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Inhaled siRNA therapy has a unique potential for treatment of severe lung diseases, such as cystic fibrosis (CF). Nevertheless, a drug delivery system tackling lung barriers is mandatory to enhance gene silencing efficacy in the airway epithelium. We recently demonstrated that lipid-polymer hybrid nanoparticles (hNPs), comprising a poly(lactic-co-glycolic) acid (PLGA) core and a lipid shell of dipalmitoyl phosphatidylcholine (DPPC), may assist the transport of the nucleic acid cargo through mucus-covered human airway epithelium. To study in depth the potential of hNPs for siRNA delivery to the lungs and to investigate the hypothesized benefit of PEGylation, here, an siRNA pool against the nuclear factor-kappa B (siNF kappa B) was encapsulated inside hNPs, endowed with a non-PEGylated (DPPC) or a PEGylated (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) or DSPE-PEG) lipid shell. Resulting hNPs were tested for their stability profiles and transport properties in artificial CF mucus, mucus collected from CF cells, and sputum samples from a heterogeneous and representative set of CF patients. Initial information on hNP properties governing their interaction with airway mucus was acquired by small-angle X-ray scattering (SAXS) studies in artificial and cellular CF mucus. The diffusion profiles of hNPs through CF sputa suggested a crucial role of lung colonization of the corresponding donor patient, affecting the mucin type and content of the sample. Noteworthy, PEGylation did not boost mucus penetration in complex and sticky samples, such as CF sputa from patients with polymicrobial colonization. In parallel, in vitro cell uptake studies performed on mucus-lined Calu-3 cells grown at the air-liquid interface (ALI) confirmed the improved ability of non-PEGylated hNPs to overcome mucus and cellular lung barriers. Furthermore, effective in vitro NF kappa B gene silencing was achieved in LPS-stimulated 16HBE14o-cells. Overall, the results highlight the potential of non-PEGylated hNPs as carriers for pulmonary delivery of siRNA for local treatment of CF lung disease. Furthermore, this study provides a detailed understanding of how distinct models may provide different information on nanoparticle interaction with the mucus barrier.

β-glucocerebrosidase is a lysosomal hydrolase involved in the catabolism of the sphingolipid glucosylceramide. Biallelic loss of function mutations in this enzyme are responsible for the onset of Gaucher disease, while monoallelic β-glucocerebrosidase mutations represent the first genetic risk factor for Parkinson's disease. Despite this evidence, the molecular mechanism linking the impairment in β-glucocerebrosidase activity with the onset of neurodegeneration in still unknown. In this frame, we developed two in vitro neuronal models of β-glucocerebrosidase deficiency, represented by mouse cerebellar granule neurons and human-induced pluripotent stem cells-derived dopaminergic neurons treated with the specific β-glucocerebrosidase inhibitor conduritol B epoxide. Neurons deficient for β-glucocerebrosidase activity showed a lysosomal accumulation of glucosylceramide and the onset of neuronal damage. Moreover, we found that neurons react to the lysosomal impairment by the induction of their biogenesis and exocytosis. This latter event was responsible for glucosylceramide accumulation also at the plasma membrane level, with an alteration in lipid and protein composition of specific signaling microdomains. Collectively, our data suggest that β-glucocerebrosidase loss of function impairs the lysosomal compartment, establishing a lysosome-plasma membrane axis responsible for modifications in the plasma membrane architecture and possible alterations of intracellular signaling pathways, leading to neuronal damage.

Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4’s broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.

The delivery of a dexamethasone formulation directly into the lung appears as an appropriate strategy to strengthen the systemic administration, reducing the dosage in the treatment of lung severe inflammations. For this purpose, a hyaluronic acid-dexamethasone formulation was developed, affording an inhalable reconstituted nanosuspension suitable to be aerosolized. The physico-chemical and biopharmaceutical properties of the formulation were tested: size, stability, loading of the spray-dried dry powder, reconstitution capability upon redispersion in aqueous media. Detailed structural insights on nanoparticles after reconstitution were obtained by light and X-ray scattering techniques. (1) The size of the nanoparticles, around 200 nm, is in the proper range for a possible engulfment by macrophages. (2) Their structure is of the core-shell type, hosting dexamethasone nanocrystals inside and carrying hyaluronic acid chains on the surface. This specific structure allows for nanosuspension stability and provides nanoparticles with muco-inert properties. (3) The nanosuspension can be efficiently aerosolized, allowing for a high drug fraction potentially reaching the deep lung. Thus, this formulation represents a promising tool for the lung administration via nebulization directly in the pipe of ventilators, to be used as such or as adjunct therapy for severe lung inflammation.

