Giovina Vianale

Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma

Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelium, is expressed in malignant pleural mesothelioma (MM). The present report examines the effect of VEGF on MM growth. Four MM cell lines produced significantly higher VEGF levels than normal mesothelial cells (1946±14 pg/ml vs. 180±17 pg/ml; p<0.001). In addition, MM cells expressed the tyrosine kinase‐related VEGF receptors Flt‐1 and KDR. Recombinant human VEGF phosphorylated both Flt‐1 and KDR and increased proliferation of all four MM cell lines in a dose‐dependent fashion. Neutralizing antibodies against either VEGF, Flt‐1 or KDR significantly reduced MM cellular proliferation. In addition, expression of VEGF, Flt‐1, and KDR was observed in MM biopsies. Moreover, higher VEGF levels were found in the pleural effusions of MM patients than in the effusions of patients with non‐malignant pleural disease (1885.7±894.9 pg/ml vs. 266.9±180.5 pg/ml; p<0.001). Linear regression analysis showed a significant inverse correlation between serum VEGF levels and MM patient survival (r=0.72; p<0.01). No correlation was found between tumour vessel density and either serum (r=0.26; p=0.42) or pleural effusion (r=0.35; p=0.26) VEGF levels. These results indicate that VEGF, via activation of its tyrosine kinase receptors, may be a key regulator of MM growth. In addition, VEGF production could have an impact on patient survival, not only by promoting tumour angiogenesis but also by directly stimulating tumour growth. Copyright

Cytokines and T cells in atopic dermatitis

Atopic dermatitis (AD) is an inflammatory disorder of the skin characterized by an impaired immune response. Several effector T cell subsets, such as pro-inflammatory cells like Th9, Th17 and Th22 cells, expressing high levels of IL-9, IL-17 and IL-22, together with the anti-inflammatory, immuno-modulating Treg cells constitutively producing IL-10, seem to play a role in this condition. IL-9 and IL-9 receptors are significantly increased in lesional AD skin compared to normal control skin. In addition, some polymorphisms in IL-9 and IL-9r genes have been associated with AD. The role of IL-17 and IL-17-producing T cells remains under debate and conflicting data are available. IL-22-producing T-cells seem to correlate with the severity of the AD. The number and function of Treg cells, producing IL-10, have been widely investigated in AD with conflicting results. Other studies suggest that high levels of IL-31 or low levels of IL-21 might be involved in the pathogenesis of AD. This review was undertaken in order to provide a better understanding of the relevance of certain cytokines in AD. We have analysed the new insight into the pathogenesis of AD, with special attention to those cytokines produced by the different T cell subpopulations.

Effects of palladium nanoparticles on the cytokine release from peripheral blood mononuclear cells of palladium-sensitized women

Objective: To investigate the effects of palladium (Pd) nanoparticles on cytokine release from peripheral blood mononuclear cells (PBMCs) of control or Pd-sensitized nonatopic women. Methods: TNF-&agr;, IL-5, IL-10 and IFN-&ggr; release and/or expression from PBMCs incubated in presence of 5 to 10 nm Pd0 nanoparticles or PdIV salt (potassium hexachloropalladate) were determined by enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction analysis. Transmission electronmicroscopy was performed. Results: In lipopolysaccharide-stimulated PBMCs from controls, Pd salt inhibited IFN-&ggr; and IL-10 release, whereas Pd nanoparticles enhanced IFN-&ggr; release and inhibited TNF-&agr; secretion. In lipopolysaccharide-stimulated PBMCs from Pd-sensitized women showing high IFN-&ggr; release, Pd nanoparticles inhibited TNF-&agr; release and Pd salt IL-10 release. TNF-&agr; and IFN-&ggr; release and messenger RNA expression were correlated. Transmission electronmicroscopy demonstrated uptake of nanoparticles in the endocytic compartment and activation of autophagy. Conclusions: Palladium ions and nanoparticles exert different effects in vitro on the expression and release of cytokines.

The effect of deproteinized bovine bone on osteoblast growth factors and proinflammatory cytokine production

OBJECTIVE To test the ability of Bio-Oss in inducing growth factors and proinflammatory cytokines that may have a role in inflammation after grafting, bone resorption, remodeling and in the homeostasis of osteoblasts. MATERIAL AND METHODS Normal human osteoblasts were seeded in Petri dishes containing granules of Bio-Oss, cells were harvested after confluency and RNA was extracted. Reverse transcriptase polymerase chain reaction was performed using specific primers for osteonectin, bone sialoprotein (BSP), bone morphogenetic protein (BMP)-2 and BMP-7, platelet-derived growth factor (PDGF), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and integrin beta1. Glycerol-3-phosphate dehydrogenase was used as the housekeeping gene and normal human osteoblasts grown on Petri dishes without Bio-Oss granules were used as negative controls. RESULTS Osteoblast grown on Bio-Oss showed a normal RNA expression of osteonectin, integrin beta1 and PDGF. However, compared with control osteoblasts it showed a reduced expression of BSP, BMP-2 and BMP-7, IL-6 and TNF-alpha. CONCLUSIONS Our findings further support the evidence that Bio-Oss is an excellent biomaterial that does not enhance the production of proinflammatory cytokines.

