Behaviour of SH-SY5Y neuroblastoma cell line grown in different media and on different chemically modified substrates.

Among the parameters that can be tested in experiments on neuronal cell culture the use of different culture media and substrates represents a powerful assay to influence cell adhesion and differentiation. In this work, plasma-enhanced-chemical vapour depositions (PE-CVD) from acrylic acid and allylamine vapours have been performed to deposit coatings bearing oxygen (O)- and nitrogen (N)-containing functional groups on polyethylenetherephtalate (PET) surface. Human neuroblastoma SH-SY5Y cells were grown on plasma modified substrates and in presence of media containing different amount of fetal calf serum (FCS) or in serum-free medium containing cAMP. Our results showed that N-containing substrates improved cell adhesion, while the neurites sprouting was influenced by cell culture media. Interestingly, the presence of carboxylic groups on the modified surface can influence the expression of a differentiation marker, neurofilament-200 (NF-H), in cells grown in serum-containing media.

Lipid-based systems loaded with PbS nanocrystals: near infrared emitting trackable nanovectors.

Hydrophobic PbS nanocrystals (NCs) emitting in the near infrared spectral region were encapsulated in the core of micelles and in the bilayer of liposomes, respectively, to form polyethylene glycol (PEG)-grafted phospholipids. The phospholipid-based functionalization process of PbS NCs required the replacement of the pristine capping ligand at the NC surface with thiol molecules. The procedures carried out for two systems, micelles and liposomes, using PEG-modified phospholipids were carefully monitored by optical, morphological and structural investigations. The hydrodynamic diameter and the colloidal stability of both micelles and liposomes loaded with PbS NCs were evaluated using Dynamic Light Scattering (DLS) and ζ-potential experiments, and both were satisfactorily stable in physiological media. The cytotoxicity of the resulting PbS NC-loaded nanovectors was assessed by the in vitro investigation on Saos-2 cells, indicating that the toxicity of the PbS NC loaded liposomes was lower than that of the micelles with the same NC cargo, which is reasonable due to the different overall composition of the two prepared nanocarriers. Finally, the cellular uptake in the Saos-2 cells of both the NC containing systems was evaluated by means of confocal microscopy studies by exploiting a visible fluorescent phospholipid and demonstrating the ability of both luminescent nanovectors to be internalized. The obtained results show the great potential of the prepared emitting nanoprobes for imaging applications in the second biological window.

STK11 mutations in Peutz-Jeghers syndrome and sporadic colon cancer.

A potential tumor suppressor gene, STK11 , encoding a serine threonine kinase, has recently been identified on chromosome 19p13. Germ-line mutations of this gene have been found in patients with Peutz-Jeghers syndrome (PJS). To further investigate the relevance of STK11 mutations in PJS, we analyzed its coding sequence in nine patients and identified two deletions and three missense mutations. Because intestinal carcinomas have been observed to develop in association with PJS, we analyzed tumors from 71 patients for allelic deletions (loss of heterozygosity) and STK11 gene mutations, to elucidate the etiological role of STK11 gene in sporadic colorectal cancer. Loss of heterozygosity, evaluated using the microsatellite D19S886, was observed in 10 of 52 informative cases. No somatic mutations were detected except for a missense alteration in one tumor. Our data indicate the heterogeneity of PJS and the infrequent involvement of the STK11 gene in colorectal cancer.

Oestrogen receptors and microsatellite instability in colorectal carcinoma patients.

About 10-15% of sporadic colorectal cancers show microsatellite instability (MIN), a mutator phenotype of mismatch repair genes. It seems that oestrogens may inhibit the pathway to colorectal carcinoma which involves a mismatch repair deficiency. Oestrogen receptorial status was evaluated in the neoplastic tissue and uninvolved surrounding mucosa of 17 MIN-positive and 33 MIN-negative tumours using an immunoenzymatic assay. MIN status was examined using the polymerase chain reaction and specific microsatellite markers. MIN was significantly associated with very low levels of oestrogen receptor in tumour tissue. Our findings suggest that MIN-positive tumours might lose a possible oestrogenic modulation mechanism.