Sphingosine-1-phosphate (S1P) is a crucial mediator involved in the progression of different cancers, including glioblastoma multiforme (GBM), the most frequent and deadly human brain tumor, characterized by extensive invasiveness and rapid cell growth. Most of GBMs overexpress the epidermal growth factor receptor (EGFR), and we investigated the possible link between S1P and EGFR signaling pathways, focusing on its role in GBM survival, using the U87MG human cell line overexpressing EGFR (EGFR+). We previously demonstrated that EGFR+ cells have higher levels of extracellular S1P and increased sphingosine kinase-1 (SK1) activity than empty vector expressing cells. Notably, we demonstrated that EGFR+ cells are resistant to temozolomide (TMZ), the standard chemotherapeutic drug in GBM treatment, and the inhibition of SK1 or S1P receptors made EGFR+ cells sensitive to TMZ, moreover, exogenous S1P reverted this effect, thus involving extracellular S1P as a survival signal in TMZ resistance in GBM cells. In addition, both PI3K/AKT and MAPK inhibitors markedly reduced cell survival, suggesting that the enhanced resistance to TMZ of EGFR+ cells is dependent on the increased S1P secretion, downstream of the EGFR-ERK-SK1-S1P pathway. Altogether, our study provides evidence of a functional link between S1P and EGFR signaling pathways enhancing the survival properties of GBM cells.

The highly conserved Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is a key target for next-generation vaccines against blood-stage malaria. PfCyRPA constitute the core of a ternary complex, including the reticulocyte binding-like homologous protein 5 (PfRh5) and the Rh5-interacting protein (PfRipr), and is fundamental for merozoite invasion of erythrocytes. In this study, we show that monoclonal antibodies (mAbs) specific to PfCyRPA neutralize the in vitro growth of Ghanaian field isolates as well as numerous laboratory-adapted parasite lines. We identified subsets of mAbs with neutralizing activity that bind to distinct sites on PfCyRPA and that in combination potentiate the neutralizing effect. As antibody responses against multiple merozoite invasion proteins are thought to improve the efficacy of blood-stage vaccines, we also demonstrated that combinations of PfCyRPA- and PfRh5 specific mAbs act synergistically to neutralize parasite growth. Yet, we identified prominent strain-dependent neutralization potencies, which our results suggest is independent of PfCyRPA expression level and polymorphism, demonstrating the importance of addressing functional converseness when evaluating blood-stage vaccine candidates. Finally, our results suggest that blood-stage vaccine efficacy can be improved by directing the antibody response towards defined protective epitopes on multiple parasite antigens.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is expressed at the apical plasma membrane (PM) of different epithelial cells. The most common mutation responsible for the onset of cystic fibrosis (CF), F508del, inhibits the biosynthesis and transport of the protein at PM, and also presents gating and stability defects of the membrane anion channel upon its rescue by the use of correctors and potentiators. This prompted a multiple drug strategy for F508delCFTR aimed simultaneously at its rescue, functional potentiation and PM stabilization. Since ganglioside GM1 is involved in the functional stabilization of transmembrane proteins, we investigated its role as an adjuvant to increase the effectiveness of CFTR modulators. According to our results, we found that GM1 resides in the same PM microenvironment as CFTR. In CF cells, the expression of the mutated channel is accompanied by a decrease in the PM GM1 content. Interestingly, by the exogenous administration of GM1, it becomes a component of the PM, reducing the destabilizing effect of the potentiator VX-770 on rescued CFTR protein expression/function and improving its stabilization. This evidence could represent a starting point for developing innovative therapeutic strategies based on the co-administration of GM1, correctors and potentiators, with the aim of improving F508del CFTR function.

Human primary bronchial epithelial cells differentiated in vitro represent a valuable tool to study lung diseases such as cystic fibrosis (CF), an inherited disorder caused by mutations in the gene coding for the Cystic Fibrosis Transmembrane Conductance Regulator. In CF, sphingolipids, a ubiquitous class of bioactive lipids mainly associated with the outer layer of the plasma membrane, seem to play a crucial role in the establishment of the severe lung complications. Nevertheless, no information on the involvement of sphingolipids and their metabolism in the differentiation of primary bronchial epithelial cells are available so far. Here we show that ceramide and globotriaosylceramide increased during cell differentiation, whereas glucosylceramide and gangliosides content decreased. In addition, we found that apical plasma membrane of differentiated bronchial cells is characterized by a higher content of sphingolipids in comparison to the other cell membranes and that activity of sphingolipids catabolic enzymes associated with this membrane results altered with respect to the total cell activities. In particular, the apical membrane of CF cells was characterized by high levels of ceramide and glucosylceramide, known to have proinflammatory activity. On this basis, our data further support the role of sphingolipids in the onset of CF lung pathology. Electronic supplementary material The online version of this article (10.1007/s10719-020-09935-x) contains supplementary material, which is available to authorized users.

