Enforcing dendritic cell vaccines by manipulating the MHC II antigen presentation pathway

Les vaccins à base de cellules dendritiques (DCs) constituent une avenue très populaire en immunothérapie du cancer. Alors que ces cellules peuvent présenter des peptides exogènes ajoutés au milieu, l’efficacité de chargement de ces peptides au le complexe majeur d'histocompatibilité (CMH) de classe II est limitée. En effet, la majorité des molécules du CMH II à la surface des DCs sont très stable et l’échange de peptide spontané est minime. Confinée aux vésicules endosomales, HLA-DM (DM) retire les peptides des molécules du CMH II en plus de leur accorder une conformation réceptive au chargement de peptides. Il est possible, cependant, de muter le signal de rétention de DM de façon à ce que la protéine s’accumule en surface. Nous avons émis l’hypothèse que ce mutant de DM (DMY) sera aussi fonctionnel à la surface que dans la voie endosomale et qu’il favorisera le chargement de peptides exogènes aux DCs. Nous avons utilisé un vecteur adénoviral pour exprimer DMY dans des DCs et avons montrer que la molécule augmente le chargement de peptides. L’augmentation du chargement peptidique par DMY est autant qualitatif que quantitatif. DMY améliore la réponse T auxiliaire (Th) du coté Th1, ce qui favorise l’immunité anti-cancer. Du côté qualitatif, le chargement de peptides résulte en des complexes peptide-CMHII (pCMH) d’une conformation supérieure (conformère). Ce conformère (Type A) est le préféré pour la vaccination et DMY édite avec succès les complexes pCMH à la surface en éliminant ceux de type B, lesquels sont indésirables. La fonction de DM est régulée par HLA-DO (DO). Ce dernier inhibe l’habilité de DM à échanger le peptide CLIP (peptide dérivée de la chaîne invariante) en fonction du pH, donc dans les endosomes tardifs. Mes résultats indiquent que la surexpression de DO influence la présentation des superantigènes (SAgs) dépendants de la nature du peptide. Il est probable que DO améliore indirectement la liaison de ces SAgs au pCMH dû à l’accumulation de complexe CLIP-CMH, d’autant plus qu’il neutralise la polarisation Th2 normalement observée par CLIP. Ensemble, ces résultats indiquent que DMY est un outil intéressant pour renforcer le chargement de peptides exogènes sur les DCs et ainsi générer des vaccins efficaces. Un effet inattendu de DO sur la présentation de certains SAgs a aussi été observé. Davantage de recherche est nécessaire afin de résoudre comment DMY et DO influence la polarisation des lymphocytes T auxiliaires. Cela conduira à une meilleure compréhension de la présentation antigénique et de son étroite collaboration avec le système immunitaire.