Improved osteoblast cell affinity on plasma-modified 3-D extruded PCL scaffolds.

Cellular adhesion and proliferation inside three-dimensional synthetic scaffolds represent a major challenge in tissue engineering. Besides the surface chemistry of the polymers, it is well recognized that scaffold internal architecture, namely pore size/shape and interconnectivity, has a strong effect on the biological response of cells. This study reports for the first time how polycaprolactone (PCL) scaffolds with controlled micro-architecture can be effectively produced via bioextrusion and used to enhance the penetration of plasma deposited species. Low-pressure nitrogen-based coatings were employed to augment cell adhesion and proliferation without altering the mechanical properties of the structures. X-ray photoelectron spectroscopy carried out on different sections of the scaffolds indicates a uniform distribution of nitrogen-containing groups throughout the entire porous structure. In vitro biological assays confirm that plasma deposition sensitively promotes the activity of Saos-2 osteoblast cells, leading to a homogeneous colonization of the PCL scaffolds.

The study of specific and nonspecific hepatoma cells behavior by means of plasma-treated substrates.

Physical-chemical surface modifications represent a formidable tool to drive a suitable cell behavior on materials intended to be used in the biomedical field. Plasma processes are among the more powerful methods utilized to modify the surface of materials without altering their bulk intrinsic properties. In particular, by means of plasma treatment processes it is possible to graft chemical functional groups on polymer substrate. Functional groups grafted on the surface can improve per se cell adhesion and can also represent suitable anchor sites for biomolecule immobilization. The aim of this work was to determine the effect of plasma treatment and biomolecule immobilization on Polystyrene (PS) Petri dishes on the behavior of a human hepatocellular carcinoma cell line (HepG2). For this aim Petri dishes were grafted with N-containing groups in order to obtain grafted N-functionalities, to be used as anchor groups for the immobilization of galactosamine. In this way two different modified surfaces, NH(3) grafted polystyrene (PS-NH(3)) and polystyrene owing galactosamine moieties (PS-NH(3)-GalNH(2)), have been obtained. Differences in cell morphology, urea and plasma Fibronectin (pFN) production were clearly observed on HepG2 seeded on PS-NH(3) and PS-NH(3)-GalNH(2). These results highlight the role of specific and non specific cell response in the in vitro study of materials intended to be used for biomedical purposes.

A nanoscale fluorocarbon coating on PET surfaces improves the adhesion and growth of cultured coronary endothelial cells.

Plasma deposition was applied to deposit smooth and nanostructured fluorocarbon coatings on polyethylene terephthalate substrates, with the aim to obtain surfaces with identical chemical composition but different roughness to improve the endothelialization process on PET surfaces. We found that increased roughness was associated with enhanced endothelial cell response, as shown by the ability of cells to grow and adhere to nanostructures. We also observed specific interaction of filopodia protruding from the cell membrane with individual nanostructures, leading to increased cell attachment, spreading and cell viability. Among the modified surfaces, one termed PET-tfl90 emerged as the one capable of best sustaining the formation of a confluent monolayer of endothelial cells. In conclusion, PET modified by nanostructured fluorocarbon film represents an improved graft material, over conventional PET, for endothelial cell adhesion and growth.

CD24, a glycosylphosphatidylinositol-anchored molecules is transiently expressed during the development of human central nervous system and is a marker of human neural cell lineage tumors.

CD24 is a glycoprotein with an unusual structure consisting of a small protein core extensively glycosylated and linked to the outer surface of the plasma membrane by a glycosylphosphatidylinositol (GPI) lipid anchor. Its murine homolog mCD24 is transiently expressed during the development and differentiation of the hematopoietic and neural cell lineages. We have searched for the expression of CD24 in the developing and in the mature human brain as well as in a wide range of neuroectodermal tumors. Neuroblastomas, a subgroup of tumors able to maturate from undifferentiated features towards mature ganglioneuromas, were more extensively studied. Immunohistochemical studies demonstrated that CD24 is transiently expressed by neurons during human brain development. In neuroectodermal tumors, CD24 is a marker of neuronal tumors. Furthermore, in neuroblastomas, CD24 expression decreases as tumors differentiate. In non-neuronal neuroectodermal tumors, CD24 expression is mostly absent. When present, it correlates with the emergence of anaplastic histological features. Reverse transcriptase -polymerase chain reaction (RT-PCR) demonstrated the presence of an unique transcript identical in both hematopoietic, developing and tumoral nervous tissue. RT-PCR and in situ hydridization techniques showed that CD24 expression is transcriptionally regulated. Interestingly, Western blot analysis demonstrated differential CD24 isoforms according to the tissue (hematopoietic versus nervous), the differentiation status, and the origin of neuroblastomas likely reflecting variations in the extent of glycosylation. This indicates an additional level of regulation of CD24 involving post-translational modifications.

