Desenvolvimento de imunógeno bacteriano de Pseudomonas aeruginosa conjugado ao toxóide tetânico

Resumo: Pseudomonas aeruginosa e uma bacteria gram-negativa responsavel por diversas doencas e manifestacoes clinicas. Sua virulencia permite a infeccao e colonizacao de individuos pertencentes a grupos de risco, como imunocomprometidos, queimados e portadores de fibrose cistica. O presente estudo visa o desenvolvimento de uma vacina a base de Lipopolissacarideo (LPS), antigeno consagrado em diversos protocolos de imunizacao. Este antigeno foi conjugado a proteina, constituindo uma vacina conjugada. Vacinas conjugadas sao imunogenos nao-proteicos que, ao serem ligados a uma proteina, adquirem uma nova propriedade: geracao de memoria imunologica. A proteina utilizada no estudo e o toxoide tetanico (toxina inativada do Clostridium tetani, TT). Ainda, capacita neonatos e criancas a interagirem com um antigeno contra o qual nao responderiam normalmente. A tecnica de conjugacao utilizada foi a aminacao redutiva direta, avaliada como simples e de baixo custo. Atraves da adaptacao do protocolo de Shen e colaboradores, foi possivel conjugar o Lipopolissacarideo ao toxoide tetanico (LPS-TT). Avaliou-se a resposta in vivo atraves de aplicacoes intraperitoneais do imunogenos em camundongos Swiss, atraves de dois protocolos: o primeiro avalia a formacao de anticorpos anti-pseudomonas pela imunizacao com LPS, LPS e TT ou LPS-TT. O segundo avalia a formacao de anticorpos utilizando LPS, LPS e TT, ou LPS-TT apos aplicacao intraperitoneal de bacterina (celula inativada de P. aeruginosa). Os grupos que foram imunizados com LPS e TT e LPS-TT (20 e 40 ƒÊg/ml) apresentaram resposta de anticorpos aglutinantes superior ao controle LPS

A new landscape of host–protozoa interactions involving the extracellular vesicles world

This version is free to view and download for private research and study only. Not for re-distribution, re-sale or use in derivative works. © Cambridge University Press 2018Extracellular vesicles (EVs) are released by a wide number of cells including blood cells, immune system cells, tumour cells, adult and embryonic stem cells. EVs are a heterogeneous group of vesicles (~30–1000 nm) including microvesicles and exosomes. The physiological release of EVs represents a normal state of the cell, raising a metabolic equilibrium between catabolic and anabolic processes. Moreover, when the cells are submitted to stress with different inducers or in pathological situations (malignancies, chronic diseases, infectious diseases.), they respond with an intense and dynamic release of EVs. The EVs released from stimulated cells vs those that are released constitutively may themselves differ, both physically and in their cargo. EVs contain protein, lipids, nucleic acids and biomolecules that can alter cell phenotypes or modulate neighbouring cells. In this review, we have summarized findings involving EVs in certain protozoan diseases. We have commented on strategies to study the communicative roles of EVs during host–pathogen interaction and hypothesized on the use of EVs for diagnostic, preventative and therapeutic purposes in infectious diseases. This kind of communication could modulate the innate immune system and reformulate concepts in parasitism. Moreover, the information provided within EVs could produce alternatives in translational medicine.Peer reviewedFinal Accepted Versio

Peptidylarginine Deiminase Inhibition Abolishes the Production of Large Extracellular Vesicles From

is a microaerophilic protozoan that is an important etiologic agent of diarrhea worldwide. There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which modulate the physiopathology of giardiasis. Here we describe new features of EV production, revealing its capacity to shed two different enriched EV populations: large (LEV) and small extracellular vesicles (SEV) and identified relevant adhesion functions associated with the larger population. Proteomic analysis revealed differences in proteins relevant for virulence and host-pathogen interactions between the two EV subsets, such as cytoskeletal and anti-oxidative stress response proteins in LEVS. We assessed the effect of two recently identified inhibitors of EV release in mammalian cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release from . The compounds were both able to effectively reduce EV shedding, the PAD-inhibitor specifically affecting the release of LEVs and reducing parasite attachment to host cells . Our results suggest that LEVs and SEVs have a different role in host-pathogen interaction, and that treatment with EV-inhibitors may be a novel treatment strategy for recurrent giardiasis. [Abstract copyright: Copyright © 2020 Gavinho, Sabatke, Feijoli, Rossi, da Silva, Evans-Osses, Palmisano, Lange and Ramirez.

