Dendritic cell differentiation blocked by primary effusion lymphoma-released factors is partially restored by inhibition of P38 MAPK.

To better understand the molecular mechanisms underlying the dendritic cell (DC) defects in cancer, we analyzed which signaling pathway is implicated in the abnormal monocyte differentiation into DC determined by the presence of Primary effusion lymphoma (PEL) released factors. Our results indicate that the DC, obtained in this condition, together with phenotypic abnormalities and reduced allostimulatory function, showed hyperphosphorylation of signal transducer and activator of transcription 3 (STAT3) and p38 mitogen-activated protein kinase (MAPK) molecules, in comparison to the DC differentiated in the absence of PEL-released factors. The inhibition of p38 MAPK but not of STAT3 phosphorylation, with specific inhibitors, was able to revert the effect of the PEL-released factors on the DC phenotype. This study suggests that p38 MAPK signaling pathway is an important contributor to the abnormal differentiation of DC in PEL.

Designing novel pH-sensitive non-phospholipid vesicle: characterization and cell interaction.

In this work, we report the preparation, the characterization and interaction with cells of novel pH-sensitive non-phospholipid vesicle formulations, from a non-ionic surfactant mixed with cholesterol (CHOL) and his derivative cholesteryl hemisuccinate (CHEMS), as pH-sensitive molecule. This molecule, can destabilize the vesicle lipid bilayer when exposed to an acidic environment, with a subsequent release of vesicular content, enhancing the cytoplasmatic delivery of drugs to target cells. Vesicles were characterized by static and dynamic light scattering, in order to evaluate their dimensions, bilayer thickness and vesicle stability. Membrane permeability changes were determined by the release of entrapped hydroxypyrene-1,3,6-trisulfonic acid (HPTS). Also diphenylhesatriene (DPH) fluorescence anisotropy and zeta potential measurements were used to evidence the pH sensitivity. Furthermore vesicles were characterized by means of electronic microscopy after freeze-fracture. The interaction of non-lipid vesicles containing different fluorescent dyes with Raw 264.7, mouse monocite macrophage, were analyzed by flow cytometric analysis. The obtained results indicate that the pH-sensitive vesicular structures show good plasma stability and relevant pH-sensitivity. Moreover this formulation was able to interact with target membranes (i.e. plasma or endosomal membrane) and to release the encapsulated material into the cytoplasm.

New vesicular ampicillin-loaded delivery systems for topical application: characterization, in vitro permeation experiments and antimicrobial activity.

In this paper, the experimental conditions for preparing ampicillin-loaded surfactant vesicles (SVs) are described. Our studies are focused on the potential use of a vesicular polymeric dispersion as ampicillin delivery system for topical application. The main components of the formulation are uncharged and charged SVs loaded with ampicillin and dispersed in a gellan solution. The following issues are addressed: the drug encapsulation efficiency (e.e.), the kinetic of drug release from the delivery systems, the antimicrobial activity of vesicle-entrapped ampicillin. The in vitro permeation experiments through a synthetic lipophilic barrier (Silastic) and through porcine skin are carried out to evaluate the potential use as a dermal formulation. The use of both a synthetic and a biological membrane allows to discriminate between the effects related to variations of thermodynamic parameters and those correlated to biological factors. The release rate of ampicillin is increased by encapsulation in neutral and negatively charged SVs and the permeation rate was slowed by dispersion of drug-loaded SVs in gellan solution. Finally, studies of antimicrobial activity on prepared systems evidenced that ampicillin encapsulated in SVs exhibit a higher activity than the free drug.

Lidocaine-loaded non-ionic surfactant vesicles: characterization and in vitro permeation studies.

