Regina Tavano

CD28 and Lipid Rafts Coordinate Recruitment of Lck to the Immunological Synapse of Human T Lymphocytes

In T lymphocytes, the Src family kinase Lck associates lipid rafts and accumulates at the immunological synapse (IS) during T cell stimulation by APCs. Using CD4- or CD28-deficient murine T cells, it was suggested that recruitment of Lck to the IS depends on CD4, whereas CD28 sustains Lck activation. However, in human resting T cells, CD28 is responsible for promoting recruitment of lipid rafts to the IS by an unknown mechanism. Thus, we performed a series of experiments to determine 1) whether Lck is recruited to the IS through lipid rafts; and 2) whether Lck recruitment to the IS of human resting T cells depends on CD4 or on CD28 engagement. We found that CD28, but not CD4, stimulation induced recruitment of Lck into detergent-resistant domains as well as its accumulation at the IS. We also found that Lck recruitment to the IS depends on the CD28 COOH-terminal PxxPP motif. Thus, the CD28-3A mutant, generated by substituting the prolines in positions 208, 211, and 212 with alanines, failed to induce Lck and lipid raft accumulation at the synapse. These results indicate that CD28 signaling orchestrates both Lck and lipid raft recruitment to the IS to amplify T cell activation.

Human monocytes/macrophages are a target of Neisseria meningitidis Adhesin A (NadA).

Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial‐monocyte association, but a NadA‐positive strain induced a significantly higher amount of TNF‐α and IL‐8 compared with the parental NadA‐negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadAΔ351–405, a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL‐8, IL‐6, MCP‐1, and MIP‐1α and low levels of the main vasoactive mediators TNF‐α and IL‐1. NadAΔ351–405 also inhibited monocyte apoptosis and determined its differentiation into a macrophage‐like phenotype.

Water-soluble peptide-coated nanoparticles: control of the helix structure and enhanced differential binding to immune cells.

The stabilizing action of C(α)-tetrasubstituted α-amino acids inserted into a sequence of short peptides allowed for the first time the preparation of water-soluble nanoparticles of different materials coated with a helix-structured undecapeptide. This peptide coating strongly favors nanoparticle uptake by human immune system cells.

Stem-cell therapy in an experimental model of pulmonary hypertension and right heart failure: Role of paracrine and neurohormonal milieu in the remodeling process

BACKGROUND In this study we investigated the effect of human amniotic fluid stem (hAFS) cells and rat adipose tissue stromal vascular fraction GFP-positive cell (rSVC-GFP) therapy and the contribution of the paracrine and neurohormonal milieu to cardiac and pulmonary vascular remodeling in a rat model of pulmonary hypertension (PH) and right heart failure (RHF). METHODS Sprague-Dawley rats were injected with monocrotaline (MCT). Four million hAFS or rSVC-GFP cells were injected via the tail vein 3 weeks after MCT. RHF was confirmed by RV hypertrophy/dilation and by brain natriuretic peptide (BNP) level. Cytokine profile was assessed by Multiplex array. Stem cell (SC) differentiation was studied by immunofluorescence. RESULTS MCT rats showed eccentric RV hypertrophy with increased RV dilation (measured as right ventricular mass/right ventricular volume [RVM/RVV]: MCT, 1.46 ± 0.12; control, 2.33 ± 0.24; p = 0.01), and increased RV hypertrophy (measured as LVM/RVM: MCT, 1.58 ± 0.06; control, 2.83 ± 0.1; p < 0.00001), increased BNP (MCT, 5.2 ± 1.2; control, 1.5 ± 0.1; p < 0.001) and both pro- and anti-inflammatory cytokines. SC produced a fall of BNP (hAFS, 2.1 ± 0.7; rSVC-GFP, 1.98 ± 1.3; p < 0.001) and pro-inflammatory cytokines. Positive RV remodeling with decreased RV dilation (RVM/RVV: hAFS, 1.87 ± 0.44; rSVC-GFP, 2.12 ± 0.24; p < 0.03 and p < 0.05 vs MCT) and regression of RV hypertrophy (LVM/RVM: hAFS, 2.06 ± 0.08; rSVC-GFP, 2.16 ± 0.08; p < 0.00001 vs MCT) was seen together with a decrease in medial wall thickness of pulmonary arterioles (hAFS, 35.33 ± 2.78%; rSVC-GFP, 37.15 ± 2.92%; p = 0.0001 vs MCT). CONCLUSIONS SC engrafted in the lung, heart and skeletal muscle modulated the pro- and anti-inflammatory cytokine milieu, and produced a positive neurohormonal response. This was accompanied by positive cardiac and pulmonary vascular remodeling, with formation mainly of new vascular cells.

