Giuseppe Bardi

A Crucial Role for the p110δ Subunit of Phosphatidylinositol 3-Kinase in B Cell Development and Activation

Mice lacking the p110δ catalytic subunit of phosphatidylinositol 3-kinase have reduced numbers of B1 and marginal zone B cells, reduced levels of serum immunoglobulins, respond poorly to immunization with type II thymus-independent antigen, and are defective in their primary and secondary responses to thymus-dependent antigen. p110δ−/− B cells proliferate poorly in response to B cell receptor (BCR) or CD40 signals in vitro, fail to activate protein kinase B, and are prone to apoptosis. p110δ function is required for BCR-mediated calcium flux, activation of phosphlipaseCγ2, and Brutons tyrosine kinase. Thus, p110δ plays a critical role in B cell homeostasis and function.

Functional motor recovery from brain ischemic insult by carbon nanotube-mediated siRNA silencing

Stroke is the second cause of death worldwide with ischemic stroke accounting for 80% of all stroke insults. Caspase-3 activation contributes to brain tissue loss and downstream biochemical events that lead to programmed cell death after traumatic brain injury. Alleviation of symptoms following ischemic neuronal injury can be potentially achieved by either genetic disruption or pharmacological inhibition of caspases. Here, we studied whether silencing of Caspase-3 using carbon nanotube-mediated in vivo RNA interference (RNAi) could offer a therapeutic opportunity against stroke. Effective delivery of siRNA directly to the CNS has been shown to normalize phenotypes in animal models of several neurological diseases. It is shown here that peri-lesional stereotactic administration of a Caspase-3 siRNA (siCas 3) delivered by functionalized carbon nanotubes (f-CNT) reduced neurodegeneration and promoted functional preservation before and after focal ischemic damage of the rodent motor cortex using an endothelin-1 induced stroke model. These observations illustrate the opportunity offered by carbon nanotube-mediated siRNA delivery and gene silencing of neuronal tissue applicable to a variety of different neuropathological conditions where intervention at well localized brain foci may offer therapeutic and functional benefits.

The T cell chemokine receptor CCR7 is internalized on stimulation with ELC, but not with SLC

ELC and SLC are potent agonists for CCR7, a receptor of up‐most importance for the regulation of the homing and traffic of lymphocytes into and within secondary lymphoid tissues. We have studied the effects of both chemokines on receptor re‐distribution in T lymphocytes and other CCR7‐bearing cells by flow cytometry and by assessing receptor mediated functions. In this paper we show thatELC and SLC differ fundamentally in the ability to induce the internalization of their receptor. ELC induced a rapid time‐ and concentration‐dependent internalization of CCR7 and markedly decreasedthe ability of CCR7‐bearing cells to respond to a second stimulation. No receptor internalization, by contrast, was observed on stimulation with SLC. Receptors that were internalized on stimulationwith ELC were re‐expressed when the cells were washed. Re‐expression of receptors and consequent re‐activation of the cells was prevented in the presence of ELC, but was not affected in the presence of SLC. These findings could explain how T lymphocytes that enter lymphoid tissues in response to SLC produced by high‐endothelial venules can subsequently migrate in response to SLC and ELC expressed within the T cell areas.

In vivo distribution and toxicity of PAMAM dendrimers in the central nervous system depend on their surface chemistry.

Dendrimers have been described as one of the most tunable and therefore potentially applicable nanoparticles both for diagnostics and therapy. Recently, in order to realize drug delivery agents, most of the effort has been dedicated to the development of dendrimers that could internalize into the cells and target specific intracellular compartments in vitro and in vivo. Here, we describe cell internalization properties and diffusion of G4 and G4-C12 modified PAMAM dendrimers in primary neuronal cultures and in the CNS of live animals. Confocal imaging on primary neurons reveals that dendrimers are able to cross the cell membrane and reach intracellular localization following endocytosis. Moreover, functionalization of PAMAMs has a dramatic effect on their ability to diffuse in the CNS tissue in vivo and penetrate living neurons as shown by intraparenchymal or intraventricular injections. 100 nM G4-C12 PAMAM dendrimer already induces dramatic apoptotic cell death of neurons in vitro. On the contrary, G4 PAMAM does not induce apoptotic cell death of neural cells in the sub-micromolar range of concentration and induces low microglia activation in brain tissue after a week. Our detailed description of dendrimer distribution patterns in the CNS will facilitate the design of tailored nanomaterials in light of future clinical applications.

Pluronic-coated carbon nanotubes do not induce degeneration of cortical neurons in vivo and in vitro.

Carbon nanotubes (CNTs) are nanodevices with important potential applications in biomedicine such as drug and gene delivery. Brain diseases with no current therapy could be candidates for CNT-based therapies. Little is known about toxicity of CNTs and of their dispersion factors in the brain. Here we show that multiwall CNTs (MWCNTs) coated with Pluronic F127 (PF127) surfactant can be injected in the mouse cerebral cortex without causing degeneration of the neurons surrounding the site of injection. We also show that, contrary to previous reports on lack of PF127 toxicity on cultured cell lines, concentrations of PF127 as low as 0.01% can induce apoptosis of mouse primary cortical neurons in vitro within 24 hours. However, the presence of MWCNTs can avoid PF127-induced apoptosis. These results suggest that PF127-coated MWCNTs do not induce apoptosis of cortical neurons. Moreover, the presence of MWCNTs can reduce PF127 toxicity.