Role of EFNBs and EphB4 in T cell development and function

Eph kinases are the largest family of cell surface receptor tyrosine kinases. The ligands of Ephs, ephrins (EFNs), are also cell surface molecules. Ephs interact with EFNs and the receptors and ligands transmit signals in both directions, i.e., from Ephs to EFNs and from EFNs to Ephs. Ephs and EFNs are widely involved in various developmental, physiological pathophysiological processes. Our group and others have reported the roles of Ephs/EFNs in the immune system. To further investigate the function of EphBs/EFNBs in T cell development and responses, we generated EFNB1, EFNB2, EphB4 conditional gene knockout (KO) mice and EFNB1/2 double KO mice. In the projects using EFNB1 and EFNB2 knockout mice, we specifically deleted EFNB1 or EFNB2 in T cells. The mice had normal size and cellularity of the thymus and spleen as well as normal T cell subpopulations in these organs. The bone marrow progenitors from KO mice and WT mice repopulated the host lymphoid organs to similar extents. The activation and proliferation of KO T cells was comparable to that of control mice. Naïve KO CD4 cells differentiated into Th1, Th2, Th17 and Treg cells similar to naïve control CD4 cells. In EFNB2 KO mice, we observed a significant relative increase of CD4CD8 double negative thymocytes in the thymus. Flowcytometry analysis revealed that there was a moderate increase in the DN3 subpopulation in the thymus. This suggests that EFNB2 is involved in thymocyte development. Our results indicate that the functions of EFNB1 and EFNB2 in the T cell compartment could be compensated by each other or by other members of the EFN family, and that such redundancy safeguards the pivotal roles of EFNB1 and EFNB2 in T cell development and function. In the project using EFNB1/B2 double knockout (dKO) model, we revealed a novel regulatory function of EFNb1 and EFNb2 in stabilizing IL-7Rα expression on the T cell surface. IL-7 plays important roles in thymocyte development, T cell homeostasis and survival. IL-7Rα undergoes internalization upon IL-7 binding. In the dKO mice, we observed reduced IL-7Rα expression in thymocytes and T cells. Moreover, the IL-7Rα internalization was accelerated in dKO CD4 cells upon IL-7 stimulation. In T cell lymphoma cell line, EL4, over-expression of either EFNB1 or EFNB2 retarded the internalization of IL-7Rα. We further demonstrated compromised IL-7 signaling and homeostatic proliferation of dKO T cells. Mechanism study using fluorescence resonance energy transfer and immunoprecipitation demonstrated that physical interaction of EFNB1 and EFNB2 with IL-7Rα was likely responsible for the retarded IL-7Rα internalization. In the last project, using medullary thymic epithelial cell (mTEC)-specific EphB4 knockout mice, we investigated T cell development and function after EphB4 deletion in mTEC. EphB4 KO mice demonstrated normal thymic weight and cellularity. T cell development and function were not influenced by the EphB4 deletion. Lastly, the KO mice developed normal delayed type hypersensitivity. Overall, our results suggest that comprehensive cross interaction between Eph and EFN family members could compensate function of a given deleted member in the T cell development, and only simultaneous deletion of multiple EFNBs will reveal their true function in the immune system. In fact, such redundancy signifies vital roles of Ephs and EFNs in the immune system.

Amino acids substitutions in σ1 and μ1 outer capsid proteins of a Vero cell-adapted mammalian orthoreovirus are required for optimal virus binding and disassembly

In a recent study, the serotype 3 Dearing strain of mammalian orthoreovirus was adapted to Vero cells; cells that exhibit a limited ability to support the early steps of reovirus uncoating and are unable to produce interferon as an antiviral response upon infection. The Vero cell-adapted virus (VeroAV) exhibits amino acids substitutions in both the σ1 and μ1 outer capsid proteins but no changes in the σ3 protein. Accordingly, the virus was shown not to behave as a classical uncoating mutant. In the present study, an increased ability of the virus to bind at the Vero cell surface was observed and is likely associated with an increased ability to bind onto cell-surface sialic acid residues. In addition, the kinetics of μ1 disassembly from the virions appears to be altered. The plasmid-based reverse genetics approach confirmed the importance of σ1 amino acids substitutions in VeroAV's ability to efficiently infect Vero cells, although μ1 co-adaptation appears necessary to optimize viral infection. This approach of combining in vitro selection of reoviruses with reverse genetics to identify pertinent amino acids substitutions appears promising in the context of eventual reovirus modification to increase its potential as an oncolytic virus.

Cell entry by enveloped viruses: redox considerations for HIV and SARS-coronavirus

For enveloped viruses, genome entry into the target cell involves two major steps: virion binding to the cell-surface receptor and fusion of the virion and cell membranes. Virus-cell membrane fusion is mediated by the virus envelope complex, and its fusogenicity is the result of an active virus-cell interaction process that induces conformation changes within the envelope. For some viruses, such as influenza, exposure to an acidic milieu within the cell during the early steps of infection triggers the necessary structural changes. However, for other pathogens which are not exposed to such environmental stress, activation of fusogenicity can result from precise thiol/disulfide rearrangements mediated by either an endogenous redox autocatalytic isomerase or a cell-associated oxidoreductase. Study of the activation of HIV envelope fusogenicity has revealed new knowledge about how redox changes within a viral envelope trigger fusion. We discuss these findings and their implication for anti-HIV therapy. In addition, to compare and contrast the situation outlined for HIV with an enveloped virus that can fuse with the cell plasma membrane independent of the redox status of its envelope protein, we review parallel data obtained on SARS coronavirus entry.