Procoagulant properties of bare and highly PEGylated vinyl-modified silica nanoparticles.

AIMS Undesired alterations of the blood clotting balance may follow the intravascular injection of nanotherapeutics/diagnostics. Here, we tested the procoagulant activity of synthetic amorphous silica (SAS) and organically modified silica (ORMOSIL) nanoparticles (NPs) and whether a high-density polyethylene glycol coating minimizes these effects. MATERIALS & METHODS Hageman factor- and tissue factor-dependent activation of human blood/plasma coagulation, and binding to human monocytes, endothelial cells and platelets were quantified in vitro using naked and PEGylated ORMOSIL-NPs. Their effects were compared with those of SAS-NPs, present in many industrial products, and of poly(lactic-co-glycolic acid)- and small unilamellar vesicles-NPs, already approved for use in humans. RESULTS Both SAS-NPs and ORMOSIL-NPS presented a significant procoagulant activity. However, highly PEGylated ORMOSIL-NPs were particularly averse to the interaction with the soluble factors and cellular elements that may lead to intravascular blood coagulation. CONCLUSION Stealth, highly PEGylated ORMOSIL-NPs with a poor procoagulant activity can be used as starting blocks to design hemocompatible nanomedical-devices.

Highly PEGylated silica nanoparticles: “ready to use” stealth functional nanocarriers

The peculiar properties of silica nanoparticles make them well suited to the development of smart nanomaterials for medicine. Here we report a new procedure to prepare doped and PEGylated silica-based nanoparticles. Thus, a complex multifunctional system is obtained simply by a one-pot reaction followed by a straightforward purification procedure. Control of the nanoparticles final size is obtained by carefully choosing reactants and conditions. Moreover, reactive functional groups necessary to allow subsequent bioconjugation are easily introduced in the PEG coating by the same procedure. Unprecedented high-density surface coating is obtained and this successfully stabilizes the nanoparticles against aggregation both in saline solution and in the presence of serum proteins. The absence of cytotoxicity and “stealth” behaviour toward phagocytic capture by human macrophages has been demonstrated and this enables such nanosystems as candidates for the development of drug delivery agents.

The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA- OMVs, without further stimulating their proinflammatory activity on circulating monocytes.

Hypervirulent MenB causing fatal human infections frequently display the oligomeric‐coiled coil adhesin NadA, a 45‐kDa intrinsic outer membrane protein implicated in binding to and invasion of respiratory epithelial cells. A recombinant soluble mutant lacking the 10‐kDa COOH terminal membrane domain (NadAΔ351–405) also activates human monocytes/macrophages/DCs. As NadA is physiologically released during sepsis as part of OMVs, in this study, we tested the hypothesis that NadA+ OMVs have an enhanced or modified proinflammatory/proimmune action compared with NadA– OMVs. To do this we investigated the activity of purified free NadAΔ351–405 and of OMVs from MenB and Escherichia coli strains, expressing or not full‐length NadA. NadAΔ351–405 stimulated monocytes and macrophages to secrete cytokines (IL‐1β, TNF‐α, IL‐6, IL‐12p40, IL‐12p70, IL‐10) and chemokines (IL‐8, MIP‐1α, MCP‐1, RANTES), and full‐length NadA improved MenB OMV activity, preferentially on macrophages, and only increased cytokine release. NadAΔ351–405 induced the lymphocyte costimulant CD80 in monocytes and macrophages, and NadA+ OMVs induced a wider set of molecules supporting antigen presentation (CD80, CD86, HLA‐DR, and ICAM‐1) more efficiently than NadA– OMVs only in macrophages. Moreover, membrane NadA effects, unlike NadAΔ351–405 ones, were much less IFN‐γ‐sensitive. The activity of NadA‐positive E. coli OMVs was similar to that of control OMVs. NadA in MenB OMVs acted at adhesin concentrations ∼106 times lower than those required to stimulate cells with free NadAΔ351–405.

