Paracoccidioides brasilinsis-Induced Migration of Dendritic Cells and Subsequent T-Cell Activation in the Lung-Draining Lymph Nodes

Paracoccidioidomycosis is a mycotic disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb), that starts with inhalation of the fungus; thus, lung cells such as DC are part of the first line of defense against this microorganism. Migration of DC to the lymph nodes is the first step in initiating T cell responses. The mechanisms involved in resistance to Pb infection are poorly understood, but it is likely that DC play a pivotal role in the induction of effector T cells that control Pb infection. In this study, we showed that after Pb Infection, an important modification of lung DC receptor expression occurred. We observed an increased expression of CCR7 and CD103 on lung DC after infection, as well as MHC-II. After Pb infection, bone marrow-derived DC as well lung DC, migrate to lymph nodes. Migration of lung DC could represent an important mechanism of pathogenesis during PCM infection. In resume our data showed that Pb induced DC migration. Furthermore, we demonstrated that bone marrow-derived DC stimulated by Pb migrate to the lymph nodes and activate a T helper (Th) response. To the best of our knowledge, this is the first reported data showing that Pb induces migration of DC and activate a T helper (Th) response.

Supramolecular assemblies of rifampicin and cationic bilayers: preparation, characterization and micobactericidal activity

Background: Cationic bilayers based on the inexpensive synthetic lipid dioctadecyldimethylammonium bromide (DODAB) have been useful as carriers for drug delivery, immunoadjuvants for vaccines and active antimicrobial agents. Methods: Rifampicin (RIF) or isoniazid (ISO) interacted with DODAB bilayer fragments (BF) or large vesicles (LV). Dispersions were evaluated by dynamic light-scattering for zeta-average diameter (Dz) and zeta-potential (zeta) analysis; dialysis for determination of drug entrapment efficiency; plating and CFU counting for determination of cell viability of Mycobacterium smegmatis or tuberculosis, minimal bactericidal concentration (MBC) and synergism index for DODAB/drug combinations. Results: DODAB alone killed micobacteria over a range of micromolar concentrations. RIF aggregates in water solution were solubilised by DODAB BF. RIF was incorporated in DODAB bilayers at high percentiles in contrast to the leaky behavior of ISO. Combination DODAB/RIF yielded MBCs of 2/2 and 4/0.007 mu g/mL against Mycobacterium smegmatis or Mycobacterium tuberculosis, respectively. Synergism indexes equal to 0.5 or 1.0, indicated synergism against the former and independent action, against the latter species. Conclusions: In vitro, DODAB acted effectively both as micobactericidal agent and carrier for rifampicin. The novel assemblies at reduced doses may become valuable against tuberculosis.

Dendritic Cells Transfected with scFv from Mab 7.B12 Mimicking Original Antigen gp43 Induces Protection against Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), which primarily attacks lung tissue. Dendritic cells (DCs) are able to initiate a response in naive T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv) encoding a single chain variable fragment (scFv) of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM) model.

Modification of composition of a nanoemulsion with different cholesteryl ester molecular species: Effects on stability, peroxidation, and cell uptake

Purpose: Use of lipid nanoemulsions as carriers of drugs for therapeutic or diagnostic purposes has been increasingly studied. Here, it was tested whether modifications of core particle constitution could affect the characteristics and biologic properties of lipid nanoemulsions. Methods: Three nanoemulsions were prepared using cholesteryl oleate, cholesteryl stearate, or cholesteryl linoleate as main core constituents. Particle size, stability, pH, peroxidation of the nanoemulsions, and cell survival and uptake by different cell lines were evaluated. Results: It was shown that cholesteryl stearate nanoemulsions had the greatest particle size and all three nanoemulsions were stable during the 237-day observation period. The pH of the three nanoemulsion preparations tended to decrease over time, but the decrease in pH of cholesteryl stearate was smaller than that of cholesteryl oleate and cholesteryl linoleate. Lipoperoxidation was greater in cholesteryl linoleate than in cholesteryl oleate and cholesteryl stearate. After four hours' incubation of human umbilical vein endothelial cells (HUVEC) with nanoemulsions, peroxidation was minimal in the presence of cholesteryl oleate and more pronounced with cholesteryl linoleate and cholesteryl stearate. In contrast, macrophage incubates showed the highest peroxidation rates with cholesteryl oleate. Cholesteryl linoleate induced the highest cell peroxidation rates, except in macrophages. Uptake of cholesteryl oleate nanoemulsion by HUVEC and fibroblasts was greater than that of cholesteryl linoleate and cholesteryl stearate. Uptake of the three nanoemulsions by monocytes was equal. Uptake of cholesteryl oleate and cholesteryl linoleate by macrophages was negligible, but macrophage uptake of cholesteryl stearate was higher. In H292 tumor cells, cholesteryl oleate showed the highest uptakes. HUVEC showed higher survival rates when incubated with cholesteryl stearate and smaller survival with cholesteryl linoleate. H292 survival was greater with cholesteryl stearate. Conclusion: Although all three nanoemulsion types were stable for a long period, considerable differences were observed in size, oxidation status, and cell survival and nanoemulsion uptake in all tested cell lines. Those differences may be helpful in protocol planning and interpretation of data from experiments with lipid nanoemulsions.