Our research on topical application of lidocaine-loaded non-ionic surfactant vesicles (NSVs) was prompted by the great interest on new delivery systems for local anaesthetics. This study is focused on a novel formulation of NSVs entrapping lidocaine in the form of a free base (LID) and a hydrochloride (LIDHCl). NSVs were prepared from polyoxyethylene sorbitan monolaurate (Tween20) and cholesterol. The effect of vesicle composition and environmental pH condition (8.6-5.5) on drug encapsulation efficiency (e.e.) was investigated. Experimental strategies involved: freeze-fracture, microscopy technique, dynamic light scattering, permeation through Silastic and mouse abdominal skin, in vitro release kinetics of vesicle-entrapped drugs, fluorescence quenching analyses. Diffusion experiments showed that the flux of charged lidocaine through Silastic membrane was possible only after the vesicle encapsulation. Permeation through mouse abdominal skin of LIDHCl loaded vesicles showed a higher flux and a shorter lag time with respect to classical liposome formulations, while LID permeation rate was quite similar for NSV and liposome formulations. Vesicles were also prepared in the presence of dicetylphosphate (DCP) and N-cetylpyridinium chloride (CP) to obtain negatively and positively charged vesicles respectively, but in this case the e.e. of the drug was negligible. The possible reason of the remarkable lower e.e. observed with charged vesicles was investigated by means of fluorescence quenching experiments.

Fibroblast growth factor 10 induces proliferation and differentiation of human primary cultured keratinocytes.

Fibroblast growth factor 10 is a novel member of the fibroblast growth factor family, which is involved in morphogenesis and epithelial proliferation. It is highly homologous to the keratinocyte growth factor (or fibroblast growth factor 7), a key mediator of keratinocyte growth and differentiation. Both fibroblast growth factor 10 and keratinocyte growth factor bind with high affinity to the tyrosine kinase keratinocyte growth factor receptor. Here we analyzed the effect of fibroblast growth factor 10 on primary cultures of human keratinocytes, grown in chemically defined medium, and we compared the proliferative and differentiative cell responses to fibroblast growth factor 10 with those induced by keratinocyte growth factor and epidermal growth factor. Cell counting, 5-bromo-2'-deoxyuridine incorporation, and western blot analysis showed that fibroblast growth factor 10, similarly to keratinocyte growth factor, not only is a potent mitogen for human keratinocytes, but also promotes the expression of both early differentiation markers K1 and K10 and late differentiation marker filaggrin in response to the Ca2+ signal, and seems to sustain the proliferative activity in suprabasal stratified cells. Immunoprecipitation/western blot analysis revealed that fibroblast growth factor 10, similarly to keratinocyte growth factor, is able to induce tyrosine phosphorylation of keratinocyte growth factor receptor and of cellular substrates such as PLCgamma.

Expression of GM3 microdomains on the surfaces of murine fibroblasts correlates with inhibition of cell proliferation.

The expression and surface distribution of monosialoganglioside GM3 on the plasma membranes of NIH3T3 fibroblasts cultured at semiconfluence were analyzed by immunofluorescence as well as by immunogold electron microscopy on thin sections and surface replicas. The GM3 expression was highly variable from cell to cell and the distribution of the ganglioside on the positive cells appeared punctate. Quantitative immunogold electron microscopy showed the existence of well-defined GM3 clusters of different sizes scattered all over the cell surfaces. Double immunofluorescence analysis of 5-bromo-2'-deoxyuridine incorporation to identify proliferating cells and of GM3 expression indicated that most of the GM3-positive cells appear unable to synthesize DNA and demonstrated a growth-dependent expression of GM3.

Glycosphingolipid domains on cell plasma membrane.

In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.

Increased number of caveolae and caveolin-3 overexpression in Duchenne muscular dystrophy.

Caveolae are small pockets or invaginations localized at the plasma membrane. Caveolins are the principal protein components of caveolae and play an important structural role in the formation of caveolae membranes. Here, we studied by freeze fracture and immunological techniques the spatial organization of caveolae at the muscle cell plasma membrane and the expression of caveolin-3 in Duchenne muscular dystrophy (DMD) muscle fibers. In DMD muscle, we found an increased number of caveolae at the sarcolemma that corresponds to an overexpression of caveolin-3 by immunohistochemistry and by Western blot analysis. These findings suggest a possible role for caveolae and caveolin-3 in the pathogenesis of DMD.