We show that glioblastoma multiform (GBM) cells overexpressing the constitutively active form of the epidermal growth factor receptor [epidermal growth factor receptor variant III (EGFRvIII) and U87MG human GBM cell line overexpressing EGFRvIII (EGFR+) cells] possess greater invasive properties and have higher levels of extracellular sphingosine-1-phosphate (S1P) and increased sphingosine kinase-1 (SK1) activity than the empty vector-expressing cells. Notably, the inhibition of SK1 or S1P receptors decreases the invasiveness of EGFR+ cells. Moreover, EGFR and MEK1 inhibitors reduce both SK1 activation and cell invasion, suggesting that the enhanced invasiveness observed in the EGFR+ cells depends on the increased S1P secretion, downstream of the EGFRvIII-ERK-SK1-S1P pathway. Altogether, the results of the present study indicate that, in GBM cells, EGFRvIII is connected with the S1P signaling pathway to enhance cell invasiveness and tumor progression.

Prostate cancer (PC) is one of the most common leading causes of cancer-related death in men. Currently, the main therapeutic approaches available for PC are based on the androgen deprivation and on radiotherapy. However, despite these treatments being initially effective in cancer remission, several patients undergo recurrence, developing a most aggressive and resistant PC.Emerging evidence showed that abiraterone acetate drug will reduce PC recurrence by a mechanism independent of the inhibition of Cytochrome P450 17α-hydroxylase/17,20-lyase. Here we describe the involvement in the abiraterone-mediated PC cell death of a particular class of bioactive lipids called sphingolipids (SL). Sphingolipids are components of plasma membrane (PM) that organize macromolecular complexes involved in the control of several signaling pathways including the tumor cell death induced by radiotherapy. Here, we show for the first time that both in androgen-sensitive and insensitive PC cells abiraterone and ionizing radiation induce a reorganization of the plasma membrane SL composition. This event is triggered by activation of the PM-associated glycohydrolases that induce the production of cytotoxic ceramide by the in situ hydrolyses of glycosphingolipids. Taken together our data open a new scenario on the SL involvement in the therapy of PC.

In the frame of a research program aimed to explore the relationship between chirality of iminosugars and their therapeutic potential, herein we report the synthesis of N-akyl l -deoxyiminosugars and the evaluation of the anti-inflammatory properties of selected candidates for the treatment of Pseudomonas aeruginosa infections in Cystic Fibrosis (CF) lung disease. Target glycomimetics were prepared by the shortest and most convenient approach reported to date, relying on the use of the well-known PS-TPP/I2 reagent system to prepare reactive alkoxyalkyl iodides, acting as key intermediates. Iminosugars ent-1-3 demonstrated to efficiently reduce the inflammatory response induced by P. aeruginosa in CuFi cells, either alone or in synergistic combination with their d -enantiomers, by selectively inhibiting NLGase. Surprisingly, the evaluation in murine models of lung disease showed that the amount of ent -1 required to reduce the recruitment of neutrophils was 40-fold lower than that of the corresponding d -enantiomer. The remarkably low dosage of the l -iminosugar, combined with its inability to act as inhibitor for most glycosidases, is expected to limit the onset of undesired effects, which are typically associated with the administration of its d -counterpart. Biological results herein obtained place ent -1 and congeners among the earliest examples of l -iminosugars acting as anti-inflammatory agents for therapeutic applications in Cystic Fibrosis.

Developing a vaccine againstPlasmodium falciparummalaria has been challenging, primarily due to high levels of antigen polymorphism and a complex parasite lifecycle. Immunization with theP.falciparummerozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 has been shown to give rise to growth inhibitory and synergistic antisera. Therefore, these five merozoite proteins are considered to be promising candidates for a second-generation multivalent malaria vaccine. Nevertheless, little is known about IgG and IgM responses to these antigens in populations that are naturally exposed toP.falciparum. In this study, serum samples from clinically immune adults and malaria exposed children from Ghana were studied to compare levels of IgG and IgM specific for PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5. All five antigens were found to be specifically recognized by both IgM and IgG in serum from clinically immune adults and from children with malaria. Longitudinal analysis of the latter group showed an early, transient IgM response that was followed by IgG, which peaked 14 days after the initial diagnosis. IgG levels and parasitemia did not correlate, whereas parasitemia was weakly positively correlated with IgM levels. These findings show that IgG and IgM specific for merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 are high in children duringP.falciparummalaria, but that the IgM induction and decline occurs earlier in infection than that of IgG.

The base excision repair DNA N-glycosylase MBD4 (also known as MED1), an interactor of the DNA mismatch repair protein MLH1, plays a central role in the maintenance of genomic stability of CpG sites by removing thymine and uracil from G:T and G:U mismatches, respectively. MBD4 is also involved in DNA damage response and transcriptional regulation. The interaction with other proteins is likely critical for understanding MBD4 functions. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (˜66 kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitro sumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Nitroso-N-methylurea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.