Polyelectrolyte-protein complexation driven by charge regulation

The interplay between the biocolloidal characteristics (especially size and charge), pH, salt concentration and the thermal energy results in a unique collection of mesoscopic forces of importance to the molecular organization and function in biological systems. By means of Monte Carlo simulations and semi-quantitative analysis in terms of perturbation theory, we describe a general electrostatic mechanism that gives attraction at low electrolyte concentrations. This charge regulation mechanism due to titrating amino acid residues is discussed in a purely electrostatic framework. The complexation data reported here for interaction between a polyelectrolyte chain and the proteins albumin, goat and bovine alpha-lactalbumin, beta-lactoglobulin, insulin, k-casein, lysozyme and pectin methylesterase illustrate the importance of the charge regulation mechanism. Special attention is given to pH congruent to pI where ion-dipole and charge regulation interactions could overcome the repulsive ion-ion interaction. By means of protein mutations, we confirm the importance of the charge regulation mechanism, and quantify when the complexation is dominated either by charge regulation or by the ion-dipole term.

Antimicrobial Activity of Diterpenes from Viguiera arenaria against Endodontic Bacteria

Six pimarane-type diterpenes isolated from Viguiera arenaria Baker and two semi-synthetic derivatives were evaluated in vitro against a panel of representative microorganisms responsible for dental root canal infections. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia, Prevotella buccae, Fusobacterium nucleatum, Bacteroides fragilis, Actinomyces naeslundii, Actinomyces viscosus, Peptostreptococcus micros, Enterococcus faecalis and Aggregatibacter actinomycetemcomitans. The compounds ent-pimara-8(14), 15-dien-19-oic acid, its sodium salt and ent-8(14), 15-pimaradien-3 beta-ol were the most active, displaying MIC values ranging from 1 to 10 mu g mL(-1). The results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new chemicals for use as a complement to instrumental endodontic procedures.

Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA

Background: Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and.AfcrzA mutant strains. Results: We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively). Decreased mRNA abundance in the Delta crzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 mu M, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl(2) 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP

Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes

Background: The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally. Results: We developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 mu g of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-gamma and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 mu g). Conclusion: Our objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.

T Helper 1-Inducing Adjuvant Protects against Experimental Paracoccidioidomycosis

Immunostimulatory therapy is a promising approach to improving the treatment of systemic fungal infections such as paracoccidioidomycosis (PCM), whose drug therapy is usually prolonged and associated with toxic side effects and relapses. The current study was undertaken to determine if the injection of a T helper (Th) 1-stimulating adjuvant in P. brasiliensis infected mice could have a beneficial effect on the course of experimental PCM. For this purpose, mice were infected and treated with complete Freund's adjuvant (CFA), a well-established Th1 experimental inductor, or incomplete Freund's adjuvant (IFA - control group) on day 20 postinfection. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by absence of epithelioid cell granulomas and yeast cells, whereas the control mice presented multiple sites of focal epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and nonviable yeast cells. In addition, CFA administration induced a 2.4 log reduction (>99%) in the fungal burden when compared to the control group, and led to an improvement of immune response, reversing the immunosuppression observed in the control group. The immunotherapy with Th1-inducing adjuvant, approved to be used in humans, might be a valuable tool in the treatment of PCM and potentially useful to improve the clinical cure rate in humans.

