Determinants of biological activity of nanomaterials in innate immunity cells: the role of aspect ratio, environmental contaminants and protein corona

As defined by the European Union, “ ’Nanomaterial’ (NM) means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or agglomerate, where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm ” (2011/696/UE). Given their peculiar physico-chemical features, nanostructured materials are largely used in many industrial fields (e.g. cosmetics, electronics, agriculture, biomedical) and their applications have astonishingly increased in the last fifteen years. Nanostructured materials are endowed with very large specific surface area that, besides making them very useful in many industrial processes, renders them very reactive towards the biological systems and, hence, potentially endowed with significant hazard for human health. For these reasons, in recent years, many studies have been focused on the identification of toxic properties of nanostructured materials, investigating, in particular, the mechanisms behind their toxic effects as well as their determinants of toxicity. This thesis investigates two types of nanostructured TiO2 materials, TiO2 nanoparticles (NP), which are yearly produced in tonnage quantities, and TiO2 nanofibres (NF), a relatively novel nanomaterial. Moreover, several preparations of MultiWalled Carbon Nanotubes (MWCNT), another nanomaterial widely present in many products, are also investigated.- Although many in vitro and in vivo studies have characterized the toxic properties of these materials, the identification of their determinants of toxicity is still incomplete. The aim of this thesis is to identify the structural determinants of toxicity, using several in vitro models. Specific fields of investigation have been a) the role of shape and the aspect ratio in the determination of biological effects of TiO2 nanofibres of different length; b) the synergistic effect of LPS and TiO2 NP on the expression of inflammatory markers and the role played therein by TLR-4; c) the role of functionalization and agglomeration in the biological effects of MWCNT. As far as biological effects elicited by TiO2 NF are concerned, the first part of the thesis demonstrates that long TiO2 nanofibres caused frustrated phagocytosis, cytotoxicity, hemolysis, oxidative stress and epithelial barrier perturbation. All these effects were mitigated by fibre shortening through ball-milling. However, short TiO2 NF exhibited enhanced ability to activate acute pro-inflammatory effects in macrophages, an effect dependent on phagocytosis. Therefore, aspect ratio reduction mitigated toxic effects, while enhanced macrophage activation, likely rendering the NF more prone to phagocytosis. These results suggest that, under in vivo conditions, short NF will be associated with acute inflammatory reaction, but will undergo a relatively rapid clearance, while long NF, although associated with a relatively smaller acute activation of innate immunity cells, are not expected to be removed efficiently and, therefore, may be associated to chronic inflammatory responses. As far as the relationship between the effects of TiO2 NP and LPS, investigated in the second part of the thesis, are concerned, TiO2 NP markedly enhanced macrophage activation by LPS through a TLR-4-dependent intracellular pathway. The adsorption of LPS onto the surface of TiO2 NP led to the formation of a specific bio-corona, suggesting that, when bound to TiO2 NP, LPS exerts a much more powerful pro-inflammatory effect. These data suggest that the inflammatory changes observed upon exposure to TiO2 NP may be due, at least in part, to their capability to bind LPS and, possibly, other TLR agonists, thus enhancing their biological activities. Finally, the last part of the thesis demonstrates that surface functionalization of MWCNT with amino or carboxylic groups mitigates the toxic effects of MWCNT in terms of macrophage activation and capability to perturb epithelial barriers. Interestingly, surface chemistry (in particular surface charge) influenced the protein adsorption onto the MWCNT surface, allowing to the formation of different protein coronae and the tendency to form agglomerates of different size. In particular functionalization a) changed the amount and the type of proteins adsorbed to MWCNT and b) enhanced the tendency of MWCNT to form large agglomerates. These data suggest that the different biological behavior of functionalized and pristine MWCNT may be due, at least in part, to the different tendency to form large agglomerates, which is significantly influenced by their different capability to interact with proteins contained in biological fluids. All together, these data demonstrate that the interaction between physico-chemical properties of nanostructured materials and the environment (cells + biological fluids) in which these materials are present is of pivotal importance for the understanding of the biological effects of NM. In particular, bio-persistence and the capability to elicit an effective inflammatory response are attributable to the interaction between NM and macrophages. However, the interaction NM-cells is heavily influenced by the formation at the nano-bio interface of specific bio-coronae that confer a novel biological identity to the nanostructured materials, setting the basis for their specific biological activities.

Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease?

Biomolecules are susceptible to many different post-translational modifications that have important effects on their function and stability, including glycosylation, glycation, phosphorylation and oxidation chemistries. Specific conversion of aspartic acid to its isoaspartyl derivative or arginine to citrulline leads to autoantibody production in models of rheumatoid disease, and ensuing autoantibodies cross-react with native antigens. Autoimmune conditions associate with increased activation of immune effector cells and production of free radical species via NADPH oxidases and nitric oxide synthases. Generation of neo-antigenic determinants by reactive oxygen and nitrogen species ROS and RNS) may contribute to epitope spreading in autoimmunity. The oxidation of amino acids by peroxynitrite, hypochlorous acid and other reactive oxygen species (ROS) increases the antigenicity of DNA, LDL and IgG, generating ligands for which autoantibodies show higher avidity. This review focuses on the evidence for ROS and RNS in promoting the autoimmune responses observed in diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It considers the evidence for ROS/RNS-induced antigenicity arising as a consequence of failure to remove or repair ROS/RNS damaged biomolecules and suggests that an associated defect, probably in T cell signal processing or/or antigen presentation, is required for the development of disease.

The second intracellular loop of the calcitonin gene-related peptide receptor provides molecular determinants for signal transduction and cell surface expression

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to Gs, but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells:Studies with chimeric and other peptides

1. Structure-activity relationships for the binding of human α-calcitonin gene-related peptide 8-37 (hαCGRP8-37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (hαCGRP stimulation of adenylate cyclase). 2. On SK-N-MC cells the potency order was hαCGRP8-37 > hαCGRP19-37 = AC187 > rat amylin8-37 > hα[Tyr0]-CGRP28-37 (apparent pKBS of 7.49 ± 0.25, 5.89 ± 0.20, 6.18 ± 0.19, 5.85 ± 0.19 and 5.25 ± 0.07). The SK-N-MC receptor appeared CGRP1-like. 3. On Col 29 cells, only hαCGRP8-37 of the above compounds was able to antagonize the actions of hαCGRP (apparent pKB = 6.48 ± 0.28). Its receptor appeared CGRP2-like. 4. hα[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal α-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than hαCGRP8-37. 5. On SK-N-MC cells, hαCGRP(8-18, 28-37) (M433) and mastoparan-hαCGRP28-37 (M432) had apparent pKBS of 6.64 ± 0.16 and 6.42 ± 0.26, suggesting that residues 19-27 play a minor role in binding. The physico-chemical properties of residues 8-18 may be more important than any specific side-chain interactions. 6. M433 was almost as potent as hαCGRP8-37 on Col 29 cells (apparent pKB = 6.17 ± 0.20). Other antagonists were inactive.

The selectivity and structural determinants of peptide antagonists at the CGRP receptor of rat, L6 myocytes

1. Potency orders were determined for a series of agonists and antagonists on the calcitonin gene-related peptide (CGRP) receptor of rat L6 myocytes. The agents tested were all shown to have been active against CGRP, amylin or adrenomedullin receptors. 2. AC187 had a PIC50 Of 6.8 ± 0.10, making it 14 fold less potent as an antagonist than CGRP8-37 (PIC50, 7.95 ± 0.14). Amyline8-37 was equipotent to AC187 (PIC50, 6.6 ± 0.16) and CGRP19-37 was a fold less potent than either (pIC50 6.1 ± 0.24). 3. [Ala11]-CGRP8-37 was 6 fold less potent than CGRP8-37, (pIC50 7.13 ± 0.14), whereas [Ala18] CGRP8-37 was approximately equipotent to CGRP8-37 (pIC50, 7.52 ± 0.15). However, [Ala11,Ala18]- CGRP8-37 was over 300 fold less potent than CGRP8-37 (pIC50, 5.30 ± 0.04). 4. [Tyr0]-CGRP28-37, amylin19-37 and adrenomedullin22-52 were inactive as antagonists at concentrations of up to 1 μM. 5. Biotinyl-human α-CGRP was 150 fold less potent than human α-CGRP itself (EC50 values of 48 ± 17 nM and 0.31 ± 0.13 nM, respectively). At 1 μM, [Cys(acetomethoxy)(2'7)]-CGRP was inactive as an agonist. 6. These results confirm a role for Arg11 in maintaining the high affinity binding of CGRP8-37. Arg18 is of less direct significance for high affinity binding, but it may be important in maintaining the amphipathic nature of CGRP and its analogues.

