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Strategies for endosomal escape and access to the cell nucleus are highly sought for nanocarriers to deliver their load efficiently following endocytosis. In this work, we have studied the uptake and intracellular trafficking of a polycationic polyamidoamine (PAA) endowed with a luminescent Ru complex, Ru-PhenAN, that shows unique trafficking to the cell nucleus. Live cell imaging confirmed the capacity of this polymer to access the nucleus, excluding artifacts due to cell fixation, and clarified that the mechanism of escape is light-triggered and relies on the presence of the Ru complexes and their capacity to absorb light and act as photosensitizers for singlet oxygen production. These results open up the possibility to use PAA-ruthenium complexes for targeted light-triggered delivery of genetic material or drugs to the cytosol and nucleus.

Self-assembly of biomolecules such as peptides, nucleic acids or their analogues affords supramolecular objects, exhibiting structures and physical properties dependent on the amino-acid or nucleobase composition. Conjugation of the peptide diphenylalanine (FF) to peptide nucleic acids triggers formation of self-assembled structures, mainly stabilized by interactions between FF. In this work we report formation of homogeneous chiral fibers upon self-assembly of the hybrid composed of the tetraphenylalanine peptide (4F) conjugated to the PNA dimer adenine-thymine (at). In this case nucleobases seem to play a key role in determining the morphology and chirality of the fibers. When the PNA "at" is replaced by guanine-cytosine dimer "gc", disordered structures are observed. Spectroscopic characterization of the self-assembled hybrids, along with AFM and SEM studies is reported. Finally, a structural model consistent with the experimental evidence has also been obtained, showing how the building blocks of 4Fat arrange to give helical fibers.

The adsorption of biomacromolecules is a fundamental process that can alter the behaviour and adverse effects of nanoparticles (NPs) in natural systems. While the interaction of NPs with natural molecules present in the environment has been described, their biological impacts are largely unknown. Therefore, this study aims to provide a first evidence of the influence of biomolecules sorption on the toxicity of cerium oxide nanoparticles (CeO2NPs) towards the freshwater bivalve Dreissena polymorpha. To this aim, we compared naked CeO2NPs and coated with alginate and chitosan, two polysaccharides abundant in aquatic environments. Mussels were exposed to the three CeO2NPs (naked, chitosan- and alginate-coated) up to 14 days at 100 μg L−1, which is a concentration higher than the environmental one predicted for this type of NP. A suite of biomarkers related to oxidative stress and energy metabolism was applied, and metabolomics was also carried out to identify metabolic pathways potentially targeted by CeO2NPs. Results showed that the coating with chitosan reduced NP aggregation and increased the stability in water. Nonetheless, the Ce accumulation in mussels was similar in all treatments. As for biological effects, all three types of CeO2NPs reduced significantly the level of reactive oxygen species and the activity of superoxide dismutase, glutathione peroxidase and glutathione-S-transferase. The effect was more pronounced in individuals exposed to CeO2NPs coated with alginate, which also significantly induced the activity of the electron transport system. Metabolomics analysis of amino acid metabolism showed modulation only in mussels treated with CeO2NPs coated with alginate. In this group, 25 metabolites belonging to nucleotides, lipids/sterols and organic osmolytes were also modulated, suggesting that the nanoparticles affect energetic metabolism and osmoregulation of mussels. This study highlights the key role of the interaction between nanoparticles and natural molecules as a driver of nanoparticle ecotoxicity.

It is known that, for marine coastal ecosystems, pollution and global warming are among the most threatening factors. Among emerging pollutants, nanoparticles (NPs) deserve particular attention as their possible adverse effects are significantly influenced by environmental factors such as salinity, pH and temperature, as well as by their ability to interact with other contaminants. In this framework, the present study aimed to evaluate the potential interactions between CeO2 NPs and the toxic classic metal mercury (Hg), under current and warming conditions. The marine bivalve Mytilus galloprovincialis was used as biological model and exposed to CeO2 NPs and Hg, either alone or in combination, for 28 day at 17 °C and 22 °C. A suite of biomarkers related to energetic metabolism, oxidative stress/damage, redox balance, and neurotoxicity was applied in exposed and non-exposed (control) mussels. The Hg and Ce accumulation was also assessed. Results showed that the exposure to CeO2 NPs alone did not induce toxic effects in M. galloprovincialis. On the contrary, Hg exposure determined a significant loss of energetic metabolism and a general impairment in biochemical performances. Hg accumulation in mussels was not modified by the presence of CeO2 NPs, while the biochemical alterations induced by Hg alone were partially canceled upon co-exposure with CeO2 NPs. The temperature increase induced loss of metabolic and biochemical functions and the effects of temperature prevailed on mussels exposed to pollutants acting alone or combined.