Lysosomal accumulation of undegraded materials is a common feature of lysosomal storage diseases, neurodegenerative disorders, and the aging process. To better understand the role of lysosomal storage in the onset of cell damage, we used human fibroblasts loaded with sucrose as a model of lysosomal accumulation. Sucrose-loaded fibroblasts displayed increased lysosomal biogenesis followed by arrested cell proliferation. Notably, we found that reduced lysosomal catabolism and autophagy impairment led to an increase in sphingolipids ( i.e., sphingomyelin, glucosylceramide, ceramide, and the gangliosides GM3 and GD3), at both intracellular and plasma membrane (PM) levels. In addition, we observed an increase in the lysosomal membrane protein Lamp-1 on the PM of sucrose-loaded fibroblasts and a greater release of the soluble lysosomal protein cathepsin D in their extracellular medium compared with controls. These results indicate increased fusion between lysosomes and the PM, as also suggested by the increased activity of lysosomal glycosphingolipid hydrolases on the PM of sucrose-loaded fibroblasts. The inhibition of β-glucocerebrosidase and nonlysosomal glucosylceramidase, both involved in ceramide production resulting from glycosphingolipid catabolism on the PM, partially restored cell proliferation. Our findings indicate the existence of a new molecular mechanism underlying cell damage triggered by lysosomal impairment.-Samarani, M., Loberto, N., Solda, G., Straniero, L., Asselta, R., Duga, S., Lunghi, G., Zucca, F. A., Mauri, L., Ciampa, M. G., Schiumarini, D., Bassi, R., Giussani, P., Chiricozzi, E., Prinetti, A., Aureli, M., Sonnino, S. A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.

Sphingolipids (SLs) are amphiphilic molecules mainly associated with the external leaflet of eukaryotic plasma membrane, and are structural membrane components with key signaling properties. Since the beginning of the last century, a large number of papers described the involvement of these molecules in several aspects of cell physiology and pathology. Several lines of evidence support the critical role of SLs in inflammatory diseases, by acting as anti- or pro-inflammatory mediators. They are involved in control of leukocyte activation and migration, and are recognized as essential players in host response to pathogenic infection. We propose here a critical overview of current knowledge on involvement of different classes of SLs in inflammation, focusing on the role of simple and complex SLs in pathogen-mediated inflammatory response.

The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p amp;lt; 0.01) Gb3 ganglioside (-50 +/- 3%) and sphingosine 1-phosphate (S1P, -40 +/- 4%), which ended up to reduction in cell motility (-46 +/- 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility. Funding Agencies|Lions Club Milano ai Cenacoli

Cystic fibrosis (CF) is the most common autosomal genetic recessive disease caused by mutations of gene encoding for the cystic fibrosis transmembrane conductance regulator. Patients with CF display a wide spectrum of symptoms, the most severe being chronic lung infection and inflammation, which lead to onset of cystic fibrosis lung disease. Several studies indicate that sphingolipids play a regulatory role in airway inflammation. The inhibition and downregulation of GBA2, the enzyme catabolizing glucosylceramide to ceramide, are associated with a significant reduction of IL-8 production in CF bronchial epithelial cells. Herein, we demonstrate that GBA2 plays a role in the proinflammatory state characterizing CF cells. We also report for the first time that Pseudomonas aeruginosa infection causes a recruitment of plasma membrane-associated glycosphingolipid hydrolases into lipid rafts of CuFi-1-infected cells. This reorganization of cell membrane may be responsible for activation of a signaling cascade, culminating in aberrant inflammatory response in CF bronchial epithelial cells upon bacterial infection. Taken together, the presented data further support the role of sphingolipids and their metabolic enzymes in controlling the inflammatory response in CF.

Outbreaks of Yellow Fever occur regularly in endemic areas of Africa and South America frequently leading to mass vaccination campaigns straining the availability of the attenuated Yellow Fever vaccine, YF-17D. The WHO has recently decided to discontinue regular booster-vaccinations since a single vaccination is deemed to confer life-long immune protection. Here, we have examined humoral (neutralizing antibody) and cellular (CD8 and CD4 T cell) immune responses in primary and booster vaccinees (the latter spanning 8 to 36 years after primary vaccination). After primary vaccination, we observed strong cellular immune responses with T cell activation peaking ≈2 weeks and subsiding to background levels ≈ 4 weeks post-vaccination. The number of antigen-specific CD8+ T cells declined over the following years. In >90% of vaccinees, in vitro expandable T cells could still be detected >10 years post-vaccination. Although most vaccinees responded to a booster vaccination, both the humoral and cellular immune responses observed following booster vaccination were strikingly reduced compared to primary responses. This suggests that pre-existing immunity efficiently controls booster inoculums of YF-17D. In a situation with epidemic outbreaks, one could argue that a more efficient use of a limited supply of the vaccine would be to focus on primary vaccinations.

