Physical and chemical characterization of representative samples of recycled rubber from end-of-life tires.

A large number of end-of-life tires (ELTs) were sampled and classified by type, age and origin to obtain recycled rubber samples representative of the materials placed on the Italian market. The selected recycled tire rubber samples were physically and chemically characterized and a chemometric approach was used to determine correlations. The polycyclic aromatic hydrocarbons (PAHs) content was correlated to the aromaticity index and a model was built to establish the H-Bay aromaticity index (H-Bay) from the PAH concentrations. ELT of different origin and age produced in non-European countries generally had higher PAH content and a higher H-Bay index. H-Bay values of all the samples were lower than the REACH limits and old tires had higher aromatic content than recent ones, possibly due to the replacement of aromatic oils in tire production.

Dynamic measurement of newly formed carbonyl compounds in vapors from electronic cigarettes.

Recently, the formation of carbonyl compound within e-cigarettes usage has been reported. The aim of this study was to develop a new analytical method for the direct analysis of carbonyl compounds in vaporized liquids. Two different types of e-cigarettes and different puff's duration have been evaluated, using a modified smoking machine for vapor generation. An isotopic dilution approach, based on deuterated internal standard addition to the e-liquid before filling the e-cigarette tank, has been developed. Carbonyl compounds have been sampled in vapors using a direct, simple, solid-phase microextraction technique with on-fiber derivatization. Related oximes have been analyzed by gas chromatography/mass spectrometry technique. Results confirmed that new carbonyl compounds are formed during the vaping process, and that formation depends both from the heating device and from puffing topography.

A simple headspace gas chromatography/mass spectrometry method for the quantitative determination of the release of the antioxidants butylated hydroxyanisole and butylated hydroxytoluene from chewing gum.

RATIONALE: Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are widely used to prevent oxidation and rancidity in foodstuffs, pharmaceutical preparations and cosmetic formulations. Although their safety has been thoroughly investigated, possible endocrine side-effects have been suggested. A useful method for the determination of BHA and BHT in foods is needed to estimate their daily intake through the diet. METHODS: We selected commercial chewing gums as a model of a complex food matrix and developed a new method based on gas chromatography/mass spectrometry. This allows the determination of 130 pg/gum of BHA and 9 pg/gum of BHT. RESULTS: Analysis of different chewing gums from the European market indicated that the two antioxidants were never used together and that the content of BHA was in the range of 220-348 µg/gum and BHT ranged from 278 up to 479 µg/gum. These amounts correspond to 86-157 mg/kg gum for BHA and 170-185 mg/kg gum for BHT, and are both within the maximum levels established by the European Food Safety Authority. Chewing a piece of gum for 15 min resulted in the release of up to 28% of BHA, but no release of BHT was detectable. CONCLUSIONS: A new, simple and rapid method for the determination of BHA and BHT in chewing gums was described. This analytical method, based on headspace sampling, did not require the extraction of antioxidants from chewing gum samples, assuring the absence of any gum material contaminants that might affect the instrumentation. It is also automatable, employing a sequential automatic sampler. This method could be of interest to academic researchers and to food industrialists looking for a new methodological approach for BHA and BHT determination in foodstuffs with complex matrices. Copyright © 2017 John Wiley & Sons, Ltd.

Risk assessment for the Italian population of acetaldehyde in alcoholic and non-alcoholic beverages.

Acetaldehyde is a naturally-occurring carcinogenic compound, present in different food items, especially in alcoholic beverages. The aims of this study were to measure acetaldehyde concentration in different beverages consumed in Italy and to estimate the potential cancer risk. The analytical procedure was based on headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS), using the isotopic dilution method. The margin of exposure (MOE) approach of the European Food Safety Authority (EFSA) was used for risk characterisation. The highest concentrations (median, min-max) were detected in grappa samples (499, 23.4-1850mg/l), followed by fruit-based liqueurs and spirits (62.0, 5.23-483mg/l) and wine (68.0, 18.1-477mg/l); the lowest were detected in gin (0.91, 0.78-1.90mg/l). The lowest MOE was estimated for high wine consumers (69). These results suggest that regulatory measures and consumer guidance may be necessary for acetaldehyde in beverages.

