Induction of pro-inflammatory response of the placental trophoblast by Plasmodium falciparum infected erythrocytes and TNF.

ABSTARCT: Plasmodium falciparum placental malaria is characterized by the sequestration of infected erythrocytes (IEs) in the placental intervillous space via adherence to chondroitin sulphate A (CSA), production of inflammatory molecules, and leukocytes infiltration. Previous reports suggest that the syncytiotrophoblast (ST) immunologically responds to IEs contact. This study explores the inflammatory response induced in BeWo cells by adherence of IEs and TNFstimulation. METHODS: A non-syncitialized BeWo cells (trophoblast model) were used to evaluate its response to CSA-adherents IEs (FCB1csa, FCB2csa, FCR3csa, 3D7csa) and TNF stimulation. Expression of membrane ICAM-1 (mICAM-1) receptor in BeWo cells was quantified by flow cytometry and the IL-8, IL-6 and soluble ICAM-1 (sICAM-1) concentrations were quantified by enzyme-linked immunosorbentassay (ELISA) in BeWo stimulated supernatants. RESULTS: BeWo cells stimulated with TNF and CSA-adherents IEs of FCB1csa and 3D7csa (strains with higher adhesion) increase the expression of ICAM-1 on the surface of cells and the secretion of immune factors IL-8, IL-6 and sICAM-1. This inflammatory response appears to be related to the level of adherence of IEs because less adherent strains do not induce significant changes. CONCLUSIONS: It was found that BeWo cells responds to CSA-IEs and to TNF favouring a placental pro-inflammatory environment, evidenced by increases in the expression of membrane mICAM-1 and release of soluble ICAM-1, as well as the IL-8 and IL-6 secretion. The expression of ICAM-1 in BeWo cells might be associated to an increase in leukocyte adhesion to the trophoblast barrier, promoting greater inflammation, while the sICAM-1 release could be a protection mechanism activated by trophoblastic cells, in order to regulate the local inflammatory response

Métodos proteómicos aplicados al estudio de la malaria: plasmodium falciparum

La malaria es una de las enfermedades parasitarias que causa  mayor  impacto en la salud pública en países en desarrollo. La secuenciación del genoma de Plasmodium falciparum y el desarrollo de la proteómica han permitido un gran avance en el conocimiento de la biología del parasito. La proteómica ha permitido caracterizar cualitativa y cuantitativamente la expresión de proteínas del parásito y ha proveído información de la expresión relativa de proteínas bajo condiciones de stress como presión por antimaláricos.  Dada la complejidad de su ciclo de vida, el cual se lleva a cabo en el hospedero vertebrado y el mosquito, se ha caracterizado la expresión de proteínas para cada estadio del parásito; con el fin de determinar el proteoma que media diversos procesos metabólicos, fisiológicos, energéticos. Técnicas de electroforesis bidimensional, cromatografía liquida y espectrometría de masas,  han sido útiles para evaluar los efectos de antimaláricos sobre la expresión de proteínas del parasito y caracterizar el proteoma de diferentes formas y organelas de P.falciparum.  El propósito de esta revisión es  presentar el estado del arte de los avances en  proteómica aplicada al estudio de la malaria,  y exponer las diferentes estrategias experimentales empleadas para el estudio del proteoma del parásito con el fin de exponer las ventajas y desventajas de cada una de estas metodologías a partir de los resultados obtenidos en los artículos revisados en este manuscrito

Nuevas vías de permeabilidad y regulación del ph intracelular como posibles blancos terapéuticos en plasmodium falciparum

Actualmente, existe una necesidad sentida para el desarrollo de nuevos fármacos antimaláricos o de compuestos conocidos dirigidos contra blancos terapéuticos diferentes a los afectados por los medicamentos usuales. Son diversos los blancos que pueden ser aprovechados en Plasmodium, y la alteración de parámetros fisiológicos como el pH y el transporte de solutos pueden explicar la muerte del parásito cuando se usan compuestos antiplasmodiales, lo que representa una opción para el desarrollo de nuevas alternativas antiparasitarias. El propósito de esta revisión es por tanto, proporcionar una visión general de los efectos causados por esteroides, discutiendo el caso específico de los esteroides antiplasmodiales aislados de Solanum nudum y revisar dos procesos fisiológicos importantes en el parásito como posibles blancos terapéuticos, la modificación de permeabilidad del eritrocito infectado y el mantenimiento del pH intracelular de Plasmodium