The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested., Author Summary Live attenuated yellow fever vaccine (YF-17D) is an efficient and generally safe vaccine. Nevertheless, in recent years the reporting of serious adverse effects together with the given limitations in the use of this live vaccine in certain risk groups has spurred an interest in developing a more generally applicable and safer alternative. Using an adenovector platform and recombinant vaccines targeting both structural and non-structural YF antigens, we now demonstrate that non-replicating adenobased vaccines may be used to induce a state of host immunity, which like YF-17D vaccination encompasses both major arms of the adaptive immune system. Furthermore, in a murine challenge model, adenovector induced protection fully matched that induced by the current vaccine. Taken together our results strongly suggest that adenovectored vaccines targeting structural and non-structural viral antigens represent a viable and safe alternative to the existing live, attenuated YF vaccine.

We detected significant levels of β-glucosidase, β-galactosidase, sialidase Neu3 and sphingomyelinase activities associated with the plasma membrane of fibroblasts from normal and Niemann-Pick subjects and of cells from breast, ovary, colon and neuroblastoma tumors in culture. All of the cells subjected to ionizing radiations showed an increase of the activity of plasma membrane β-glucosidase, β-galactosidase and sialidase Neu3, in addition of the well known increase of activity of plasma membrane sphingomyelinase, under similar conditions. Human breast cancer cell line T47D was studied in detail. In these cells the increase of activity of β-glucosidase and β-galactosidase was parallel to the increase of irradiation dose up to 60 Gy and continued with time, at least up to 72 h from irradiation. β-glucosidase increased up to 17 times and β-galactosidase up to 40 times with respect to control. Sialidase Neu3 and sphingomyelinase increased about 2 times at a dose of 20 Gy but no further significant differences were observed with increase of radiation dose and time. After irradiation, we observed a reduction of cell proliferation, an increase of apoptotic cell death and an increase of plasma membrane ceramide up to 3 times, with respect to control cells. Tritiated GM3 ganglioside has been administered to T47D cells under conditions that prevented the lysosomal catabolism. GM3 became component of the plasma membranes and was transformed into LacCer, GlcCer and ceramide. The quantity of ceramide produced in irradiated cells was about two times that of control cells.

Glioblastoma is a deadly cancer with intrinsic chemoresistance. Understanding this property will aid in therapy. Glucosylceramide synthase (GCS) is associated with resistance and poor outcome; little is known about glioblastomas. In glioblastoma cells, temozolomide and paclitaxel induce ceramide increase, which in turn promotes cytotoxicity. In drug-resistant cells, both drugs are unable to accumulate ceramide, increased expression and activity of GCS is present, and its inhibitors hinder resistance. Resistant cells exhibit cross-resistance, despite differing in marker expression, and cytotoxic mechanism. These findings suggest that GCS protects glioblastoma cells against autophagic and apoptotic death, and contributes to cell survival under chemotherapy.

The plasma membrane-associated sialidase NEU3 plays crucial roles in regulation of transmembrane signaling, and its aberrant up-regulation in various cancers contributes to malignancy. However, it remains uncertain how NEU3 is naturally activated and locates to plasma membranes, because of its Triton X-100 requirement for the sialidase activity in vitro and its often changing subcellular location. Among phospholipids examined, we demonstrate that phosphatidic acid (PA) elevates its sialidase activity 4 to 5 times at 50 μM in vitro at neutral pH and promotes translocation to the cell surface and cell migration through Ras-signaling in HeLa and COS-1 cells. NEU3 was found to interact selectively with PA as assessed by phospholipid array, liposome coprecipitation, and ELISA assays and to colocalize with phospholipase D (PLD) 1 in response to epidermal growth factor (EGF) or serum stimulation. Studies using tagged NEU3 fragments with point mutations identified PA- and calmodulin (CaM)-binding sites around the N terminus and confirmed its participation in translocation and catalytic activity. EGF induced PLD1 activation concomitantly with enhanced NEU3 translocation to the cell surface, as assessed by confocal microscopy. These results suggest that interactions of NEU3 with PA produced by PLD1 are important for regulation of transmembrane signaling, this aberrant acceleration probably promoting malignancy in cancers.