Morphological analysis of the interaction of charged surfactant vesicles (SVs) with human cultured cells.

We analyzed the binding and fusogenic properties of surfactant vesicles (SVs), composed of ionic and nonionic surfactants and cholesterol, with the surface of different human lymphoid cells. The influence of charge on SVs-cell interaction was evaluated by monitoring the presence of fluorescent sodium calcein artificially entrapped in the vesicles using optical fluorescence microscopy and laser scanning confocal microscopy. Our results clearly indicate that only negatively charged vesicles bind and fuse with the plasma membrane of human lymphoid cells, and the number of SVs bound to the cell surface was variable among the positive cells. Thin section electron microscopy illustrated that the fusogenic events of SVs with the cell plasma membrane mostly occurred at smooth and nonvillous regions of the cell surface. Taken together, our results suggest that binding and fusion of SVs with the cell plasma membrane might be dependent on interactions with specific membrane components that preferentially recognize negatively charged SVs.

Selective localization of matrix metalloproteinase 9, beta1 integrins, and human lymphocyte antigen class I molecules on membrane vesicles shed by 8701-BC breast carcinoma cells.

The shedding of membrane vesicles from the cell surface is a vital process considered to be involved in cell-cell and cell-matrix interactions and in tumor progression. By immunoelectron microscopic analysis of surface replicas of 8701-BC human breast carcinoma cells, we observed that membrane vesicles shed from plasma membranes contained densely clustered gelatinase B [matrix metalloproteinase 9 (MMP-9)], beta1 integrins, and human lymphocyte antigen class I molecules. By contrast, alpha-folate receptor was uniformly distributed on the smooth cell membrane and shedding areas. Both cell surface clustering of selected molecules and membrane vesicle release were evident only when cells were cultured in the presence of serum. Vesicle shedding occurred preferentially at the edge or along narrow protrusions of the cell. Specific accumulation of proMMP-9 and active forms of MMP-9 in shed vesicles was also demonstrated by gelatin zymography. In addition, Western blotting analysis showed the presence of a large amount of proMMP-9/tissue inhibitor of metalloproteinase 1 complex. The release of selected areas of plasma membranes enriched with MMP-9 and beta1 integrins indicates that membrane vesicle shedding from tumor cells plays an important role in the directional proteolysis of the extracellular matrix during cellular migration. The presence of human lymphocyte antigen class I antigens suggests a mechanism for tumor cells to escape from immune surveillance.

Modulation of vesicle shedding in 8701 BC human breast carcinoma cells.

Vesicles, shed in the extracellular medium by several kinds of normal and tumoral cells, are known to play important roles in cell-cell and cell-matrix interactions and to participate in mechanisms by which tumoral cells acquire metastatic capability and evade immune surveillance. Regulation of the shedding phenomenon and molecular mechanisms involved in extracellular vesicle production are not known and are the subject of this investigation. Fetal calf serum stimulated shedding short after its addition and its stimulatory effect was dose dependent. This effect was reduced after gelatin-Sepharose adsorption indicating a possible involvement of gelatinases on its stimulatory effect. This conclusion was confirmed by the inhibitory effect of bathophenanthroline. Shedding of membrane vesicles decreased after treatment with all trans retinoic acid, a molecule known for its capability to induce cell differentiation. Brefeldin A, an inhibitor of intracellular vesicle movements, and methylamine, an inhibitor of exocytosis, did not abolish shedding. Quercetin, an inhibitor of phosphatidyl inositol 4 kinase and 1,4 phosphatidyl inositol 5 kinase, and 8-Cl-cAMP, a site selective cAMP analogous which induces growth inhibition and differentiation, significantly decreased the amount of shed vesicles.

In vitro T cell activation in elderly individuals: failure in CD69 and CD71 expression.