Polyamide 6 (PA6) suffers from fast degradation in humid conditions due to hydrolysis of amide bonds, which limits its durability. The addition of nanotubular fillers represents a viable strategy for overcoming this issue, although the additive/polymer interface at high filler content can become privileged site for moisture accumulation. As a cost-effective and versatile material, halloysite nanotubes (HNT) were investigated to prepare PA6 nanocomposites with very low loadings (1&ndash, 45% w/w). The roles of the physicochemical properties of two differently sourced HNT, of filler functionalization with (3-aminopropyl)triethoxysilane and of dispersion techniques (in situ polymerization vs. melt blending) were investigated. The aspect ratio (5 vs. 15) and surface charge (&minus, 31 vs. &minus, 59 mV) of the two HNT proved crucial in determining their distribution within the polymer matrix. In situ polymerization of functionalized HNT leads to enclosed and well-penetrated filler within the polymer matrix. PA6 nanocomposites crystal growth and nucleation type were studied according to Avrami theory, as well as the formation of different crystalline structures (&alpha, and &gamma, forms). After 1680 h of ageing, functionalized HNT reduced the diffusion of water into polymer, lowering water uptake after 600 h up to 90%, increasing the materials durability also regarding molecular weights and rheological behavior.

We present for the first time a method for the preparation of magnetic halloysite nanotubes (HNT) by loading of preformed superparamagnetic magnetite nanoparticles (SPION) of diameter size ∼6 nm with a hydrodynamic diameter of ∼10 nm into HNT. We found that the most effective route to reach this goal relies on the modification of the inner lumen of HNT by tetradecylphosphonic acid (TDP) to give HNT–TDP, followed by the loading with preformed oleic acid (OA)-stabilized SPION. Transmission electron microscopy evidenced the presence of highly crystalline magnetic nanoparticles only in the lumen, partially ordered in chainlike structures. Conversely, attempts to obtain the same result by exploiting either the positive charge of the HNT inner lumen employing SPIONs covered with negatively charged capping agents or the in situ synthesis of SPION by thermal decomposition were not effective. HNT–TDP were characterized by infrared spectroscopy (ATR-FTIR), thermogravimetric analysis (TGA), and ζ-potential, and all of the techniques confirmed the presence of TDP onto the HNT. Moreover, the inner localization of TDP was ascertained by the use of Nile Red, a molecule whose luminescence is very sensitive to the polarity of the environment. The free SPION@OA (as a colloidal suspension and as a powder) and SPION-in-HNT powder were magnetically characterized by measuring the ZFC-FC magnetization curves as well as the hysteresis cycles at 300 and 2.5 K, confirming that the super-paramagnetic behavior and the main magnetic properties of the free SPION were preserved once embedded in SPION-in-HNT., SPION nanoparticles are selectively loaded into halloysite lumen, keeping their superparamagnetic character.

Postsynthesis ligand exchange has been employed extensively on lead halide perovskite (LHP) nanocrystals (NCs), but the complex ligand shell composition of the starting NCs prevented a clear understanding of the exchange process, and the surface chemistry of the final NCs remained poorly characterized. Here, we describe a ligand exchange strategy involving the displacement of both cationic and anionic ligands on native model systems of CsPbBr 3 NCs, which are exclusively coated with Cs-oleate. These ligands are exchanged with various quaternary ammonium bromides (R 4 NBr), and complete exchange is confirmed by nuclear magnetic resonance (NMR) spectroscopy analysis. The displacement of the native Cs-oleate ligands with proton-free R 4 NBr delivers NCs with excellent colloidal stability and near-unity PLQY, which is preserved after washing with polar solvents, over 3 weeks of storage in air, and after heating a solution of NCs to 80 °C, as confirmed by NMR analysis. The results, together with density functional theory calculations, suggest that the higher stability of quaternary ammonium capped NCs is not due to a stronger binding interaction to the surface but rather to weaker solvent-ligand interactions of R 4 NBr compared to Cs-oleate, driving the former to the surface of the NCs.