Dietary habits are major determinants of the plasma antioxidant status in healthy elderly subjects

Previous studies indicate that regular consumption of a diet rich in fruits and vegetables is associated with a lower risk for age-related diseases. The aim of the present study was to evaluate whether the often-reported age-related decrease of plasma antioxidants in man depends on differences in dietary intake or on other age- and gender-related factors. In this observational case-control study, thirty-nine community-dwelling healthy subjects aged 65 years and older consuming high intakes of fruits and vegetables daily (HI) and forty-eight healthy subjects aged 65 and older consuming low intakes of fruit and vegetables daily (LI) were enrolled. Plasma levels of retinol, tocopherols, carotenoids and malondialdehyde (MDA) as well as content of protein carbonyls in Ig G were measured. Plasma levels of retinol, tocopherols and carotenoids were significantly higher in group HI than in group LI subjects independent of age and gender. MDA levels were inversely correlated with vitamin A and α-carotene. Protein carbonyls were inversely correlated with γ-tocopherol. In the elderly, a higher daily intake of fruits and vegetables is associated with an improved antioxidant status in comparison to subjects consuming diets poor in fruits and vegetables. Modification of nutritional habits among other lifestyle changes should be encouraged to lower prevalence of disease risk factors in later life. © The Authors 2005.

Determinants of inward investment in the UK, a panel analysis

This paper develops an inter-industry model of inward investment, using a fixed effects approach. This demonstrates that when inward investment is investigated in such a framework, previous findings, relating to the specification of measures of location advantage and ownership advantages no longer hold. This also shows that there are some industries that have attracted significant inward investment over time, and continue to do so, while others are noticeably less successful. Reasons for this, and potential policy measures are briefly discussed.

Determinants of entry and exit in the foreign owned sector of UK manufacturing

This paper examines the extent to which foreign entry and exit in the UK is related to domestic industry characteristics. The units of analysis are firm numbers, and thus entry and exit at the industry level are treated as being generated by Poisson processes. This therefore uses quasimaximum likelihood estimation, to estimate entry and exit functions simultaneously. The results demonstrate that foreign entry is attracted by industry level profitability and performance, but that firm specific 'ownership' advantages are also important. The results also demonstrate that inward investors that are motivated by the desire to exploit firm-specific assets, are unlikely to be more transient than domestic firms. This however, cannot be said of those foreign entrants who are attracted to the UK by location advantage or investment incentives.

Sales manager and sales team determinants of salesperson ethical behaviour

Purpose – The purpose of this paper is to develop and test a model of the role managers and peers play in shaping salespeople's ethical behaviour. The model specifies that sales manager personal moral philosophies, whether sales managers themselves are rewarded according to the outcomes or behaviours of their salespeople, sales team job security, intra-team cooperation, and sales team tactical performance all influence sales team ethical standards. In turn, ethical standards influence the probability that sales team members will behave (un)ethically when faced with ethical dilemmas. Design/methodology/approach – The model is tested on a sample of 154 Finnish sales managers. Data were collected via mail survey. Analysis was undertaken using structural equation modelling. Findings – Ethical standards appear to be shaped by several factors; behaviour-based management controls increase ethical standards, relativist managers tend to manage less ethically-minded sales teams, job insecurity impedes the development of ethical standards, and sales teams' cooperation activity increases ethical standards. Sales teams are less likely to engage in unethical behaviour when the teams have strong ethical standards. Research limitations/implications – Cross-sectional data limits generalisability; single country data may limit the ability to generalise to different sales environments; additional measure development is needed; identification of additional antecedent factors would be beneficial. Practical implications – Sales managers should consciously develop high ethical standards in sales teams if they wish to reduce unethical behaviour. Ethical standards can be improved if sales managers change their own outward behaviour (exhibit a less relativistic ethical philosophy), foster cooperation amongst salespeople, and develop perceptions of job security. How sales managers are rewarded may shape how they approach the management of ethical behaviour in their sales teams. Originality/value – This paper appears to be the first to simultaneously examine both sales manager-specific and sales team-specific antecedents to sales team ethical standards and behaviours. As such, it provides an important base for research in this critical area.