Direct analysis of isopropylthioxanthone (ITX) in milk by high-performance liquid chromatography/tandem mass spectrometry.

A fast screening method is presented for detecting isopropylthioxanthone (ITX) contamination in milk. The method is based on direct high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analysis of milk samples. Sample preparation is limited to the addition of a deuterated ITX solution in acetonitrile that serves both as internal standard and to precipitate proteins. The method is highly accurate and sensitive. Isomeric specific analyses of 2-ITX and 4-ITX are possible at 6 microg/L levels with about 5% precision and accuracy. This approach has been used to check contamination in samples like milk, soy milk, baby milk, in their packaging material. Out of 37 milk samples analyzed, 16 were positive with concentrations ranging from 173-439 microg/L for 2-ITX and from <6 (lower than limit of quantification) to 25 microg/L for 4-ITX.

Differential effects of immunosuppressive drugs on chemokine receptor CCR7 in human monocyte-derived dendritic cells: selective upregulation by rapamycin.

BACKGROUND: Appropriate recruitment of dendritic cells (DC) at sites of inflammation and migration to secondary lymphoid organs is of critical importance for the initiation of Ag-specific immune responses. The proper localization of DC in selected tissues is guided primarily by the coordinated expression of chemokine receptors (CKR). Here we show that immunosuppressive drugs have divergent effects on the modulation of CKR in maturing DC. METHODS AND RESULTS: Dexamethazone (DEX) and IL-10 inhibited human DC migration to CCL19 in vitro and mouse DC migration to lymph nodes (LN) in vivo, by impairing CCR7 expression. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) were characterized by the inability to modulate CKR expression and migratory activity. Rapamycin (RAPA) increased DC migration to CCL19 in vitro and to LN in vivo by enhancing CCR7 expression. This effect could be mediated, in LPS-maturing DC, by the inhibition of autocrine IL-10 production. The in vivo data obtained with ex vivo RAPA treated DC were confirmed in a model of in vivo drug administration in mice, suggesting a potential clinical relevance. CONCLUSIONS: These findings demonstrate that immunosuppressive agents differently modulate the CKR switch associated with maturing DC; in particular, RAPA selectively up-regulates CCR7 and enhances the migration of differentiated DC to regional LN. This study contributes to a better understanding of the role of immunosuppressive therapy on DC migration, a potentially relevant check point of immunosuppressive treatment.

Anti-inflammatory properties of the novel antitumor agent yondelis (trabectedin): inhibition of macrophage differentiation and cytokine production.

Yondelis (Trabectedin) is a novel antitumor agent of marine origin extracted from the tunicate Ecteinascidia turbinata. This original compound is active against several human tumors including sarcoma and ovarian and breast adenocarcinoma, as evidenced in phase II clinical trials in advanced multitreated patients. Yondelis is a DNA minor groove binder that blocks cell cycle and interferes with inducible gene transcription in a selective manner. In this study, we investigated the immunomodulatory properties of Yondelis on leukocytes. Human blood monocytes were highly susceptible in vitro to its cytotoxic effect and underwent apoptosis at pharmacologically relevant concentrations (5 nmol/L), whereas lymphocytes were up to 5-fold less sensitive. Macrophages differentiated in vitro with macrophage colony-stimulating factor and tumor-associated macrophages (TAM), isolated from patients with ovarian cancer, were also susceptible. At subcytotoxic concentrations, Yondelis inhibited the in vitro differentiation of monocytes to macrophages. In tumor-treated patients, drug infusion caused a selective decrease of monocyte counts and of ex vivo macrophage differentiation. The in vitro production of two proinflammatory mediators, CCL2 and IL-6, was markedly reduced by Yondelis in monocytes, macrophages, TAM, and freshly isolated ovarian tumor cells. The chemokine CCL2 is the major determinant of monocyte recruitment at tumor sites, whereas IL-6 is a growth factor for ovarian tumors. In view of the protumor activity of TAM and of the strong association between chronic inflammation and cancer progression, the inhibitory effect of Yondelis on macrophage viability, differentiation, and cytokine production is likely to contribute to the antitumor activity of this agent in inflammation-associated human tumors.