Dendritic cells (DCs) initiate adaptive immune responses after their migration to secondary lymphoid organs. The LXR ligands/oxysterols and the RXR ligand 9-cis Retinoic Acid (9-cis RA) were shown to dampen DC migration to lymphoid organs through the inhibition of CCR7 expression. We performed transcriptomics of DCs undergoing maturation in the presence of the LXR ligand 22R-Hydroxycholesterol (22R-HC). The analysis highlighted more than 1500 genes modulated by 22R-HC treatment, including the triggering receptor expressed on myeloid cells (TREM)-1, which was found markedly up-regulated. We tested the effect of other nuclear receptor ligands (NRL) and we reported the induction of TREM-1 following RXR, RAR and VDR activation. From a functional point of view, triggering of TREM-1 induced by retinoids increased TNFα and IL-1β release, suggesting an active role of NRL-activated TREM-1(+) DCs in inflammation-driven diseases, including cancer. Consistently with this hypothesis we detected DCs expressing TREM-1 in pleural effusions and ascites of cancer patients, an observation validated by the induction of TREM-1, LXR and RAR target genes when monocyte-DCs were activated in the presence of tumor-conditioned fluids. Finally, we observed a better control of LLC tumor growth in Trem-1(−/-) bone marrow chimera mice as compared to wild type chimera mice. Future studies will be necessary to shed light on the mechanism of TREM-1 induction by distinct NRL, and to characterize the role of TREM-1(+) DCs in tumor growth.

Introduction In the search of new therapeutical strategies against non-small-cell lung cancer (NSCLC), the identification of new prognostic factors is crucial. Objectives In this study, we analyzed the prognostic value of the liver X receptor-alpha (LXR-alpha), a nuclear receptor of a family of cholesterol derivatives called oxysterols, in patients with radically resected NSCLC. Methods We retrospectively reviewed 140 stage II and III surgically treated NSCLC patients that were grouped by percentage of LXR-alpha-positive cells value above or below its median value. Tumor-related survival was evaluated as primary end point. Results The 5-year overall and tumor-related survival rates were 40% and 46%, respectively. The median percentage of LXR-alpha-positive cells was 20%. Patients with stage II NSCLC had higher LXR-alpha values than those with stage III (P = .04). Univariate analysis demonstrated that both TNM stage and LXR-alpha were significantly related to tumor-related survival (P = .006 and P = .004, respectively). The 5-year tumor-related survival rates in stage II and III NSCLC were 56% and 34%, respectively. The 5-year tumor-related survival rates in high and low LXR-alpha value were 57% and 32%, respectively. The multivariate analysis showed that both TNM stage and LXR-alpha were independent prognostic factors (P = .01 and P = .007, respectively) with hazard ratio of 1.92 and 0.49, respectively. Conclusion LXR-alpha seems to be an independent prognostic factor indicating a better survival in completely resected stage II and III NSCLC patients.

This article examines the relationship between the presence of foreign affiliates and product upgrading by Turkish manufacturing firms. The analysis suggests that Turkish firms in sectors and regions more likely to supply foreign affiliates tend to introduce more complex products, where complexity is captured using a measure developed by Hidalgo and Hausmann (2009). This finding is robust to controlling for omitted variables, sample selection and potential simultaneity bias. It is also in line with the view that inflows of foreign direct investment stimulate upgrading of indigenous production capabilities in host countries.

In this paper we explore the impact of the banking sector development on the first time export entry of small enterprises (SEs) in the Turkish manufacturing sector. By exploiting variation in the number of branches per capita across NUTS3 regions and variation in financial dependence across sectors, we support a positive and significant role of finance in fostering the access to foreign markets of SEs. This evidence is robust to the use of alternative measures, the control for omitted variables and the correction for endogeneity. We show that the banking sector reduces the incidence of sunk entry costs by providing both credit and destination-specific information. Finally, we provide original evidence on the role of the territorial diffusion of foreign banks’ branches on SEs’ exports. While no direct effect is detected, we disclose a minor and indirect effect of foreign branches working through their influence on the banking sector development at the local level.

