involving both actin and microtubule networks, maturation of the cytolytic synapse, and target cell lysis. Mechanistically, we show that Vav1 interacts with DAP10 YxNM motifs through the adaptor protein Grb2 and is required for activation of PI3K-dependent Akt signaling. Based on these findings, we propose a novel model of ITAM-independent signaling by Vav downstream of DAP10 in NK cells.     

Multiple interactions of FbsA, a surface protein from Streptococcus agalactiae, with fibrinogen: affinity, stoichiometry, and structural characterization

Streptococcus agalactiae is an etiological agent of several infective diseases in humans. We previously demonstrated that FbsA, a fibrinogen-binding protein expressed by this bacterium, elicits a fibrinogen-dependent aggregation of platelets. In the present communication, we show that the binding of FbsA to fibrinogen is specific and saturable, and that the FbsA-binding site resides in the D region of fibrinogen. In accordance with the repetitive nature of the protein, we found that FbsA contains multiple binding sites for fibrinogen. By using several biophysical methods, we provide evidence that the addition of FbsA induces extensive fibrinogen aggregation and has noticeable effects on thrombin-catalyzed fibrin clot formation. Fibrinogen aggregation was also found to depend on FbsA concentration and on the number of FbsA repeat units. Scanning electron microscopy evidentiated that, while fibrin clot is made of a fine fibrillar network, FbsA-induced Fbg aggregates consist of thicker fibers organized in a cage-like structure. The structural difference of the two structures was further indicated by the diverse immunological reactivity and capability to bind tissue-type plasminogen activator or plasminogen. The mechanisms of FbsA-induced fibrinogen aggregation and fibrin polymerization followed distinct pathways since Fbg assembly was not inhibited by GPRP, a specific inhibitor of fibrin polymerization. This finding was supported by the different sensitivity of the aggregates to the disruptive effects of urea and guanidine hydrochloride. We suggest that FbsA and fibrinogen play complementary roles in contributing to thrombogenesis associated with S. agalactiae infection.     

Root cohesion of forest species in the Italian Alps

Forests can prevent and/or mitigate hydrogeomorphic hazards in mountainous landscapes. Their effect is particularly relevant in the case of shallow landslides phenomena, where plants decrease the water content of the soil and increase its mechanical strength. Although such an effect is well known, its quantification is a relatively new challenge. The present work estimates the effect of some forest species on hillslope stability in terms of additional root cohesion by means of a model based on the classical Wu and Waldron approach (Wu in Alaska Geotech Rpt No 5 Dpt Civ Eng Ohio State Univ Columbus, USA, 1976; Waldron in Soil Sci Soc Am J 41:843–849, 1977). The model is able to account for root distribution with depth and non-simultaneous root breaking. Samples of European beech (Fagus sylvatica L.), Norway spruce (Picea abies (L.) Karst.), European larch (Larix decidua Mill.), sweet chestnut (Castanea sativa Mill.) and European hop-hornbeam (Ostrya carpinifolia Scop.), were taken from different locations of Lombardy (Northern Italy) to estimate root tensile strength, the Root Area Ratio and the root cohesion distribution in the soil. The results show that, in spite of its dramatic variability within the same species at the same location and among different locations, root cohesion can be coherently interpreted using the proposed method. The values herein obtained are significant for slope stabilisation, are consistent with the results of direct shear tests and back-analysis data, and can be used for the estimation of the stability of forested hillslopes in the Alps.