Sphingolipidoses are inherited genetic diseases due to mutations in genes encoding proteins involved in the lysosomal catabolism of sphingolipids. Despite a low incidence of each individual disease, altogether, the number of patients involved is relatively high and resolutive approaches for treatment are still lacking. The chaperone therapy is one of the latest pharmacological approaches to these storage diseases. This therapy allows the mutated protein to escape its natural removal and to increase its quantity in lysosomes, thus partially restoring the metabolic functions. Sandhoff disease is an autosomal recessive inherited disorder resulting from β-hexosaminidase deficiency and characterized by large accumulation of GM2 ganglioside in brain. No enzymatic replacement therapy is currently available, and the use of inhibitors of glycosphingolipid biosynthesis for substrate reduction therapy, although very promising, is associated with serious side effects. The chaperone pyrimethamine has been proposed as a very promising drug in those cases characterized by a residual enzyme activity. In this review, we report the effect of pyrimethamine on the recovery of β-hexosaminidase activity in cultured fibroblasts from Sandhoff patients.

In this paper, we show that the pH optimum for the plasma membrane (PM)-associated activity of four glycohydrolases (conduritol B epoxide sensitive β-glucosidase, β-glucosidase GBA2, β-hexosaminidase and β-galactosidase) measured on intact cells is acidic. Moreover, we show that drugs able to modify the efflux of protons across the PM, thus locally affecting the extracellular proton concentration close to the PM, are able to modulate the activities of these enzymes. These data strongly suggest that pH-dependent modulation of PM-associated glycohydrolases activities could be an effective way to locally modulate the cell surface glycoconjugate composition.

The glucose-dependent insulinotropic polypeptide receptor (GIPr) has been implicated in high fat diet-induced obesity and is proposed as an anti-obesity target despite an uncertainty regarding the mechanism of action. To independently investigate the contribution of the insulinotropic effects and the direct effects on adipose tissue, we generated transgenic mice with targeted expression of the human GIPr to white adipose tissue or beta-cells, respectively. These mice were then cross-bred with the GIPr knock-out strain. The central findings of the study are that mice with GIPr expression targeted to adipose tissue have a similar high fat diet -induced body weight gain as control mice, significantly greater than the weight gain in mice with a general ablation of the receptor. Surprisingly, this difference was due to an increase in total lean body mass rather than a gain in total fat mass that was similar between the groups. In contrast, glucose-dependent insulinotropic polypeptide-mediated insulin secretion does not seem to be important for regulation of body weight after high fat feeding. The study supports a role of the adipocyte GIPr in nutrient-dependent regulation of body weight and lean mass, but it does not support a direct and independent role for the adipocyte or beta-cell GIPr in promoting adipogenesis.

Gaucher disease (GD) is the most common lysosomal disorder and is caused by an inherited autosomal recessive deficiency in beta-glucocerebrosidase. This enzyme, like other glycohydrolases involved in glycosphingolipid (GSL) metabolism, is present in both plasma membrane (PM) and intracellular fractions. We analyzed the activities of CBE-sensitive beta-glucosidase (GBA1) and AMP-DNM-sensitive beta-glucosidase (GBA2) in total cell lysates and PM of human fibroblast cell lines from control (normal) subjects and from patients with GD clinical types 1, 2, and 3. GBA1 activities in both total lysate and PM of GD fibroblasts were low, and their relative percentages were similar to those of control cells. In contrast, GBA2 activities were higher in GD cells than in control cells, and the degree of increase differed among the three GD types. The increase of GBA2 enzyme activity was correlated with increased expression of GBA2 protein as evaluated by QRT-PCR. Activities of beta-galactosidase and beta-hexosaminidase in PM were significantly higher for GD cells than for control cells and also showed significant differences among the three GD types, suggesting the occurrence of cross-talk among the enzymes involved in GSL metabolism. Our findings indicate that the profiles of glycohydrolase activities in PM may provide a valuable tool to refine the classification of GD into distinct clinical types

As the majority of gliomas arise through malignant transformation of astrocytes, we aimed at investigating the interaction between malignant glioma cells and astrocytes in a co-culture experimental model. For this purpose we analyzed the expression of genes and proteins involved in tumor promotion and invasion, such as glial fibrillary acidic protein (GFAP), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of MMP-2 (TIMP-2), transforming growth factor-beta1 (TGF-beta1), secreted protein acidic and rich in cysteine (SPARC), and connexin 43 (CX43). Co-cultures of human neural stem cell-derived astrocytes and U87 MG astrocytoma cells were performed in a transwell system. Gene expression was evaluated by real-time RT-PCR, and protein analysis was performed by Western blotting, SDS-zymography, and immunofluorescence. GFAP tended to be up-regulated in astrocytes co-cultivated with U87, suggesting a reactive response induced by glioma cells. CX43 mRNA tended to be down- regulated in co-cultured astrocytes, as well as the non-phosphorylated isoform at the protein level. MMP-2 mRNA tended to be up-regulated, and MMP-2 protein levels were significantly increased in astrocytes co-cultivated with U87. TIMP-2 and SPARC mRNA decreased in astrocytes co-cultivated with U87, showing lower expression in glioma cells. By contrast, SPARC protein expression was strongly induced in supernatants of co-cultured astrocytes. TGF-beta1 was not modified. Our results suggest that U87 cells elicit phenotype modifications in the neighbouring resident astrocytes very likely mediated by soluble factors. Glioma/astrocyte interaction could possibly trigger an astrocyte phenotype modification consistent with a malignant transformation, and favouring a more permissive environment for glioma cells invasion.