A series of stigmasterol and ergosterol derivatives, characterized by the presence of oxygenated functions at C-22 and/or C-23 positions, were designed as potential liver X receptor (LXR) agonists. The absolute configuration of the newly created chiral centers was definitively assigned for all the corresponding compounds. Among the 16 synthesized compounds, 21, 27, and 28 were found to be selective LXRα agonists, whereas 20, 22, and 25 showed good selectivity for the LXRβ isoform. In particular, 25 showed the same degree of potency as 22R-HC (3) at LXRβ, while it was virtually inactive at LXRα (EC50 = 14.51 μM). Interestingly, 13, 19, 20, and 25 showed to be LXR target gene-selective modulators, by strongly inducing the expression of ABCA1, while poorly or not activating the lipogenic genes SREBP1 and SCD1 or FASN, respectively.

Carbon-based nanoparticles (CBNs) are largely distributed worldwide due to fossil fuel combustion and their presence in many consumer products. In addition to their proven toxicological effects in several biological models, attention in recent years has focussed on the role played by CBNs as Trojan-horse carriers for adsorbed environmental pollutants. This role has not been conclusively determined to date because CBNs can decrease the bioavailability of contaminants or represent an additional source of intake. Herein, we evaluated the intake, transport and distribution of one of the carbon-based powders, the so-called carbon nanopowder (CNPW), and benzo(α)pyrene, when administered alone and in co-exposure to Danio rerio embryos. Data obtained by means of advanced microscopic techniques illustrated that the “particle-specific” effect induced a modification in the accumulation of benzo(α)pyrene, which is forced to follow the distribution of the physical pollutant instead of its natural bioaccumulation. The combined results from functional proteomics and gene transcription analysis highlighted the different biochemical pathways involved in the action of the two different contaminants administered alone and when bound together. In particular, we observed a clear change in several proteins involved in the homeostatic response to hypoxia only after exposure to the CNPW or co-exposure to the mixture, whereas exposure to benzo(α)pyrene alone mainly modified structural proteins. The entire dataset suggested a Trojan-horse mechanism involved in the biological impacts on Danio rerio embryos especially due to different bioaccumulation pathways and cellular targets.

We contribute to the yet limited evidence on the relationship between trade liberalisation and a firm's product mix and diversification strategies for an emerging economy, Turkey. Lower import barriers foster firms’ specialisation in their core products. A drop in import tariffs, indeed, enhances a firm's propensity to drop fringe varieties and favours production growth of core products. More importantly, it favours firms’ specialisation in more sophisticated goods. Export tariff cuts, instead, by relaxing competitive pressure at home and lowering the cost to export, only reduce the firms’ incentive to innovate.

A dual magneto-optical nanoprobe, endowed with properties useful for photodynamic therapy, has been prepared. It is constituted by a superparamagnetic iron oxide (SPIO) core (diameter size distribution centered at ca. 10 nm), obtained by thermal decomposition of iron oleate, coated by a compact silica shell, grown in a reverse microemulsion environment. Luminescent [Re(phen)(CO)3(py)]CF3SO3 complexes were covalently anchored to the silica shell, by functionalizing the pyridine ligand with a triethoxysilane moiety. Finally, the surface of the nanoparticles (NPs) was coated with a layer of polyethylene glycol (PEG), functionalized with triethoxysilane, to improve stability and stealthiness in physiological conditions. Transmission electron microscopy of these SPIO@SiO2–Re@PEG nanocomposites showed a single population, with size distribution centered at ca. 40 nm. NPs showed nuclear relaxivity values that guarantee an appreciable contrast in magnetic resonance imaging (r2 > 30 s−1 mM−1 at frequencies higher than 5 MHz). The presence of the Re complexes imparted photoluminescence to the NPs, with blue shifted emission and higher photoluminescence quantum yields with respect to the free [Re(phen)(CO)3(py-upts)]+ complex (λem 553 vs. 580 nm, Φ 0.060 vs. 0.038, in aerated water solution). The complexes embedded into the NPs maintained a satisfactory efficiency toward 1O2 generation (quantum yields 0.21 vs. 0.26 for the free complex, as assessed using 1,5-dihydroxynaphthalene as indirect marker of the 1O2 presence). Preliminary cell penetration tests were performed on human lung adenocarcinoma A549 cells. Two photon excitation confocal microscopy showed that the NPs were easily internalized and accumulated in the perinuclear region of the cells already after 4 h of incubation. Increased cytotoxicity upon irradiation with respect to the dark was also observed, showing the potential of the nanocomposite for photodynamic therapy applications.