Use of chimeric F3-NCAM molecules to explore the properties of VASE exon in modulating polysialylation and neurite outgrowth.

Differential splicing of VASE exon in the fourth immunoglobulin (Ig) domain and attachment to the fifth Ig domain of alpha 2-8 linked sialic acid (PSA) both dramatically change, in opposite manner, Neural Cell Adhesion Molecule (NCAM) functional properties. Reciprocal patterns of VASE and PSA expression suggest that they might be mutually exclusive. Here, we tested whether informations conferring polysialylation reside in NCAM-Ig domains 4 and 5 and the influence of the VASE exon encoded sequence on this process. We also examined if the VASE sequence was still able to inhibit neurite outgrowth when presented out of its normal NCAM context. Constructs have been prepared encoding NCAM-Ig domains 4 (with or without the VASE exon) and 5 fused to the F3 molecule. Stable clones expressing the chimeric molecules or wild type F3 were then obtained in the AtT-20 cell line. Although the chimeric molecules were expressed on the cell surface none of them was bearing PSA. Thus, polysialylation cannot be conferred to proteins by addition of the NCAM-Ig domains 4 and 5 modular motif and in this molecular context, the VASE sequence is not influencing the process. These chimeric molecules, either expressed at the surface of RIN or COS cells or presented as soluble forms, were examined for their effect on neurite outgrowth. In all cases, the length of neurites of sensory neurons was significantly reduced when grown in presence of the VASE containing chimera by comparison with the chimera without VASE or wild type F3. When neurons from NCAM knock-out mice were used for the assay, the VASE inhibition could not be detected. Thus VASE is able to act as a modular motif and NCAM expressed on neurons participates in transducing its effect.

Triiodothyronine-induced shortening of chromatin repeat length in neurons cultured in a chemically defined medium.

At the time of terminal differentiation, mammalian cortical neurons undergo a dramatic change in the structural organization of their chromatin: the nucleosomal repeat length shortens from approximately 200 base pairs in fetuses to a value of 165 base pairs after birth. These events occur several days after the end of neuronal proliferation. Previously, we reported that rat cortical neurons cultured in a very selective synthetic medium were not yet programmed to these events at the end of mitotic cycles. Herein, we report that addition of triiodothyronine to neuronal cultures induces a shortening of the chromatin repeat length comparable to the natural one.

The dynamic properties of neuronal chromatin are modulated by triiodothyronine.

The effect of triiodothyronine (T3) on the rate of synthesis of nuclear proteins was studied during terminal differentiation of rat cortical neurons cultured in a serum-free medium. To this aim total and acid soluble nuclear proteins were analyzed by different electrophoretic techniques. Our results show that: 1) during maturation in vitro, neuronal nuclei undergo a dramatic change in the rate at which different classes of histones and high mobility group (HMG) proteins are synthesized; the synthetic activity, measured as incorporation of radioactive precursors into nuclear proteins, slows indeed down with age: especially evident is the decrease in core histones synthesis; at day 15, on the other hand, HMG 14 and 17 and ubiquitinated H2A (A24) are synthesized at a high rate, especially in T3-treated neurons; 2) neurons treated with T3 show, at any age tested, a higher level of lysine incorporation into nuclear proteins; 3) even if during the first days of culture neurons synthesize core histones more actively in the presence of T3, there is no accumulation of these proteins at later stages, as compared with untreated cells. Possible implications of these data and relationship with the chromatin rearrangement which accompanies neuronal terminal differentiation are discussed.