Negligible particle-specific toxicity mechanism of silver nanoparticles: the role of Ag+ ion release in the cytosol.

Toxicity of silver nanoparticles (AgNPs) is supported by many observations in literature, but no mechanism details have been proved yet. Here we confirm and quantify the toxic potential of fully characterized AgNPs in HeLa and A549 cells. Notably, through a specific fluorescent probe, we demonstrate the intracellular release of Ag(+) ions in living cells after nanoparticle internalization, showing that in-situ particle degradation is promoted by the acidic lysosomal environment. The activation of metallothioneins in response to AgNPs and the possibility to reverse the main toxic pathway by Ag(+) chelating agents demonstrate a cause/effect relationship between ions and cell death. We propose that endocytosed AgNPs are degraded in the lysosomes and the release of Ag(+) ions in the cytosol induces cell damages, while ions released in the cell culture medium play a negligible effect. These findings will be useful to develop safer-by-design nanoparticles and proper regulatory guidelines of AgNPs. From the clinical editor: The authors describe the toxic potential of silver nanoparticles (AgNP) in human cancer cell lines. Cell death following the application of AgNPs is dose-dependent, and it is mostly due to Ag+ ions. Further in vivo studies should be performed to gain a comprehensive picture of AgNP-toxicity in mammals.

Rho kinase is required for CCR7-mediated polarization and chemotaxis of T lymphocytes

We studied the role of Rho kinase and extracellular signal‐regulated kinase (ERK)‐2 in the polarization and migration of T lymphocytes in response to the CCR7 ligands EBI1 ligand chemokine (ELC; CCL19) and secondary lymphoid‐tissue chemokine (SLC; CCL21). Both Rho kinase protein isoforms are expressed in T lymphocytes. Inhibition of the Rho kinases with Y‐27632 strongly inhibited SLC‐ and ELC‐induced polarized morphology and chemotaxis of T lymphocytes. Although the chemokines induced ERK‐2 activation, the blockade of this signaling pathway showed no effect on polarization and migration. This study indicates an important role of Rho kinase in CCR7‐mediated polarization and migration of T lymphocytes, whereas ERK‐2 is not involved in these processes.

Selective targeting capability acquired with a protein corona adsorbed on the surface of 1,2-dioleoyl-3-trimethylammonium propane/DNA nanoparticles.

A possible turning point in drug delivery has been recently reached: the protein shell, which covers nanocarriers in vivo, can be used for targeting. Here, we show that nanoparticles can acquire a selective targeting capability with a protein corona adsorbed on the surface. We demonstrate that lipid particles made of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and DNA, upon interaction with human plasma components, spontaneously become coated with vitronectin that promotes efficient uptake in cancer cells expressing high levels of the vitronectin ανβ3 integrin receptor.

The biocompatibility of amino functionalized CdSe/ZnS quantum-dot-Doped SiO2 nanoparticles with primary neural cells and their gene carrying performance⋆

Nanoparticles have an enormous potential for the development of applications in biomedicine such as gene or drug delivery. We developed and characterized NH(2) functionalized CdSe/ZnS quantum dot (QD)-doped SiO(2) nanoparticles (NPs) with both imaging and gene carrier capabilities. We show that QD-doped SiO(2) NPs are internalized by primary cortical neural cells without inducing cell death in vitro and in vivo. Moreover, the ability to bind, transport and release DNA into the cell allows GFP-plasmid transfection of NIH-3T3 and human neuroblastoma SH-SY5Y cell lines. QD-doped SiO(2) NPs properties make them a valuable tool for future nanomedicine application.

Functionalized Carbon Nanotubes in the Brain: Cellular Internalization and Neuroinflammatory Responses

The potential use of functionalized carbon nanotubes (f-CNTs) for drug and gene delivery to the central nervous system (CNS) and as neural substrates makes the understanding of their in vivo interactions with the neural tissue essential. The aim of this study was to investigate the interactions between chemically functionalized multi-walled carbon nanotubes (f-MWNTs) and the neural tissue following cortical stereotactic administration. Two different f-MWNT constructs were used in these studies: shortened (by oxidation) amino-functionalized MWNT (oxMWNT-NH3 +) and amino-functionalized MWNT (MWNT-NH3 +). Parenchymal distribution of the stereotactically injected f-MWNTs was assessed by histological examination. Both f-MWNT were uptaken by different types of neural tissue cells (microglia, astrocytes and neurons), however different patterns of cellular internalization were observed between the nanotubes. Furthermore, immunohistochemical staining for specific markers of glial cell activation (GFAP and CD11b) was performed and secretion of inflammatory cytokines was investigated using real-time PCR (qRT-PCR). Injections of both f-MWNT constructs led to a local and transient induction of inflammatory cytokines at early time points. Oxidation of nanotubes seemed to induce significant levels of GFAP and CD11b over-expression in areas peripheral to the f-MWNT injection site. These results highlight the importance of nanotube functionalization on their interaction with brain tissue that is deemed critical for the development nanotube-based vector systems for CNS applications.