Distinct transcriptional programs activated by interleukin-10 with or without lipopolysaccharide in dendritic cells: induction of the B cell-activating chemokine, CXC chemokine ligand 13.

To understand the modulation of dendritic cell (DC) function by IL-10, gene expression profiling was performed by using Affymetrix technology (Santa Clara, CA) in human monocyte-derived DC treated with IL-10, alone or in combination with LPS. The modulation of selected genes was validated by real-time PCR, Northern blot, and protein production. IL-10 regulated in DC the expression of a limited number of genes, including IL-7, the receptors for transferrin and vitamin D(3), structural matrix proteins, and signal transduction elements. The combined treatment with LPS plus IL-10 modulated a number of genes comparable to LPS alone, but the expression profiles were distinct. As expected, IL-10 suppressed the expression of several LPS-inducible proinflammatory molecules. Among genes uniquely modulated by the concomitant treatment with LPS plus IL-10, phosphatidylinositol 3-kinase gamma was down-regulated while the suppressor of cytokine signaling 3, signaling lymphocytic activation molecule, regulator of G protein signaling 16, and the chemokine, CXC chemokine ligand (CXCL) 13, were up-regulated. Overall, four distinct transcriptional programs were identified, related to: 1) control of immunity and inflammation; 2) tuning of cytokine receptor and G protein-coupled receptor signaling; 3) remodeling of extracellular matrix; and 4) B cell function and lymphoid tissue neogenesis. Among the latter genes, we further demonstrate that IL-10 synergizes with TLR ligands for the production of functionally active B cell-attracting chemokine, CXCL13, in both myeloid and plasmacytoid DC. This novel finding reveals that IL-10 sustains humoral immunity by inducing the production in APCs of the chemokine, CXCL13, which amplifies B cell recruitment and promotes lymphoid tissue neogenesis.

CD40 activation of BCP-ALL cells generates IL-10-producing, IL-12-defective APCs that induce allogeneic T-cell anergy.

The use of leukemia cells as antigen-presenting cells (APCs) in immunotherapy is critically dependent on their capacity to initiate and sustain an antitumor-specific immune response. Previous studies suggested that pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells could be manipulated in vitro through the CD40-CD40L pathway to increase their immunostimulatory capacity. We extended the APC characterization of CD40L-activated BCP-ALL for their potential use in immunotherapy in a series of 19 patients. Engaging CD40 induced the up-regulation of CCR7 in 7 of 11 patients and then the migration to CCL19 in 2 of 5 patients. As accessory cells, CD40L-activated BCP-ALL induced a strong proliferation response of naive T lymphocytes. Leukemia cells, however, were unable to sustain proliferation over time, and T cells eventually became anergic. After CD40-activation, BCP-ALL cells released substantial amounts of interleukin-10 (IL-10) but were unable to produce bioactive IL-12 or to polarize TH1 effectors. Interestingly, adding exogenous IL-12 induced the generation of interferon-gamma (IFN-gamma)-secreting TH1 effectors and reverted the anergic profile in a secondary response. Therefore, engaging CD40 on BCP-ALL cells is insufficient for the acquisition of full functional properties of immunostimulatory APCs. These results suggest caution against the potential use of CD40L-activated BCP-ALL cells as agents for immunotherapy unless additional stimuli, such as IL-12, are provided.

Redox regulation of surface protein thiols: identification of integrin alpha-4 as a molecular target by using redox proteomics.