Pluripotent Reprogramming of Fibroblasts by Lentiviralmediated Insertion of SOX2, C-MYC, and TCL-1A

Reprogramming of somatic cells to pluripotency promises to boost cellular therapy. Most instances of direct reprogramming have been achieved by forced expression of defined exogenous factors using multiple viral vectors. The most used 4 transcription factors, octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and v-myc myelocytomatosis viral oncogene homolog (C-MYC), can induce pluripotency in mouse and human fibroblasts. Here, we report that forced expression of a new combination of transcription factors (T-cell leukemia/lymphoma protein 1A [TCL-1A], C-MYC, and SOX2) is sufficient to promote the reprogramming of human fibroblasts into pluripotent cells. These 3-factor pluripotent cells are similar to human embryonic stem cells in morphology, in the ability to differentiate into cells of the 3 embryonic layers, and at the level of global gene expression. Induced pluripotent human cells generated by a combination of other factors will be of great help for the understanding of reprogramming pathways. This, in turn, will allow us to better control cell-fate and apply this knowledge to cell therapy.

Helminth Coinfection Does Not Affect Therapeutic Effect of a DNA Vaccine in Mice Harboring Tuberculosis

Background: Helminthiasis and tuberculosis (TB) coincide geographically and there is much interest in exploring how concurrent worm infections might alter immune responses against bacilli and might necessitate altered therapeutic approaches. A DNA vaccine that codifies heat shock protein Hsp65 from M. leprae (DNAhsp65) has been used in therapy during experimental tuberculosis. This study focused on the impact of the co-existence of worms and TB on the therapeutic effects of DNAhsp65. Methodology/Principal Findings: Mice were infected with Toxocara canis or with Schistosoma mansoni, followed by coinfection with M. tuberculosis and treatment with DNAhsp65. While T. canis infection did not increase vulnerability to pulmonary TB, S. mansoni enhanced susceptibility to TB as shown by higher numbers of bacteria in the lungs and spleen, which was associated with an increase in Th2 and regulatory cytokines. However, in coinfected mice, the therapeutic effect of DNAhsp65 was not abrogated, as indicated by colony forming units and analysis of histopathological changes. In vitro studies indicated that Hsp65-specific IFN-gamma production was correlated with vaccine-induced protection in coinfected mice. Moreover, in S. mansoni-coinfected mice, DNA treatment inhibited in vivo TGF-beta and IL-10 production, which could be associated with long-term protection. Conclusions/Significance: We have demonstrated that the therapeutic effects of DNAhsp65 in experimental TB infection are persistent in the presence of an unrelated Th2 immune response induced by helminth infections.

A Novel Organotellurium Compound (RT-01) as a New Antileishmanial Agent

Leishmaniasis is a neglected disease and endemic in developing countries. A lack of adequate and definitive chemotherapeutic agents to fight against this infection has led to the investigation of numerous compounds. The aim of this study was to investigate the effect of RT-01, an organotellurane compound presenting biological activities, in 2 experimental systems against Leishmania amazonensis. The in vitro system consisted of promastigotes and amastigotes forms of the parasite, and the in vivo system consisted of L. amazonensis infected BALB/c mice, an extremely susceptible mouse strain. The compound proved to be toxic against promastigotes and amastigotes. The study also showed that treatment with RT-01 produces an effect similar to that treatment with the reference antimonial drug, Glucantime, in L. amazonensis infected mice. The best results were obtained following RT-01 intralesional administration (720 mu g/kg/day); mice showed significant delay in the development of cutaneous lesions and decreased numbers of parasites obtained from the lesions. Significant differences in tissue pathology consisted mainly of no expressive accumulation of inflammatory cells and well-preserved structures in the skin tissue of RT-01-treated mice compared with expressive infiltration of infected cells replacing the skin tissue in lesions of untreated mice. These findings highlight the fact that the apparent potency of organotellurane compounds, together with their relatively simple structure, may represent a new avenue for the development of novel drugs to combat parasitic diseases.