Making use of an original firm level dataset, we explore the causal impact of innovation on the manufacturing firm export activity in Turkey. We model process and product innovation as separately - through cost savings and product quality improvements, respectively - affecting the firm profitability and, consequently, the firm export propensity. This modeling choice highlights heterogeneous effects across high and low income destination markets. In a Multiple Propensity Score Matching framework, we, then, test the impact of each innovation activity and of their joint adoption. We find that only the latter fosters the first time entry into exporting, when the destination market is high income. Nevertheless, innovation positively affects the firmexport propensity. New product introduction is more rewarding than process innovation, especially for exporting to lowincome economies. Process innovation, though, strengthens the positive role of product innovation for exporting to more advanced markets.

OBJETIVO: Comparar os resultados pós-operatórios de 2 grupos de pacientes submetidos a transplante de córnea com técnicas e tempo cirúrgico diferentes, em relação à abordagem do cristalino e/ou lente intra-ocular. MÉTODOS: Neste estudo retrospectivo foram analisados 37 olhos de pacientes divididos em 2 grupos: extração de catarata, implante de lentes intra-oculares (LIO) e transplante de córnea no mesmo tempo cirúrgico - grupo 1 (G1) e extração de catarata sem implante de lentes intra-oculares no primeiro tempo cirúrgico e fixação secundária de lentes intra-oculares associada a transplante de córnea no segundo tempo cirúrgico - grupo 2 (G2). As variáveis estudadas foram: acuidade visual, pressão intra-ocular (PIO), astigmatismo refracional, astigmatismo ceratométrico e complicações pós-operatórias. RESULTADOS: Foi observado melhora da acuidade visual nos 2 grupos (G1 pPURPOSE: To compare the outcomes of two surgical techniques of penetrating keratoplasty with different surgical time, regarding the crystalline and the intraocular lens. METHODS: This retrospective study included 37 patients' eyes divided into 2 groups: extracapsular cataract extraction, posterior chamber intraocular lens implantation and penetrating keratoplasty (Group 1, G1) and transscleral fixation of posterior chamber lens and penetrating keratoplasty (Group 2, G2). The following parameters were recorded: visual acuity, intraocular pressure, refractive astigmatism, complication and keratometric astigmatism. RESULTS: Visual acuity improved in the two groups (G1 p

A production-theory approach to migration is adopted in this paper to address the role of migrant workers from extra-EU countries in Italian manufacturing firms. The adoption of flexible functional forms to model firm-level technology lets us directly derive different measures of elasticity from the coefficients of the estimated production and cost functions. The use of foreign labour is shown to affect the industry composition in favour of low skill intensive sectors and the estimated cross demand elasticities confirm the complementarity between migrant and native workers found in previous studies. However, the two labour inputs prove to be substitutes in terms of the Morishima elasticity of substitution: in general, firms tend to increase the foreign labour intensity of production in response to a decline in migrants’ wage, while the migrant to domestic labour ratio responds to changes in the domestic workers’ wage only for firms in low skill intensive sectors.

We developed an all-optical method to measure the temperature on gold (nanorods and nanostars) and magnetite nanoparticles under near-infrared and radiofrequency excitation by monitoring the excited state lifetime of Rhodamine B that lies within ≅20 nm from the nanoparticle surface. We reached high temperature sensitivity (0.029 ± 0.001 ns/ C) and low uncertainty (±0.3 C). Gold nanostars are ≅3 and ≅100 times more efficient than gold nanorods and magnetite nanoparticles in inducing localized hyperthermia. © 2013 American Chemical Society.

The new heterogeneous firm models in international economics suggest a negative impact of trade openness on the within-sector disparities, due to a restructuring process leading to a resources reallocation toward high efficient firms and the exit of less productive ones. I test this hypothesis for the Italian manufacturing sectors making use of both static and dynamic panel data models. Especially, I investigate the existence of heterogeneous effects in terms of origin of imports and I take into account of the regional heterogeneity computing the productivity dispersion indicator within each sector and regional macro-area. The analysis is implemented within a comprehensive framework controlling for other potential determinants, such as the technological factors and the domestic competition. My findings show that the competitive pressure from low income countries reduces the productivity heterogeneity across firms, while an opposing impact is detected for the exposure to trade with high income economies, and I argue that two different mechanisms are at work behind these effects.

