M. Teresa Cruz

Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons

The development of methodologies for gene transfer into the central nervous system is crucial for gene therapy of neurological disorders. In this study, different cationic liposome formulations were used to transfer DNA into C6 glioma cells and primary hippocampal and cortical neurons by varying the nature of the helper lipid (DOPE, Chol) or a mixture of DOPE and cholesterol (Chol) associated to DOTAP. In addition, the effect of the lipid/DNA (+/-) charge ratio, the association of the ligand transferrin to the lipoplexes, and the stage of differentiation of the primary cells on the levels of transfection activity, transfection efficiency, and duration of gene expression were evaluated. Mechanistic studies were also performed to investigate the route of delivery of the complexes into neurons. Our results indicate that DOTAP:Chol (1:1 mol ratio) was the best formulation to transfer a reporter gene into C6 glioma cells, primary hippocampal neurons, and primary cortical neurons. The use of transferrin-associated lipoplexes resulted in a significant enhancement of transfection activity, as compared to plain lipoplexes, which can be partially attributed to the promotion of their internalization mediated by transferrin. While for hippocampal neurons the levels of luciferase gene expression are very low, for primary cortical neurons the levels of transgene expression are high and relatively stable, although only 4% of the cells has been transfected. The stage of cell differentiation revealed to be critical to the levels of gene expression. Consistent with previous findings on the mechanisms of cell internalization, the experiments with inhibitors of the endocytotic pathway clearly indicate that transferrin-associated lipoplexes are internalized into primary neurons by endocytosis. Promising results were obtained in terms of the levels and duration of gene expression, particularly in cortical neurons when transfected with the Tf-associated lipoplexes, this finding suggesting the usefulness of these lipid-based carriers to deliver genes within the CNS.

Kinetic analysis of the initial steps involved in lipoplex–cell interactions: effect of various factors that influence transfection activity

We investigated the mode of interaction of lipoplexes (DOTAP:DOPE/DNA) with HeLa cells, focusing on the analysis of the initial steps involved in the process of gene delivery. We evaluated the effect of different factors, namely the stoichiometry of cationic lipids and DNA, the presence of serum in the cell culture medium, and the incorporation of the ligand transferrin into the lipoplexes, on the extent of binding, association and fusion (lipid mixing) of the lipoplexes with the cells. Parallel experiments were performed upon cell treatment with inhibitors of endocytosis. Our results indicate that a decrease of the net charge of the complexes (upon addition of DNA) generally leads to a decrease in the extent of binding, cell association and fusion, except for the neutral complexes. Association of transferrin to the lipoplexes resulted in a significant enhancement of the interaction processes referred to above, which correlates well with the promotion of transfection observed under the same conditions. Besides triggering internalization of the complexes, transferrin was also shown to mediate fusion with the endosomal membrane. The extent of fusion of this type of complexes was reduced upon their incubation with cells in the presence of serum, suggesting that serum components limit the transferrin fusogenic properties. Results were analyzed by using a theoretical model which allowed to estimate the kinetic parameters involved in lipoplex--cell interactions. The deduced fusion and endocytosis rate constants are discussed and compared with those obtained for other biological systems. From the kinetic studies we found a twofold enhancement of the fusion rate constant (f) for the ternary lipoplexes. We also concluded that HeLa cells yield a relatively low rate of endocytosis. Overall, our results estimate the relative contribution of fusion of lipoplexes with the plasma membrane, endocytosis and fusion with the endosomal membrane to their interactions with cells, this information being of crucial importance for the development of gene therapy strategies.

Tf-lipoplex-mediated NGF gene transfer to the CNS: neuronal protection and recovery in an excitotoxic model of brain injury

The development of efficient systems for in vivo gene transfer to the central nervous system (CNS) may provide a useful therapeutic strategy for the alleviation of several neurological disorders. In this study, we evaluated the feasibility of nonviral gene therapy to the CNS mediated by cationic liposomes. We present evidence of the successful delivery and expression of both a reporter and a therapeutic gene in the rodent brain, as evaluated by immunohistochemical assays. Our results indicate that transferrin-associated cationic liposome/DNA complexes (Tf-lipoplexes) allow a significant enhancement of transfection activity as compared to plain complexes, and that 8/1 (+/−) Tf-lipoplexes constitute the best formulation to mediate in vivo gene transfer. We demonstrated that Tf-lipoplex-mediated nerve growth factor transgene expression attenuates the morphological damages of the kainic acid-induced lesion as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) vital staining. These findings suggest the usefulness of these lipid-based vectors in mediating the delivery of therapeutic genes to the CNS.