HMGB1 (high mobility group box 1 protein) is a nuclear protein that can also act as an extracellular trigger of inflammation, proliferation and migration, mainly through RAGE (the receptor for advanced glycation end products); HMGB1-RAGE interactions have been found to be important in a number of cancers. We investigated whether HMGB1 is an autocrine factor in human glioma cells. Western blots showed HMGB1 and RAGE expression in human malignant glioma cell lines. HMGB1 induced a dose-dependent increase in cell proliferation, which was found to be RAGE-mediated and involved the MAPK/ERK pathway. Moreover, in a wounding model, it induced a significant increase in cell migration, and RAGE-dependent activation of Rac1 was crucial in giving the tumour cells a motile phenotype. The fact that blocking DNA replication with anti-mitotic agents did not reduce the distance migrated suggests the independence of the proliferative and migratory effects. We also found that glioma cells contain HMGB1 predominantly in the nucleus, and cannot secrete it constitutively or upon stimulation; however, necrotic glioma cells can release HMGB1 after it has translocated from the nucleus to cytosol. These findings provide the first evidence supporting the existence of HMGB1/RAGE signalling pathways in human glioblastoma cells, and suggest that HMGB1 may play an important role in the relationship between necrosis and malignancy in glioma tumours by acting as an autocrine factor that is capable of promoting the growth and migration of tumour cells.

In this review, the focus is on the role of salvage pathways in glycosphingolipid, particularly, ganglioside metabolism. Ganglioside de novo biosynthesis, that begins with the formation of ceramide and continues with the sequential glycosylation steps producing the oligosaccharide moieties, is briefly outlined in its enzymological and cell-topological aspects. Neo-synthesized gangliosides are delivered to the plasma membrane, where their oligosaccharide chains protrude toward the cell exterior. The metabolic fate of gangliosides after internalization via endocytosis is then described, illustrating: (a) the direct recycling of gangliosides to the plasma membrane through vesicles gemmated from sorting endosomes; (b) the sorting through endosomal vesicles to the Golgi apparatus where additional glycosylations may take place; and (c) the channelling to the endosomal/lysosomal system, where complete degradation occurs with formation of the individual sugar (glucose, galactose, hexosamine, sialic acid) and lipid (ceramide, sphingosine, fatty acid) components of gangliosides. The in vivo and in vitro evidence concerning the metabolic recycling of these components is examined in detail. The notion arises that these salvage pathways, leading to the formation of gangliosides and other glycosphingolipids, sphingomyelin, glycoproteins and glycosaminoglycans, represent an important saving of energy in the cell economy and constitute a relevant event in overall ganglioside (or glycosphingolipid, in general) turnover, covering from 50% to 90% of it, depending on the cell line and stage of cell life. Sialic acid is the moiety most actively recycled for metabolic purposes, followed by sphingosine, hexosamine, galactose and fatty acid. Finally, the importance of salvage processes in controlling the active concentrations of ceramide and sphingosine, known to carry peculiar bioregulatory/signalling properties, is discussed.

Ceramide represents an important sphingoid mediator involved in the signaling pathways that control cell proliferation, differentiation, and death. To determine whether ceramide levels correlate with the malignant progression of human astrocytomas, we investigated these levels in surgical specimens of glial tumors of low-grade and high-grade malignancy. Tumor samples obtained from 52 patients who underwent therapeutic removal of primary brain tumors were used. The tumors were classified according to standard morphologic criteria and were grouped into tumors of low-grade and high-grade malignancy. Sections of normal brain tissue adjacent to the tumor were also analyzed in 11 of the 52 patients. After extraction and partial purification, ceramide was measured by quantitative derivatization to ceramide-1-phosphate using diacylglycerol kinase and [γ-32P]ATP. Ceramide levels were significantly lower in the combined high-grade tumors compared with low-grade tumors and in both tumor groups compared with peritumoral tissue. The results indicate an inverse correlation between the amount of ceramide and tumor malignancy as assessed by both the histological grading and ganglioside pattern. Moreover, overall survival analysis of 38 patients indicates that ceramide levels are significantly associated with patient survival. The present findings indicate that ceramide is inversely associated with malignant progression of human astrocytomas and poor prognosis. The downregulation of ceramide levels in human astrocytomas emerges as a novel alteration that may contribute to glial neoplastic transformation. The low ceramide levels in high-grade tumors may provide an advantage for their rapid growth and apoptotic resistant features. This study appears to support the rationale for the potential benefits of a ceramide-based chemotherapy. GLIA 39:105–113, 2002. © 2002 Wiley-Liss, Inc.