Thiols affect a variety of cell functions, an effect known as redox regulation. We show here that treatment (1-2 h) of cells with 0.1-5 mM N-acetyl-L-cysteine (NAC) increases surface protein thiol expression in human peripheral blood mononuclear cells. This effect is not associated with changes in cellular glutathione (GSH) and is also observed with a non-GSH precursor thiol N-acetyl-D-cysteine or with GSH itself, which is not cell-permeable, suggesting a direct reducing action. NAC did not augment protein SH in the cytosol, indicating that they are already maximally reduced under normal, nonstressed, conditions. By using labeling with a non permeable, biotinylated SH reagent followed by two-dimensional gel electrophoresis and analysis by MS, we identified some of the proteins associated with the membrane that are reduced by NAC. These proteins include the following: integrin alpha-4, myosin heavy chain (nonmuscle type A), myosin light-chain alkali (nonmuscle isoform), and beta-actin. NAC pretreatment augmented integrin alpha-4-dependent fibronectin adhesion and aggregation of Jurkat cells without changing its expression by fluorescence-activated cell sorter, suggesting that reduction of surface disulfides can affect proteins function. We postulate that some of the activities of NAC or other thiol antioxidants may not only be due to free radical scavenging or increase of intracellular GSH and subsequent effects on transcription factors, but could modify the redox state of functional membrane proteins with exofacial SH critical for their activity.

Cross-linking of the mannose receptor on monocyte-derived dendritic cells activates an anti-inflammatory immunosuppressive program.

Immature monocyte-derived dendritic cells (DC) strongly express the endocytic mannose receptor (MR). Addition of a specific anti-MR mAb (clone PAM-1) for 24 h to cultures of immature DC induced phenotypical and functional maturation of the cells, assessed as up-regulation of costimulatory molecules and CD83, and chemotactic response to CCL19. A different isotype-matched anti-MR mAb (clone 19.2) had no significant effect. Engagement of MR with mAb PAM-1 induced the production of the anti-inflammatory cytokines IL-10, IL-1R antagonist, and of the nonsignaling IL-1R type II. In contrast IL-1beta, TNF, and IL-12 were not produced. PAM-1-treated DC were unable to polarize Th1 effector cells and did not secrete the chemokines CXCL10 and CCL19; in turn, they produced large amounts of CCL22 and CCL17, thus favoring the amplification of Th2 circuits. T cells cocultured with PAM-1-matured DC initially proliferated but later became anergic and behaved as suppressor/regulatory cells. Natural ligands binding to MR had differential effects. MUC III (a partially purified mucin), biglycan (a purified complex proteoglycan), and mannosylated lipoarabinomannan from Mycobacterium tuberculosis affected cytokine production with high IL-10, IL-1R antagonist, IL-1R type II, and inhibition of IL-12. In contrast, mannan, dextran, and thyroglobulin had no significant effect. In conclusion, the appropriate engagement of the MR by mAb PAM-1 and selected natural ligands elicit a secretory program in mono-derived DC characterized by a distinct profile of cytokines/chemokines with the ability to dampen inflammation and to inhibit the generation of Th1-polarized immune responses.

Human pancreatic islets produce and secrete MCP-1/CCL2: relevance in human islet transplantation.

We investigated the capacity of human islets to produce monocyte chemoattractant protein-1 (MCP-1). Primary cultures of pancreatic islets expressed and secreted MCP-1, as determined by Northern blot, immunohistochemistry, in situ hybridization, and enzyme-linked immunosorbent assay. The produced MCP-1 was biologically active as it attracted monocytes in chemotaxis assay, and chemotactic activity was almost abrogated by a neutralizing anti-MCP-1 monoclonal antibody. Expression of MCP-1 was increased by primary inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha) and lipopolysaccharide at both the mRNA and protein levels but not by glucose. However, MCP-1 did not modulate insulin secretion. MCP-1 secreted by pancreatic islets plays a relevant role in the clinical outcome of islet transplant in patients with type 1 diabetes. In fact, low MCP-1 secretion resulted as the most relevant factor for long-lasting insulin independence. This finding opens new approaches in the management of human islet transplantation. Finally, the finding that MCP-1 appears constitutively present in normal human islet beta-cells (immunohistochemistry and in situ hybridization), in the absence of an inflammatory infiltrate, suggests that this chemokine could have functions other than monocyte recruitment and opens a new link between the endocrine and immune systems.