Expression of glycoprotein 90K in human malignant pleural mesothelioma: correlation with patient survival.

The expression of the tumour‐associated glycoprotein 90K in patients with malignant pleural mesothelioma (MM) has not been described. This study used enzyme‐linked immunoassay (ELISA) to measure 90K in pleural effusions (PEs) and sera from patients with MM (n=28), lung cancer (LC) (n=14) and benign pleural disease (BPD) (n=15). Immunohistochemistry was used to investigate 90K expression in MM and LC tissue sections. The expression of 90K was further evaluated in vitro by ELISA and western blot analysis of conditioned media and cellular extracts of MM, LC and normal human mesothelial (NHM) cell cultures. Finally, the relationships between 90K expression in MM and patient age and survival were studied. The mean 90K level was significantly higher (p<0.05) in PEs of MM patients (11.0±6.6 µg/ml) than in LC (6.1±3.2 µg/ml) or BPD (6.2±5.0 µg/ml) patients. Immunohistochemistry showed a positive reaction for 90K in MM biopsy sections and positive staining limited to inflammatory infiltrates in LC sections. The level of 90K was significantly higher in cell culture media of MM than of LC or NHM (p<0.001). Bands representing proteins with molecular weight of approximately 90 kDa were detected by western blot in MM cellular extracts. An inverse correlation between PE 90K levels and MM patient age (r=−0.45; p=0.017) and a positive correlation between serum 90K levels and MM patient survival (r=0.62; p=0.006) were detected by linear regression analysis. Kaplan–Meier univariate analysis showed increased survival probability for MM patients with serum 90K level >7.3 µg/ml (log rank, p<0.05). This is the first report in MM of the expression of 90K and of its potential diagnostic and prognostic application. Copyright

mTOR Activation by PI3K/Akt and ERK Signaling in Short ELF-EMF Exposed Human Keratinocytes

Several reports suggest that ELF-EMF exposures interact with biological processes including promotion of cell proliferation. However, the molecular mechanisms by which ELF-EMF controls cell growth are not completely understood. The present study aimed to investigate the effect of ELF-EMF on keratinocytes proliferation and molecular mechanisms involved. Effect of ELF-EMF (50 Hz, 1 mT) on HaCaT cell cycle and cells growth and viability was monitored by FACS analysis and BrdU assay. Gene expression profile by microarray and qRT-PCR validation was performed in HaCaT cells exposed or not to ELF-EMF. mTOR, Akt and MAPKs expressions were evaluated by Western blot analysis. In HaCaT cells, short ELF-EMF exposure modulates distinct patterns of gene expression involved in cell proliferation and in the cell cycle. mTOR activation resulted the main molecular target of ELF-EMF on HaCaT cells. Our data showed the increase of the canonical pathway of mTOR regulation (PI3K/Akt) and activation of ERK signaling pathways. Our results indicate that ELF-EMF selectively modulated the expression of multiple genes related to pivotal biological processes and functions that play a key role in physio-pathological mechanisms such as wound healing.

Mutual regulation of TGF-β1, TβRII and ErbB receptors expression in human thyroid carcinomas

The role of EGF and TGF-β1 in thyroid cancer is still not clearly defined. TGF-β1 inhibited the cellular growth and migration of follicular (FTC-133) and papillary (B-CPAP) thyroid carcinoma cell lines. Co-treatments of TGF-β1 and EGF inhibited proliferation in both cell lines, but displayed opposite effect on their migratory capability, leading to inhibition in B-CPAP and promotion in FTC-133 cells, by a MAPK-dependent mechanism. TGF-β1, TβRII and EGFR expressions were evaluated in benign and malignant thyroid tumors. Both positivity (51.7% and 60.0% and 80.0% in FA and PTC and FTC) and overexpression (60.0%, 77.7% and 75.0% in FA, PTC and FTC) of EGFR mRNA correlates with the aggressive tumor behavior. The moderate overexpression of TGF-β1 and TβRII mRNA in PTC tissues (61.5% and 62.5%, respectively), counteracted their high overexpression in FTC tissues (100% and 100%, respectively), while EGFR overexpression was similar in both carcinomas. Papillary carcinomas were positive to E-cadherin expression, while the follicular carcinomas lose E-cadherin staining. Our findings of TGF-β1/TβRII and EGFR overexpressions together with a loss of E-cadherin observed in human follicular thyroid carcinomas, and of increased migration ability MAPK-dependent after EGF/TGF-β1 treatments in the follicular thyroid carcinoma cell line, reinforced the hypothesis of a cross-talk between EGF and TGF-β1 systems in follicular thyroid carcinomas phenotype.