Proinflammatory effects of bare and PEGylated ORMOSIL-, PLGA- and SUV-NPs on monocytes and PMNs and their modulation by f-MLP

AIMS We wanted to test the proinflammatory effects of vinyltriethoxysilane-based organically modified silica nanoparticles (ORMOSIL-NPs) in vitro on blood leukocytes. MATERIALS & METHODS Cell selectivity, cytokines/chemokines and O(2) (-) production were analyzed using nonpolyethylene glycol (PEG)ylated and PEGylated ORMOSIL-NPs, poly(lactic-co-glycolic acid) (PLGA)-NPs and small unilamellar vesicles (SUV)-NPs. RESULTS ORMOSIL-NPs mostly bound to monocytes while other NPs to all leukocyte types similarly. Cell capture of PEGylated-NPs decreased strongly (ORMOSIL), moderately (PLGA) and weakly (SUV). Bare ORMOSIL-NPs effectively stimulated the production of IL-1β/IL-6/TNF-α/IL-8 by monocytes and of IL-8 by polymorphonuclear leukocytes (PMNs). NP PEGylation inhibited such effects only partially. Formyl-methionine-leucine phenylalanine (f-MLP) further increased the release of cytokines/chemokines by monocytes/PMNs primed with bare and PEGylated ORMOSIL-NPs. PEGylated SUV-NPs, bare and PEGylated ORMOSIL- and PLGA-NPs sensitize PMNs and monocytes to secrete O(2) (-) upon f-MLP stimulation. CONCLUSION ORMOSIL-NPs are preferentially captured by circulating monocytes but stimulate both monocytes and PMNs per se or by sensitizing them to another agonist (f-MLP). PEG-coating confers stealth effects but does not completely eliminate leukocyte activation. Safe nanomedical applications require the evaluation of both intrinsic and cooperative proinflammatory potential of NPs.

Substitution of the arginine/leucine residues in apidaecin Ib with peptoid residues: effect on antimicrobial activity, cellular uptake, and proteolytic degradation.

Several aspects of the mechanism of action of Pro-rich antimicrobial peptides, together with their low toxicity in mammalian cells, make them good candidates for the development of new antibiotic agents. We investigated the effect induced in the insect antimicrobial peptide apidaecin Ib by the replacement of a single arginine/leucine residue with a N-substituted glycine. The resulting peptoid-peptide hybrids are more resistant to proteolysis and devoid of any significant cytotoxic activity, but moving the [NArg]residue from the N- to the C-terminal end of the molecule progressively reduces the antibacterial activity. Cell uptake experiments in E. coli cells suggest that the loss of antibacterial activity of [NArg(17)]apidaecin is a consequence of its inability to translocate into bacterial cells. Conversely, apidaecin and its peptoid-peptide hybrids are able to cross the plasma membrane in eukaryotic cells and to diffuse in the cytosol, although their translocating ability is far less effective than that of other known cell permeant peptides.

Targeted delivery of photosensitizers: efficacy and selectivity issues revealed by multifunctional ORMOSIL nanovectors in cellular systems

PEGylated and non-PEGylated ORMOSIL nanoparticles prepared by microemulsion condensation of vinyltriethoxy-silane (VTES) were investigated in detail for their micro-structure and ability to deliver photoactive agents. With respect to pure silica nanoparticles, organic modification substantially changes the microstructure and the surface properties. This in turn leads to a modulation of both the photophysical properties of embedded photosensitizers and the interaction of the nanoparticles with biological entities such as serum proteins. The flexibility of the synthetic procedure allows the rapid preparation and screening of multifunctional nanosystems for photodynamic therapy (PDT). Selective targeting of model cancer cells was tested by using folate, an integrin specific RGD peptide and anti-EGFR antibodies. Data suggest the interference of the stealth-conferring layer (PEG) with small targeting agents, but not with bulky antibodies. Moreover, we showed that selective photokilling of tumour cells may be limited even in the case of efficient targeting because of intrinsic transport limitations of active cellular uptake mechanisms or suboptimum localization.