Granulocyte-macrophage colony-stimulating factor activates the transcription of nuclear factor kappa B and induces the expression of nitric oxide synthase in a skin dendritic cell line

Nitric oxide (NO) produced by skin dendritic cells and keratinocytes plays an important role in skin physiology, growth and remodelling. Nitric oxide is also involved in skin inflammatory processes and in modulating antigen presentation (either enhancing or suppressing it). In this study, we found that GM‐CSF stimulates the expression of the inducible isoform of nitric oxide synthase (iNOS) in a fetal‐skin‐derived dendritic cell line (FSDC) and, consequently, increases the nitrite production from 11.9 ± 3.2 μmol/L (basal level) to 26.9 ± 4.2 μmol/L. Pyrrolidinedithiocarbamate (PDTC) inhibits nitrite production, with a half maximal inhibitory concentration (IC50) of 19.3 μmol/L and the iNOS protein expression in FSDC. In addition, western blot assays revealed that exposure of FSDC to GM‐CSF induces the phosphorylation and degradation of the inhibitor of NF‐κB (IkB), with subsequent translocation of the p50, p52 and RelB subunits of the transcription nuclear factor kappa B (NF‐κB) from the cytosol to the nucleus. Electrophoretic mobility shift assays (EMSA) showed that FSDC exposure to GM‐CSF activates the transcription factor NF‐κB. Together, these results show that GM‐CSF induces iNOS expression in skin dendritic cells by a mechanism involving activation of the NF‐κB pathway.

Contact sensitizer nickel sulfate activates the transcription factors NF-kB and AP-1 and increases the expression of nitric oxide synthase in a skin dendritic cell line

Abstract:  Nuclear factor kappa B (NF‐kB) and activating protein‐1 (AP‐1) transcription factors are ubiquitously expressed signaling molecules known to regulate the transcription of a large number of genes involved in immune responses, namely the inducible isoform of nitric oxide synthase (iNOS). In this study, we demonstrate that a fetal skin‐derived dendritic cell line (FSDC) produces nitric oxide (NO) in response to the contact sensitizer nickel sulfate (NiSO4) and increases the expression of the iNOS protein, as determined by immunofluorescence and Western blot analysis. The sensitizer NiSO4 increased cytoplasmic iNOS expression by 31.9 ± 10.3% and nitrite production, as assayed by the Griess reaction, by 27.6 ± 9.5%. Electrophoretic mobility shift assay (EMSA), showed that 30 min of FSDC exposure to NiSO4 activates the transcription factor NF‐kB by 58.2 ± 7.0% and 2 h of FSDC exposure to NiSO4 activates the transcription factor AP‐1 by 26.0 ± 1.4%. Together, these results indicate that NiSO4 activates the NF‐kB and AP‐1 pathways and induces iNOS expression in skin dendritic cells.

Dexamethasone prevents granulocyte-macrophage colony-stimulating factor-induced nuclear factor-kappaB activation, inducible nitric oxide synthase expression and nitric oxide production in a skin dendritic cell line.

AIMS: Nitric oxide (NO) has been increasingly implicated in inflammatory skin diseases, namely in allergic contact dermatitis. In this work, we investigated the effect of dexamethasone on NO production induced by the epidermal cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) in a mouse fetal skin dendritic cell line. METHODS: NO production was assessed by the method of Griess. Expression of the inducible isoform of nitric oxide synthase (iNOS) protein was evaluated by western blot analysis and immunofluorescence microscopy. Western blot analysis was also performed to evaluate cytosolic IkappaB-alpha (IkappaB-alpha) protein levels. The electrophoretic mobility shift assay was used to evaluate the activation or inhibition of nuclear factor kappa B (NF-kappaB). RESULTS: GM-CSF induced iNOS expression and NO production, and activated the transcription factor NF-kappaB. Dexamethasone inhibited, in a dose-dependent manner, NO production induced by GM-CSF. Addition of dexamethasone to the culture, 30 min before GM-CSF stimulation, significantly inhibited the cellular expression of iNOS. Dexamethasone also inhibited GM-CSF-induced NF-kappaB activation by preventing a significant decrease on the IkappaB-alpha protein levels, thus blocking NF-kappaB migration to the nucleus. CONCLUSIONS: The corticosteroid dexamethasone inhibits GM-CSF-induced NF-kappaB activation, iNOS protein expression and NO production. These results suggest that dexamethasone is a potent inhibitor of intracellular events that are involved on NO synthesis, in skin dendritic cells.

Prospective phospholipid markers for skin sensitization prediction in keratinocytes: a phospholipidomic approach.