Job strain in different types of employment affects the immune response

The immune system, in cooperation with neuroendocrine functions, defends from cancer and infections mainly by the activity of blood natural killer (NK) cells. Blood NK activity may be influenced by the type of employment since work is the central part of life; moreover, job stress is a situation affecting both neuroendocrine and immune systems. This study examines anxiety (by STAI 1 and 2), job strain (by the Karaseks JCQ) and blood NK activity (by an in vitro radio-isotopic method) of 134 male workers. These men, over 38 years old with stable employment, were working in factories, in construction yards, in offices, as hospital attendants or as self-employed craftsmen. Workers in factories and in construction yards, with high job strain, showed lower NK activity, while office employees, with low job demand, and craftsmen with low anxiety and elevated decision latitude, showed higher NK activity; the level of NK activity of the hospital attendants was between the other groups. In conclusion, this study confirms that the type of employment, related to job stress, affects blood NK activity. Moreover, blood NK activity may be used in the bio-monitoring of workers at high risk.

AGE- AND BRAIN AREAS-RELATED EXPRESSION OF CYTOKINES, CHOLINESTERASES, AND NICOTINIC ACETYLCHILINE RECEPTORS

Background: Changes in endocytosis in Alzheimer’s disease (AD) were originally identified more than 15 years ago but it was not known whether they were causative or an effect of the disease. More recent studies on the genetics of AD have identified several genes encoding endocytic proteins which are associated with a small, increased risk of developing AD, strongly suggesting that changes in endocytosis are indeed involved in the aetiology of the disease. One of these genes encodes for the protein phosphatidylinositol binding clathrin assembly protein (PICALM). The ‘Amyloid Hypothesis’ suggests that the build-up of amyloid-beta (Ab) is a primary causal event in AD. The Ab peptide is cleaved from amyloid precursor protein (APP) by the band gsecretases, primarily after APP has undergone endocytosis and within the endosomal/lysosomal system. We examined whether decreasing the expression of PICALM affected the processing of APP into Ab to further our understanding of the importance of the role of this protein in the pathophysiology of AD.Methods: Levels of PICALMwere depleted by siRNA using oligofectamine for 48 hours in human brain-derived H4 neuroglioma cells that endogenously express APP. Cells were lysed and media collected to detect intraand extracellular protein levels of endocyticrelated proteins, APP and APP metabolites including Ab. Levels of endocytosis were quantified using ALEXA 488-conjugated transferrin as a marker of clathrin-mediated endocytosis and quantified using flow cytometry. Results: PICALM levels were depleted by more than 85% with siRNA. Levels of intracellular APP, intracellular b-CTF and secreted sAPP b (APP fragments produced by b-secretase cleavage) were significantly reduced suggesting an inhibition of the amyloidogenic pathway. Meanwhile levels of transferrin uptake were significantly reduced in PICALM siRNA-treated cells after 15 and 30 minutes suggesting an inhibition of endocytosis. Conclusions: These results suggest that in cells with endogenous levels of APP, representative of levels of APP in AD in man, PICALM can modulate the amyloidogenic processing of APP to Ab, primarily via its effect on levels and rates of endocytosis.

Abstract C58: Humanization and characterization of an anti-ErbB-3 murine monoclonal antibody.

Background: The signaling network downstream of the human epidermal growth factor receptors ErbB-1 and ErbB-2 have been extensively targeted by cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance which is often associated with reactivation of the ErbB-3/PI3K-Akt axis. This may be overcome by ErbB-3 ligands that abrogates receptor-mediated signaling. Methods: the murine MP-RM-1 monoclonal antibody that specifically binds ErbB-3 ectodomain was chimerized by replacing the murine constant regions with the human constant regions and humanized by gratfing Complementarity Determining Regions from the murine into a human antibody framework using a computer assisted molecular modelling. Recombinant genes were placed into the pCDNA3.1 expression vector and transfected into Chinese Hamster Ovary-S cells. Antibodies were screened on the basis of their ability to inhibit Neuregulin-1 (NRG-1)-induced activation of the ErbB-3/PI3K-Akt axis in IR-8 human melanoma cells (Table 1). Four antibody variants, i.e., cMP-RM-1 #1, hMP-RM-1 #6, hMP-RM-1 #10, and hMP-RM-1 #20, were selected and further tested for their ability to promote ErbB-3 downregulation and to inhibit in vitro and in vivo growth of tumor cells. Results: Four chimeric (c) and sixteen humanized (h) antibody variants were obtained. All variants showed to possess antigen binding affinity comparable to that of their parental antibody. Initial screening identified one chimeric (cMP-RM-1 #1) and three humanized (hMP-RM-1 #6, hMP-RM-1 #10, hMP-RM-1 #20) antibodies as the most effective in inhibiting ErbB-3/Akt phosphorylation and promotion of ErbB-3 downregulation. Further testing revealed the antibodies potently inhibited colony formation ability of tumor cells and halted growth of melanoma and ovarian cancer xenografts in nude mice. Treatment with the antibodies was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Conclusions: This successful production of chimeric/humanized antibodies possessing antitumor activity in vitro and in vivo may provide novel candidates for ErbB-3-targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C58.