Phylogenetic relationships within the speciose family Characidae (Teleostei: Ostariophysi: Characiformes) based on multilocus analysis and extensive ingroup sampling

Background: With nearly 1,100 species, the fish family Characidae represents more than half of the species of Characiformes, and is a key component of Neotropical freshwater ecosystems. The composition, phylogeny, and classification of Characidae is currently uncertain, despite significant efforts based on analysis of morphological and molecular data. No consensus about the monophyly of this group or its position within the order Characiformes has been reached, challenged by the fact that many key studies to date have non-overlapping taxonomic representation and focus only on subsets of this diversity. Results: In the present study we propose a new definition of the family Characidae and a hypothesis of relationships for the Characiformes based on phylogenetic analysis of DNA sequences of two mitochondrial and three nuclear genes (4,680 base pairs). The sequences were obtained from 211 samples representing 166 genera distributed among all 18 recognized families in the order Characiformes, all 14 recognized subfamilies in the Characidae, plus 56 of the genera so far considered incertae sedis in the Characidae. The phylogeny obtained is robust, with most lineages significantly supported by posterior probabilities in Bayesian analysis, and high bootstrap values from maximum likelihood and parsimony analyses. Conclusion: A monophyletic assemblage strongly supported in all our phylogenetic analysis is herein defined as the Characidae and includes the characiform species lacking a supraorbital bone and with a derived position of the emergence of the hyoid artery from the anterior ceratohyal. To recognize this and several other monophyletic groups within characiforms we propose changes in the limits of several families to facilitate future studies in the Characiformes and particularly the Characidae. This work presents a new phylogenetic framework for a speciose and morphologically diverse group of freshwater fishes of significant ecological and evolutionary importance across the Neotropics and portions of Africa.

Use of Cassava Peel as Carbon Source for Production of Amylolytic Enzymes by Aspergillus niveus

Aspergillus niveus produced high levels of alpha-amylase and glucoamylase in submerged fermentation using the agricultural residue cassava peel as a carbon source. In static conditions, the amylase production was substantially greater than in the agitated condition. The optimized culture conditions were initially at pH 5.0, 35 degrees C during 48 hours. Amylolytic activity was still improved (50%) with a mixture of cassava peel and soluble starch in the proportion 1:1 (w/w). The crude extract exhibited temperature and pH optima approximately 70 degrees C and 4.5, respectively. Amylase activity was stable for 1 h at 60 degrees C, and at pH values between 3.0 and 7.0. The enzyme hydrolysed preferentially maltose, starch, penetrose, amylose, isomaltose, maltotriose, glycogen and amylopectin, and not hydrolysed cyclodextrin (alpha and beta), trehalose and sucrose. In the first hour of reaction on soluble starch, the hydrolysis products were glucose and maltose, but after two hours of hydrolysis, glucose was the unique product formed, confirming the presence in the crude extract of an alpha-amylase and a glucoamylase.

Genetic variability of the Amazon River prawn Macrobrachium amazonicum (Decapoda, Caridea, Palaemonidae)

The freshwater prawn Macrobrachium amazonicum is widely distributed in South America, and occupies habitats with a wide range of salinities. Several investigations have revealed the existence of wide intraspecific variability among different populations, although the understanding of this variability is still fragmentary and incomplete. We compared and characterized inland and coastal populations of M. amazonicum from Brazil, using molecular data (16S and COI mtDNA) to describe the degree of variability, structure, and relationships among them. Genetic divergence rates among populations showed variability at the intraspecific level. All the analyses evidenced significant genetic divergence among populations, structuring them in three groups: I-inland waters of the Amazonian Hydrographic Region (HR); II-Parana/Paraguay HR; and III-coastal systems of northern and northeastern Brazil. Phylogenetic reconstructions revealed that the populations form a single monophyletic clade, which supports their characterization as a single species. Clade I was a sister clade of that formed by clades II and III, which were themselves sister clades. Populations from Sertaozinho/Miguelopolis and Avare, introduced into the state of Sao Paulo, may have originated from natural populations in the states of Mato Grosso do Sul and Para, respectively. Geographical isolation probably contributed to the observed variation, and if this isolation continues. M. amazonicum may undergo speciation within its broad geographical distribution. The sequences obtained here can be used as name-tags for population identification, and the DNA barcodes are useful to identify the origin of specimens used in different freshwater-prawn cultures or introduced populations of unknown origin.