Partial Characterization of Venom from the Colombian Spider Phoneutria Boliviensis (Aranae:Ctenidae)

We report on the first studies on the characterization of venom from Phoneutria boliviensis (Aranae:Ctenidae) (F. O. Pickard-Cambridge, 1897), done with Colombian species. After the electrostimulation extraction process, the venom showed physicochemical properties corresponding to a colorless and water-soluble liquid with a density of 0.86 mg/mL and 87% aqueous content. P. boliviensis venom and RP-HPLC fractions showed hemolytic activity and hydrolyzed the synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid, indicating the presence of phospholipases A2 enzymes. The electrophoretic profile showed an important protein content with molecular masses below 14 kDa, and differences between male and female protein content were also revealed. The RP-HPLC venom profile exposes differences between males and female content consistent with the electrophoretic profile. Five fractions collected from the RP-HPLC displayed significant larvicidal activity. Mass analysis indicates the presence of peptides ranging from 1047.71 to 3278.07 Da. Two peptides, Ctenitoxin-Pb48 and Ctenitoxin-Pb53, were partially identified using HPLC-nESI-MS/MS, which showed a high homology with other Ctenitoxins (family Tx3) from Phoneutria nigriventer, Phoneutria keyserlingi and Phoneutria reidyi affecting voltage-gated calcium receptors (Cav 1, 2.1, 2.2 and 2.3) and NMDA-glutamate receptors

Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species

Antiplasmodial effect of the venom of Crotalus durissus cumanensis, crotoxin complex and Crotoxin B

2082-11 Embargo por política editorialThe antiplasmodial activity of phospholipases A2 (PLA2) isolated from different animals has been studied. We explored the in vitro anti Plasmodium falciparum effect of a fraction containing crotoxin, Crotoxin B and whole venom of the rattlesnake Crotalus durissus cumanensis. Fraction II (crotoxin complex) was obtained by size exclusion chromatography, whereas Crotoxin B was purified by RP-HPLC. The whole venom is active against the parasite at concentrations of 0.17 ± 0.03 μg/ml, fraction II at 0.76 ± 0.17 μg/ml and Crotoxin B at 0.6 ± 0.04 μg/ml. Differences were observed in the cytotoxic activity against peripheral mononuclear cells, with Crotoxin B exhibiting the highest cytotoxicity. The concentration of Crotoxin B required to exert cytotoxic activity was higher than that required to exert antiplasmodial activity. Lethality in mice confirmed the higher toxicity and neurotoxicity of whole venom and fraction II, whereas Crotoxin B was not lethal at the doses tested. These results suggest the potential of Crotoxin B as a lead compound for antimalarial activity.Departamento Administrativo de Ciencia, Tecnología e Innovación/[111540820526]/COLCIENCIAS/ColombiaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

An acidic phospholipase A2 with antibacterial activity from Porthidium nasutum snake venom

Snake venoms are complex mixtures of proteins among which both basic and acidic phospholipases A2 (PLA2s) can be found. Basic PLA2s are usually responsible for major toxic effects induced by snake venoms, while acidic PLA2s tend to have a lower toxicity. A novel PLA2, here named PnPLA2, was purified from the venom of Porthidium nasutum by means of RP-HPLC on a C18 column. PnPLA2 is an acidic protein with a pI of 4.6, which migrates as a single band under both non-reducing and reducing conditions in SDS-PAGE. PnPLA2 had a molecular mass of 15,802.6 Da, determined by ESI-MS. Three tryptic peptides of this protein were characterized by HPLC-nESI-MS/MS, and N-terminal sequencing by direct Edman degradation showing homology to other acidic PLA2s from viperid venoms. PnPLA2 displayed indirect hemolytic activity in agarose erythrocyte-egg yolk gels and bactericidal activity against Staphylococcus aureus in a dose-dependent manner, with a MIC and MBC of 32 μg/mL. In addition, PnPLA2 showed a potent inhibitory effect on platelet aggregation with doses up to 40 μg/mL. This acidic PLA2, in contrast to basic enzymes isolated from other viperid snake venoms, was not cytotoxic to murine skeletal muscle myoblasts C2C12. This is the first report on a bactericidal protein of Porthidium nasutum venom.Departamento Administrativo de Ciencia, Tecnología e Innovación /[1115-459-21441]/COLCIENCIAS/ColombiaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP