A Meeting of East and West: Can Eastern-Influenced Therapies be Effective in the Treatment of Stress and Mood Disorders?

Given that the human brain is plastic and that structural alterations have been seen in monks who meditate on a regular basis, the question arises of whether these two facts are actually related. Furthermore, if this is in fact the case, would it be possible to apply these findings to the public? In this paper I will present the different conditions that induce neuroplasticity as well as give an overview of meditation and the ways that it is practiced nowadays. To this end I will argue that if monks are able to alter the structure of their brains and the brain is naturally inclined to heal itself then incorporating eastern practices, such as mindfulness and imagery, into western therapies could benefit patients suffering from mood disorders and, in particular, stress.

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.

Aspectos estruturais, farmacológicos e biológicos de fucanas da alga marrom sargassum vulgare

The present study examines the chemical composition and their effects on free radicals, inflammation, angiogenesis, coagulation, VEGF effects and cellular proliferation of a polysaccharides from alga Sargassum vulgare. The sulfated polysaccharide was extracted from brown seaweed by proteolysis with enzymes maxataze. The presence of proteins and sugars were observed in crude polysaccharides. Fractionation of this crude extract was made with growing concentration of acetone (0.3-1.5 v) and produced four groups of polysaccharides. Anionic polysaccharides from brown seaweed Sargassum vulgare, SV1and PSV1 were fractionated (SV1) and purified (PSV1), and displayed with high total sugars and sulfate content and very low level of protein. This fucan SV1 contains low levels of protein and high carbohydrate and sulfate content. This polysaccharides prolonged activated partial thromboplastin time (aPTT) at 50 μg (>240 s). SV1 was found to have no effect on prothrombin time (PT), corresponding to the extrinsic pathway of coagulation. SV1 exhibits high antithrombotic action in vivo, with a concentration ten times higher than heparin. Polysaccharides from S. vulgare promoted direct inhibition enzymatic activity of thrombin and stimulated enzymatic activity of FXa. SV1 showed optimal inhibitory activity of thrombin (50.2±0.28%) at a concentration of 25 μg/mL. Its antioxidant action on scavenging radicals by DPPH was (22%), indicating the polymer has no cytotoxic action (hemolytic) on ABO and Rh blood types in different erythrocyte groups and displays strong anti-inflammatory action on all concentrations tested in the carrageenan-induced paw edema model, demonstrated by reduced edema and cellular infiltration. Angiogenesis is a dynamic process of proliferation and differentiation. It requires endothelial proliferation, migration, and tube formation. In this context, endothelial cells are a preferred target for several studies and therapies. The antiangiogenic efficacy of polysaccharides was examined in vivo in the chick chorioallantoic membrane (CAM) model by using fertilized eggs. Decreases in the density of the capillaries were assessed and scored. The results showed that SV1 and PSV1 have an inhibitory effect on angiogenesis. These results were also confirmed by inhibition tubulogenesis in rabbit aorta endothelial cell (RAEC) in matrigel. These compounds were assessed in Apoptosis assay (Annexin V - FITC / PI) and cell viability by MTT assay of RAEC. These polysaccharides do not affect the viability and do not have apoptotic or necrotic action. RAEC cell when incubated with SV1 and PSV1showed inhibition of VEGF secretion, observed when compounds were incubated at 25, 50 and 100 μg/μL. The VEGF secretion with the RAEC cell line for 24 h, was more effective for PSV1 at 50 μg/μL(71.4%) than SV1 100 μg/μL (75.9%). SV1 and PSV1 had an antiproliferative action (47%) against tumor cell line HeLa. Our results indicate that these sulfated polysaccharides have antiangiogenic and antitumoral actions

Kinetics of cellular component in inflammatory response induced by different stimuli in the swim bladder of pacu, Piaractus mesopotamicus Holmberg 1887 (Characidae)

The present study assessed the kinetics of cell accumulation at the site of inflammation induced by thioglycolate, Escherichia coli lipopolysaccharide (LPS) and heat-inactivated Aeromonas hydrophila, in the pacu, Piaractus mesopotamicus (Characidae), swim bladder. A quantitative, as well as qualitative, assessment was done of all the cells present in the exudate at 6, 24, and 48 h (n = 8) after inoculation of inflammatory agents. The results show that the thioglycolate was the irritant to induce higher total inflammatory cell accumulation when compared to the control group, 6 h after insult (P < 0.05). Inoculation of heat-inactivated Aeromonas hydrophila induced progressive accumulation of total inflammatory cells, with cell number peaking after 24 h and being significantly higher than observed in the other groups (P < 0.05). Injection of LPS also induced greater cell accumulation when compared to the control group (P < 0.05), although in lower numbers than those induced by the other two irritants. All irritants injected induced significantly greater accumulation of lymphocytes and thrombocytes when compared to the control group (P < 0.05).

Humoral and cellular immune response generated by different vaccine programs before and after Salmonella Enteritidis challenge in chickens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

Interactions of Paracoccidioides brasiliensis with host cells: recent advances

Host-fungal interactions are inherently complex and dynamic. In order to identify new microbial targets and develop more effective anti-fungal therapies, it is important to understand the cellular and molecular mechanisms of disease. Paracoccidioidomycosis provokes a variety of clinical symptoms, and Paracoccidioides brasiliensis can reach many tissues, but primarily attacks the lungs. The ability of the pathogen to interact with the host surface structures is essential to further colonization, invasion, and growth. Epithelial cells may represent the first host barrier or the preferential site of entry of the fungus. For this reason, interactions between P. brasiliensis and Vero/A549 epithelial cells were evaluated, with an emphasis on the adherence, induction of cytoskeletal alterations, and differential signaling activity of the various surface molecules. The adhesion to and invasion of epithelial cells by P. brasiliensis may represent strategies employed to thwart the initial host immune response, and may help in the subsequent dissemination of the pathogen throughout the body.

New antimicrobial therapies used against fungi present in subgingival sites-A brief review

Although the main reservoir of Candida spp. is believed to be the buccal mucosa, these microorganisms can coaggregate with bacteria in subgingival biofilm and adhere to epithelial cells. The treatment of periodontal disease includes scaling and root planning (SR?) associated with proper oral hygiene. However, some patients may have negative responses to different therapeutic procedures, with a continuous loss of insertion, so the use of antimicrobials is needed as an adjuvant to SRP treatment. The use of a broad-spectrum antibiotic, such as tetracycline and metronidazole, as an aid in periodontal treatment has also been a factor for the development of superinfections by resistant bacteria and Candida species, even in patients with HIV. In the dental practice, the most commonly used antifungals are nystatin and fluconazole. However, the introduction of new drugs like the next generation of azoles is essential before the onset of emergent species in periodontal disease. Plants are good options for obtaining a wide variety of drugs. This alternative could benefit a large population that uses plants as a first treatment option. Plants have been used in medicine for a long time and are extensively used in folk medicine, because they represent an economic alternative, are easily accessible and are applicable to various diseases. Herein, we briefly review the literature pertaining the presence of Candida sp. in periodontal pockets, the conventional antifungal resistance and new therapies that include natural antifungal agents are reviewed. (C) 2011 Elsevier Ltd. All rights reserved.