The metabolic fate of exogenous [3H]sphingosine was investigated in five types of cultured cells: primary cultures of neurons and astrocytes, murine and human neuroblastoma cells and human skin fibroblasts. After administration of 40 nM [3-3H]sphingosine into a cell-conditioned medium containing fetal calf serum, all cell types rapidly and efficiently incorporated the long-chain base in a time-dependent fashion. In all cases, after a 120 min pulse, the amount of radioactivity taken up was in the range of the endogenous sphingosine content. However, unchanged [3H]sphingosine represented only a very minor portion of the label incorporated into cells throughout the pulse period (10–120 min), indicating rapid and efficient sphingosine metabolism in these cells. Most of the [3H]sphingosine taken up was metabolically processed, either by degradation (assessed as 3H2O release into the culture medium) or by N-acylation (mainly to radioactive ceramide, sphingomyelin, neutral glycolipids and gangliosides). [3H]Sphingosine 1-phosphate accounted for less than 2% of the total radioactivity incorporated in all cases. Throughout the pulse period and in all cell types, 3H-labelled organic metabolites largely prevailed over 3H2O, indicating that N-acylation is the major metabolic fate of sphingosine in these cells under apparently physiological conditions. These results are consistent with the notion that sphingosine has a rapid turnover in the cells studied, and indicate that regulation of the basal level of this bioactive molecule occurs mainly through N-acylation.

The involvement of ceramide in the differentiation of two neuroblastoma cell lines, Neuro2a and SH-SY5Y, and cerebellar granule cells in primary culture was investigated. The following results were obtained: (a) the cellular content of ceramide markedly increased with induced differentiation of Neuro2a cells (inducers: RA, FCS deprivation), SH-SY5Y cells (inducers: RA, PMA), and spontaneous differentiation of cerebellar granule cells; (b) all the investigated cells in the differentiated form displayed a higher ability to produce ceramide from exogenously administered [3H]Sph-SM and expressed a higher content of neutral sphingomyelinase and, in the case of cerebellar granule cells, also of acidic sphingomyelinase; (c) inhibition of ceramide biosynthesis by Fumonisin B1 blocked the process of differentiation in Neuro2a and cerebellar granule cells; and (d) treatments capable of enhancing ceramide level (administration of sphingosine or C2-Ceramide) induced differentiation in both Neuro2a and SH-SY5Y cells. The data obtained support the notion that ceramide plays a general biomodulatory role in neural cell differentiation.

Cerebellar granule cells in culture were subjected to a pulse (0.5–4 h)-chase (0–4 h) of 10 −6 M [ 3 H]ganglioside GM1 carrying the radioactive label at the level of NeuAc ([ 3 H-NeuAc]GM1), Sph ([ 3 H-Sph]GM1) or Gal ([ 3 H-GallGMl) and the formed [ 3 H]metabolites were determined. With all forms of [ 3 H]GM1, there was formation of [ 3 H]catabolites, including [ 3 H]H 2 O and [ 3 H]biosynthetic products obtained by recycling of [ 3 H]NeuAc, [ 3 H]Sph and [ 3 H]Gal released during intralysosomal ganglioside degradation (salvage processes). Much higher amounts of [ 3 MH 2 O were produced from [ 3 H-Gal]GM1 than [ 3 H-Sph]GM1 and [ 3 H-NeuAc]GM1; conversely, more products from salvage processes (polysialogangliosides GD1a, GDlb, GTlb, O-acetylated GT1b, protein-bound radioactivity) were obtained with [ 3 H-NeuAc]GM1; than the two other forms of [ 3 H]GM1, Liberated [ 3 H]NeuAc produced 10-fold less tritiated water and 10-fold higher salvage products than [ 3 H]Gal. Using [ 3 H-NeuAc]GM1; granule cells appeared to metabolize 7.7% of membrane-incorporated exogenous GM1 per hour with a high degree of NeuAc recycling and the calculated metabolic half-life was 6.5 h.

DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular patterns that increase activation of the innate immune system. Importantly, viral-vectored vaccines that act through the induction of one or more of these factors also may benefit from cytokine coadministration and increased antigen presentation. In order to increase immunogenicity to the level achieved with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even with these modifications, it is likely that the primary use of DNA vaccines may be as primers for viral-vectored vaccines, rather than as single agents. This review discusses the approaches used to enhance DNA vaccine immunogenicity, with a primary focus on fusion strategies that enhance antigen presentation.