Tumor-derived oxysterols recruit protumor neutrophils in an LXR-independent, CXCR2-dependent manner, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression., Tumor-infiltrating immune cells can be conditioned by molecules released within the microenvironment to thwart antitumor immune responses, thereby facilitating tumor growth. Among immune cells, neutrophils play an important protumorigenic role by favoring neoangiogenesis and/or by suppressing antitumor immune responses. Tumor-derived oxysterols have recently been shown to favor tumor growth by inhibiting dendritic cell migration toward lymphoid organs. We report that tumor-derived oxysterols recruit protumor neutrophils in a liver X receptor (LXR)–independent, CXCR2-dependent manner, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression. We demonstrate that interfering with the oxysterol–CXCR2 axis delays tumor growth and prolongs the overall survival of tumor-bearing mice. These results identify an unanticipated protumor function of the oxysterol–CXCR2 axis and a possible target for cancer therapy.

We study the relationship between offshoring and job stability in Italy in the period 1995–2001 by using an administrative dataset on manufacturing workers. We find that the international fragmentation of production negatively affects job stability. Service offshoring and material purchases from developed countries foster job-to-job transitions within manufacturing of all workers and white collars, respectively. However, the most detrimental effects for job stability come from material offshoring to low income countries which drives blue collar workers out of manufacturing. Therefore, policy interventions should especially focus on this latter category of workers more exposed to fragmentation processes and foreign competition.

A series of [Re 2(μ-ER) 2(CO) 6(μ-pydz) ] complexes have been synthesized (E = S, R = C 6H 5, 2; E = O, R = C 6F 5, 3; C 6H 5, 4; CH 3, and 5; H, 6), starting either from [Re(CO) 5O 3SCF 3] (for 2 and 4), [Re 2(μ-OR) 3(CO) 6] - (for 3 and 5), or [Re 4(μ 3-OH) 4(CO) 12] (for 6). Single-crystal diffractometric analysis showed that the two μ-phenolato derivatives (3 and 4) possess an idealized C 2 symmetry, while the μ-benzenethiolato derivative (2) is asymmetrical, because of the different conformation adopted by the phenyl groups. A combined density functional and time-dependent density functional study of the geometry and electronic structure of the complexes showed that the lowest unoccupied molecular orbital (LUMO) and LUMO+1 are the two lowest-lying π* orbitals of pyridazine, whereas the highest occupied molecular orbitals (HOMOs) are mainly constituted by the "t 2g" set of the Re atoms, with a strong Re-(μ-E) π* character. The absorption spectra have been satisfactorily simulated, by computing the lowest singlet excitation energies. All the complexes exhibit one reversible monoelectronic reduction centered on the pyridazine ligand (ranging from -1.35 V to -1.53 V vs Fc +|Fc). The benzenethiolato derivative 2 exhibits one reversible two-electron oxidation (at 0.47 V), whereas the OR derivatives show two close monoelectronic oxidation peaks (ranging from 0.85 V to 1.35 V for the first peak). The thioderivative 2 exhibits a very small electrochemical energy gap (1.9 eV, vs 2.38-2.70 eV for the OR derivatives), and it does not show any photoluminescence. The complexes containing OR ligands show from moderate to poor photoluminescence, in the range of 608-708 nm, with quantum yields decreasing (ranging from 5.5% to 0.07%) and lifetimes decreasing (ranging from 550 ns to 9 ns) (3 > 4 > 6 ≈ 5) with increasing emission wavelength. The best emitting properties, which are closely comparable to those of the dichloro complex (1), are exhibited by the pentafluorophenolato derivative (3). © 2012 American Chemical Society.