Prevalence of skin inflammatory disorders has increased in recent years being estimated that 15-20% of the general population suffers from allergic contact dermatitis (ACD). Currently, the sensitizing potential of chemicals is assessed through animal tests; however growing ethical concerns and actual legislative framework impose the development of new alternative tests. Several genomic and proteomic approaches have already indicated some potential biomarkers, but lipidomic analysis was not so far explored with this purpose. A growing body of data suggests that phospholipids (PLs) play important roles in the modulation of immune responses. Therefore, this work focused in identifying changes in the PLs profile of human keratinocytes (KCs). For that, HaCaT cell line was exposed to two immune stimulators: the strong skin allergen 2,4-dinitrofluorobenzene (DNFB) and the non-allergenic stimulus LPS, and to the irritant benzalkonium chloride (BC), using off line TLC-ESI-MS, HPLC-MS and MS/MS. LPS and DNFB reduced PS class relative content, corroborating with consistent changes observed in its molecular profile. PC profile was also altered by immune stimulators. These findings suggest that PC and PS molecular species may discriminate immunogenic compounds from irritants. Analysis of such alterations may be therefore valuable in a future in vitro test platform for skin sensitization prediction.

Profiling changes triggered during maturation of dendritic cells: a lipidomic approach.

Lipids are important in several biological processes because they act as signalling and regulating molecules, or, locally, as membrane components that modulate protein function. This paper reports the pattern of lipid composition of dendritic cells (DCs), a cell type of critical importance in inflammatory and immune responses. After activation by antigens, DCs undergo drastic phenotypical and functional transformations, in a process known as maturation. To better characterize this process, changes of lipid profile were evaluated by use of a lipidomic approach. As an experimental model of DCs, we used a foetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). The results showed that LPS treatment increased ceramide (Cer) and phosphatidylcholine (PC) levels and reduced sphingomyelin (SM) and phosphatidylinositol (PI) content. Mass spectrometric analysis of a total lipid extract and of each class of lipids revealed that maturation promoted clear changes in ceramide profile. Quantitative analysis enabled identification of an increase in the total ceramide content and enhanced Cer at m/z 646.6, identified as Cer(d18:1/24:1), and at m/z 648.6, identified as Cer(d18:1/24:0). The pattern of change of these lipids give an extremely rich source of data for evaluating modulation of specific lipid species triggered during DC maturation.

Gene Delivery by Cationic Liposome–DNA Complexes Containing Transferrin or Serum Albumin

Publisher Summary This chapter discusses various methods and techniques used to prepare protein-associated lipoplexes and to evaluate their biological activity and mechanisms of interaction with cells. These approaches may constitute important tools for the development and application of protein-associated or peptide-associated lipoplexes, especially those designed for targeting purposes. The development of new approaches to improve the features of lipoplexes aiming at generating viable alternatives to viral vectors is crucial for gene therapy applications. To achieve such a goal, attempts have been made to confer viral attributes to lipoplexes—namely, through the association of certain proteins or peptides. These strategies are designed taking into consideration the various biological barriers faced by the complexes on their in vivo administration, which need to be overcome for gene expression to occur. Some important considerations are whether these improvements result in a system that, although exhibiting a satisfactory ability to mediate transfection, lead to such a complexity that could endanger its versatility and large-scale production or could limit extended/repeated in vivo use as a result of immunogenicity.

Detection of phosphatidylserine with a modified polar head group in human keratinocytes exposed to the radical generator AAPH.

Phosphatidylserine (PS) is preferentially located in the inner leaflet of the cell membrane, and translocation of PS oxidized in fatty acyl chains to the outside of membrane has been reported as signaling to macrophage receptors to clear apoptotic cells. It was recently shown that PS can be oxidized in serine moiety of polar head-group. In the present work, a targeted lipidomic approach was applied to detecting OxPS modified at the polar head-group in keratinocytes that were exposed to the radical generator AAPH. Glycerophosphoacetic acid derivatives (GPAA) were found to be the major oxidation products of OxPS modified at the polar head-group during oxidation induced by AAPH-generated radicals, similarly to previous observations for the oxidation induced by OH radical. The neutral loss scan of 58Da and a novel precursor ion scan of m/z 137.1 (HOPO3CH2COOH) allowed the recognition of GPAA derivatives in the total lipid extracts obtained from HaCaT cells treated with AAPH. The positive identification of serine head group oxidation products in cells under controlled oxidative conditions opens new perspectives and justifies further studies in other cellular environments in order to understand fully the role of PS polar head-group oxidation in cell homeostasis and disease.