A large number of T cell dysfunctions have been observed in the elderly. The most widely observed is the inability of these cells to proliferate at a level comparable to T cells from young individuals after stimulation by mitogens. To better characterize T cell impairment, we have focused on the in vitro T cell activation, analyzing by flow cytometry the activation molecules CD69 and CD71 on mitogen-stimulated lymphocytes from young and elderly subjects. The results show that the percentages of CD69+ and CD71 + T cells were significantly decreased in cultures from elderly subjects when compared to values obtained culturing cells from young individuals. The differences observed seem not due to differences in CD4 and CD8 rates in the "old' cells that underwent activation, since, following activation, the pattern of CD4 and CD8 phenotypes was the same in both groups of subjects. Signals from CD69 are relevant in controlling cytokine gene expression because its stimulation leads to interleukin-2 production and increases its receptor expression. The interaction of this cytokine with its cellular receptor is an essential requirement for T lymphocytes to express CD71 and to start proliferation. Thus, a key role in the age-associated impairment of T cell activation could be played by an ineffective modulation of CD69 expression suggesting a defect in the signal transduction pathway of the T cell receptor-CD3 complex in elderly.

Patching and capping of LFA-1 molecules on human lymphocytes.

The distribution and dynamics of LFA-1 molecules over the surface of human lymphocytes were analysed using immunogold label-fracture and fracture-flip methods. Patching and capping were induced by incubation at 37 degrees C with antibodies directed against the alpha and beta chains respectively of the heterodimeric LFA-1 molecule, and were followed by immunofluorescence. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) to link LFA-1 molecules to the cytoskeleton increased the percentage of capped cells, implying a faster and more efficient process of capping. At all times of clustering or upon phorbol ester treatment, the concentration of LFA-1 in patches and then in caps was not accompanied by a parallel concentration of membrane particles on the freeze-fractured plasma membranes. Our results support the role of the cytoskeleton in regulating the capping phenomenon and in controlling the structural organization of the plasma membranes.

Dynamics of transmembrane proteins during Sindbis virus budding.

Label-fracture and immunogold fracture-flip techniques are used to address at the ultrastructural level the dynamics of viral and cellular transmembrane proteins during the budding of Sindbis virus on the plasma membrane of infected cells. Immunolabeling with anti-Sindbis spike antibodies shows that the viral proteins are mostly in clusters, all associated with budding viruses. Ultrastructural observation of the unlabeled freeze-fractured plasma membranes shows that membrane particles aggregate over the budding viruses. These results indicate that the concentration of viral transmembrane proteins gives rise to a parallel concentration of membrane particles. Immunolabeling with anti-CD8 antibodies of cells expressing by transfection the CD8 transmembrane protein and infected with Sindbis virus shows absence of labeling on the particle aggregates over the forming virions. These findings indicate the exclusion of CD8 proteins from the portions of the membrane where budding occurs.

High-resolution surface views of human lymphocytes during capping of CD4 and HLA antigens as revealed by immunogold fracture-flip.

The surface ultrastructure of lymphocytes during capping of two transmembrane proteins is shown. As seen by fracture-flip the plasma membranes of human lymphocytes are covered by a high density of surface particles. Incubation in 30% glycerol leads to aggregation of these surface particles. Immunogold labelling shows that the transmembrane proteins bearing HLA class I and CD4 antigens are confined to the particle aggregates. These results indicate that surface particles revealed by fracture-flip represent surface protrusions of integral membrane proteins seen as intramembrane particles in freeze-fractured lymphocytes. During capping HLA or CD4 antigens aggregate into progressively larger patches and, finally, into single caps. As revealed by fracture-flip the patches/caps are seen as clearly differentiated raised platforms that are clearly and sharply demarcated relative to contiguous areas of the surface. In non-patched (non-capped) regions, the pattern of distribution and apparent density of surface particles remain unaltered. Immunogold labelling clearly demarcates patches and caps, and shows that virtually no antigen molecules remain dispersed over the non-patched (non-capped) regions. Estimates of the surface density of either HLA or CD4 antigens over the capped areas point to high planar concentrations of the transmembrane proteins that bear these antigens.