A new amphoteric copolymer, PhenISA, has been obtained by copolymerization of 4-(4′-aminobutyl)-1,10-phenanthroline (BAP) with 2-methylpiperazine and bis(acrylamido)acetic acid (BAC) (6% of phenanthroline-containing repeating units). The copolymer showed excellent solubility in water, where it self-aggregated to give clear nanoparticle suspensions (hydrodynamic diameter = 21 ± 2 nm, by dynamic light scattering (DLS) analysis). The phenanthroline pendants of the polymer stably coordinated either Re(CO) 3+ or Ru(phen)22+ fragments, affording luminescent Re-PhenISA, Re-Py-PhenISA, and Ru-PhenISA polymer complexes, emitting from triplet metal-to-ligand charge transfer ( 3MLCT) excited states (with λem = 608, 571, and 614 nm, respectively, and photoluminescence quantum yields φem = 0.7%, 4.8%, and 4.1%, in aerated water solution, respectively). DLS analyses indicated that the polymer complexes maintained the nanosize of PhenISA. All the complexes were stable under physiological conditions (pH 7.4, 0.15 M NaCl) in the presence of an excess of the ubiquitous competitor cysteine. In vitro viability assays showed no toxicity of Re-Py-PhenISA and Ru-PhenISA complexes, at concentrations in the range of 0.5-50 μM (calculated on the metal-containing unit), toward HEK-293 (human embryonic kidney) cells. A preliminary investigation of internalization in HEK-293 cells, by means of fluorescence confocal microscopy, showed that Ru-PhenISA enters cells via an endocytic pathway and, subsequently, homogeneously diffuse within the cytoplasm across the vesicle membranes. © 2012 American Chemical Society.

Sterol metabolism has recently been linked to innate and adaptive immune responses through liver X receptor (LXR) signaling. Whether products of sterol metabolism interfere with antitumor responses is currently unknown. Dendritic cells (DCs) initiate immune responses, including antitumor activity after their CC chemokine receptor-7 (CCR7)-dependent migration to lymphoid organs. Here we report that human and mouse tumors produce LXR ligands that inhibit CCR7 expression on maturing DCs and, therefore, their migration to lymphoid organs. In agreement with this observation, we detected CD83(+)CCR7(-) DCs within human tumors. Mice injected with tumors expressing the LXR ligand-inactivating enzyme sulfotransferase 2B1b (SULT2B1b) successfully controlled tumor growth by regaining DC migration to tumor-draining lymph nodes and by developing overt inflammation within tumors. The control of tumor growth was also observed in chimeric mice transplanted with bone marrow from mice lacking the gene encoding LXR-alpha (Nr1h3(-/-) mice) Thus, we show a new mechanism of tumor immunoescape involving products of cholesterol metabolism. The manipulation of this pathway could restore antitumor immunity in individuals with cancer. Sterol metabolism has recently been linked to innate and adaptive immune responses through liver X receptor (LXR) signaling. Whether products of sterol metabolism interfere with antitumor responses is currently unknown. Dendritic cells (DCs) initiate immune responses, including antitumor activity after their CC chemokine receptor-7 (CCR7)-dependent migration to lymphoid organs. Here we report that human and mouse tumors produce LXR ligands that inhibit CCR7 expression on maturing DCs and, therefore, their migration to lymphoid organs. In agreement with this observation, we detected CD83(+)CCR7(-) DCs within human tumors. Mice injected with tumors expressing the LXR ligand-inactivating enzyme sulfotransferase 2B1b (SULT2B1b) successfully controlled tumor growth by regaining DC migration to tumor-draining lymph nodes and by developing overt inflammation within tumors. The control of tumor growth was also observed in chimeric mice transplanted with bone marrow from mice lacking the gene encoding LXR-alpha (Nr1h3(-/-) mice) Thus, we show a new mechanism of tumor immunoescape involving products of cholesterol metabolism. The manipulation of this pathway could restore antitumor immunity in individuals with cancer.

The exploitation of the physiologic processing and presenting machinery of DCs by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. Here we show that lymphocytes genetically modified to express self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of tyrosinase-related protein 2-transduced (TRP-2-transduced) lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice. Analysis of the mechanism responsible for the induction of such an immune response allowed us to demonstrate that cross-presentation of the antigen mediated by the CD11c(+)CD8 alpha(+) DC subset had occurred. Furthermore, we demonstrated in vivo and in vitro that DCs had undergone activation upon phagocytosis of genetically modified lymphocytes, a process mediated by a cell-to-cell contact mechanism independent of CD40 triggering. Targeting and activation of secondary lymphoid organ-resident DCs endowed antigen-specific T cells with full effector functions, which ultimately increased tumor growth control and animal survival in a therapeutic tumor setting. We conclude that the use of transduced lymphocytes represents an efficient method for the in vivo loading of tumor-associated antigens on DCs.

Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) beta gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long-term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4+ phenotype. Retroviral integrations and TCR-beta gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA-deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.