Conserved hydrophobic clusters on the surface of the Caf1A usher C-terminal domain are important for F1 antigen assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A(C)). Caf1A(C) is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A(C) is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A(C) were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.

The platelet-surface thiol isomerase enzyme ERp57 modulates platelet function

Background: Thiol isomerases are a family of endoplasmic reticulum enzymes which orchestrate redox-based modifications of protein disulphide bonds. Previous studies have identified important roles for the thiol isomerases PDI and ERp5 in the regulation of normal platelet function. Objectives: Recently, we demonstrated the presence of a further five thiol isomerases at the platelet surface. In this report we aim to report the role of one of these enzymes - ERp57 in the regulation of platelet function. Methods/Results: Using enzyme activity function blocking antibodies, we demonstrate a role for ERp57 in platelet aggregation, dense granule secretion, fibrinogen binding, calcium mobilisation and thrombus formation under arterial conditions. In addition to the effects of ERp57 on isolated platelets, we observe the presence of ERp57 in the developing thrombus in vivo. Furthermore the inhibition of ERp57 function was found to reduce laser-injury induced arterial thrombus formation in a murine model of thrombosis. Conclusions: These data suggest that ERp57 is important for normal platelet function and opens up the possibility that the regulation of platelet function by a range of cell surface thiol isomerases may represent a broad paradigm for the regulation of haemostasis and thrombosis.

Improving promiscuous mammalian cell entry by the baculovirus AcMNPV

The insect baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells we show that entry is favoured by low pH and increasing the available cell surface area through transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268-281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell to cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies suggesting additional factors also govern entry.

Heterogeneous surface expression of EspA translocon filaments by Escherichia coli O157:H7 is controlled at the posttranscriptional level

Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as "effectors" such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both beta-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEEI-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEEI-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.

Platelet endothelial cell adhesion molecule-1 inhibits platelet response to thrombin and von Willebrand factor by regulating the internalization of glycoprotein Ib via AKT/glycogen synthase kinase-3/dynamin and integrin αIIbβ3

OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. APPROACH AND RESULTS: Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbβ3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of β3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3β Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. CONCLUSIONS: PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbβ3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.

Comparison of bioactivities, binding properties and intra-follicular levels of bovine follistatins

Five isoforms of follistatin (FST) (Mr 31, 33, 35, 37, 41kDa) were purified from bovine follicular fluid (bFF). Comparison of their activin- and heparan sulphate proteoglycan (HSP)-binding properties and bio-potencies in neutralization of activin-A action in vitro revealed that all five isoforms bound activin-A, but with different affinities. Only the 31kDa isoform (FST-288) bound to HSP. FST-288 also showed the greatest biopotency with 35 and 41kDa isoforms being least potent. To determine whether bovine follicle development is associated with changing intrafollicular FST and activin profiles, we analyzed bFF from dominant (DF) and subordinate (SF) follicles collected at strategic times during a synchronized estrous cycle. Total FST, activin-A and activin-AB were measured by immunoassay while individual FST isoforms were quantified by immunoblotting. Follicle diameter was positively correlated with estrogen:progesterone ratio (r=0.56) in bFF but negatively correlated with activin-A (r=-0.34), activin-AB (r=-0.80) and ‘total’ FST (r=-0.70) levels. Follicle diameter was positively correlated with abundance of the 41 kDa isoform (r=0.59) but negatively correlated with abundance of 33 and 31 kDa isoforms (r=-0.56, -0.41). Both follicle status (DF vs SF) and cycle stage affected total FST, activin-A, activin-B levels while follicle status, but not cycle stage, affected abundance of 41, 37, 33 and 31kDa FST isoforms. Collectively, these findings indicate that intrafollicular FST isoforms that differ in their ability to bind and neutralise activins and associate with cell-surface proteoglycans, show divergent changes during follicle development. Enhanced FST production may have an important negative role, either directly or via inhibition of the positive effects of activins, on follicle growth and function during follicular waves.

On spermiogenesis, sperm cell morphology and accompanying secretions of Copidognathus (Acari: Halacaridae)

The genus Copidognathus includes one-third of the species of Halacaridae described to date. This article describes spermiogenesis, sperm cell morphology and accompanying secretions from three species of Copidognathus. Initial spermatids have electron-dense cytoplasm with scattered mitochondria, a well-developed Golgi body, and nuclei with patches of heterochromatin. The cytoplasm and nuclei of these cells undergo intense swelling. The second spermatids are large electron-translucent cells, with small mitochondria in row along the remains of the endoplasmatic reticulum. In the succeeding stage, most of the cytoplasmatic structures and mitochondria have disappeared or have undergone profound transformations. Nuclei and cells elongate and chromatin begins to condense near the nuclear envelope. An acrosomal complex appears at the tip of the nucleus. The acrosomal filament is thick and runs the entire length of the nucleus. Plasmalemmal invaginations at the cell surface give rise to tubules filled with an electron-dense material. Sperm cell maturation is completed in the ventral portion of the germinal part, near the testicular lumen. As a final step in spermiogenesis, cytoplasm of the last spermatid undergoes a moderate condensation and the cariotheca disappears. Mature sperm cells were found in a matrix of ""simple"" and ""complex"" corpuscles, the latter consisting of flattened, spindle-shaped secreted bodies. Rather than in individual sperm aggregates, spermatozoa were contained in a single droplet inside the vas deferens, on a large secretion mass, structured as rows of platelets sunk in a fine grained matrix. Each mature sperm cell is covered by a thick secreted coat. In contrast to the genera Rhombognathus and other Actinotrichida, Copidognathus displays a set of features that must be regarded as apomorphic. The absence of usual mitochondria, the presence of electro-dense tubules and secretions similar to those present in Thalassarachna and Halacarellus, and the pattern of nuclear condensation are possibly shared apomorphies with these latter genera. (C) 2010 Elsevier GmbH. All rights reserved.

Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

Interaction of oligonucleotide-based amphiphilic block copolymers with cell membrane models

Oligonucleotides have unique molecular recognition properties, being involved in biological mechanisms such as cell-surface receptor recognition or gene silencing. For their use in human therapy for drug or gene delivery, the cell membrane remains a barrier, but this can be obviated by grafting a hydrophobic tail to the oligonucleotide. Here we demonstrate that two oligonucleotides, one consisting of 12 guanosine units (G(12)), and the other one consisting of five adenosine and seven guanosine (A(5)G(7)) units, when functionalized with poly(butadiene), namely PB-G(12) and PB-A(5)G(7), can be inserted into Langmuir monolayers of dipalmitoyl phosphatidyl choline (DPPC), which served as a cell membrane model. PB-G(12) and PB-A(5)G(7) were found to affect the DPPC monolayer even at high surface pressures. The effects from PB-G(12) were consistently stronger, particularly in reducing the elasticity of the DPPC monolayers, which may have important biological implications. Multilayers of DPPC and nucleotide-based copolymers could be adsorbed onto solid supports, in the form of Y-type LB films, in which the molecular-level interaction led to lower energies in the vibrational spectra of the nucleotide-based copolymers. This successful deposition of solid films opens the way for devices to be produced which exploit the molecular recognition properties of the nucleotides. (C) 2010 Elsevier Inc. All rights reserved.

Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)