DESENVOLVIMENTO DE IMUNÓGENO CONJUGADO DE LIPOPOLISSACARÍDEO DE Pseudomonas Aeruginosa E TOXÓIDE TETÂNICO

O lipopolissacarídeo (LPS) de Pseudomonas aeruginosa foi conjugado ao toxóide tetânico (TT, toxina inativada do Clostridium tetani) utilizando a técnica de aminação redutiva direta, para o desenvolvimento de uma vacina conjugada (LPS-TT). Avaliou-se a resposta imune in vivo após aplicações intraperitoneais do imunógeno LPS-TT em camundongos Swiss, administrando-se 4 aplicações de LPS-TT com intervalos entre as doses de 14 dias. Avaliou-se o título de anticorpos anti-Pseudomonas produzidos pela técnica de soroaglutinação microscópica. Os grupos que foram imunizados com LPS-TT e com LPS+TT (não conjugado quimicamente) apresentaram resposta de anticorpos aglutinantes superior ao controle imunizado apenas com LPS, e a resposta após o reforço vacinal dos animais imunizados com o conjugado LPS-TT foi 567% superior ao dos animais imunizados com LPS

Estudo das diferentes populações de vesículas extracelulares produzidas por Giardia intestinalis

Orientador: Prof. Dr. Marcel Ivan RamirezTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Defesa : Curitiba, 10/12/2020Inclui referências: p. 84-99Resumo: Giardia intestinalis, um protozoário microaerofílico, é um importante agente etiológico de infecção intestinal em todo o mundo, conhecido também por ser a "diarréia do viajante" Há evidências de que, em diversas condições, o parasita é capaz de liberar vesículas extracelulares (VEs) que modulam a fisiopatologia da giardíase. Neste trabalho, descrevemos novas características da produção de VEs de G. intestinalis, revelando sua capacidade de liberar duas populações diferentes de VEs enriquecidas: vesículas extracelulares largas (VELs) e pequenas (VEPs), assim como funções de adesão relevantes associadas à população maior. A análise proteômica revelou diferenças em proteínas importantes para a virulência e também para as interações patógeno-hospedeiro entre os dois tipos de VEs, como proteínas de resposta ao estresse antioxidante e citoesquelético em VELs. Avaliamos o efeito de dois inibidores associados à liberação de VEs em células de mamíferos, a saber, inibidores de peptidilarginina deiminases (PAD) e canabidiol (CBD), na liberação de VEs de Giardia. Os compostos foram ambos capazes de reduzir efetivamente a liberação de VEs, o inibidor de PAD afetando especificamente a liberação de VELs e reduzindo a adesao do parasita às células hospedeiras in vitro. Nossos resultados sugerem que VELs e VEPs têm um papel diferente na interação patógeno-hospedeiro, e que o tratamento com inibidores de VEs pode ser uma nova estratégia de tratamento para a giardíase. Além disso, investigaram-se os efeitos do decavanadato de sódio (doravante denominado NaV10), nicotinamida (composto I) e isonicotinamida (composto II) em células de Giardia intestinalis. A exposição dos trofozoítos de Giardia ao NaV10 e ao composto II revelou redução na viabilidade dos trofozoítos (valores de GI50 de cerca de 10 ?mol L - 1) e afetou a aderência do parasita aos tubos de cultura de poliestireno e a uma monocamada de células Vero, mesmo em baixas concentrações.Abstract: Giardia intestinalis is a microaerophilic protozoan that is an important etiologic agent of intestinal infection worldwide, also known as the "traveler diarrhea". There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which modulate the physiopathology of giardiasis. Here we describe new features of G. intestinalis EV production, revealing its capacity to shed two different enriched EV populations: large (LEV) and small extracellular vesicles (SEV) and identified relevant adhesion functions associated with the larger population. Proteomic analysis revealed differences in proteins relevant for virulence and host-pathogen interactions between the two EV subsets, such as cytoskeletal and anti-oxidative stress response proteins in LEVS .We assessed the effect of two recently identified inhibitors of EV release in mammalian cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release from Giardia. The compounds were both able to effectively reduce EV shedding, the PAD-inhibitor specifically affecting the release of LEVs and reducing parasite attachment to host cells in vitro. Our results suggest that LEVs and SEVs have a different role in host-pathogen interaction, and that treatment with EV-inhibitors may be a novel treatment strategy for recurrent giardiasis. In addition, the effects of sodium decavanadate (henceforth called NaV10), nicotinamide (compound I) and isonicotinamide (compound II) were investigated in Giardia intestinalis cells. Exposure of Giardia trophozoites to NaV10 and compound II revealed reduction in trophozoite viability (GI50 values of ca. 10 ?mol L-1) and affected the parasite adherence to both polystyrene culture tubes and a monolayer of Vero cells, even at low concentrations

Unveiling the role of EVs in anaerobic parasitic protozoa

The anaerobic or microaerophilic protozoan parasites such as the enteric human pathogens Entamoeba histolytica, Giardia intestinalis, Cryptosporidium parvum, Blastocystis hominis and urogenital tract parasites Trichomonas vaginalis are able to survival in an environment with oxygen deprivation. Despite living in hostile environments these pathogens adopted different strategies to survive within the hosts. Among them, the release of extracellular vesicles (EVs) has become an active endeavor in the study of pathogenesis for these parasites. EVs are heterogenous, membrane-limited structures that have played important roles in cellular communication, transferring information through cargo and modulating the immune system of the host. In this review, we described several aspects of the recently characterized EVs of the anaerobic protozoa, including their role in adhesion, modulation of the immune response and omics analysis to understand the potential of these EVs in the pathogenesis of these diseases caused by anaerobic parasites.Fil: Sabatke, Bruna. Universidade Federal do Paraná; BrasilFil: Gavinho, Bruno. Universidade Federal do Paraná; BrasilFil: Cóceres, Verónica Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: de Miguel, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ramirez, Marcel I.. Universidade Federal do Paraná; Brasil. Fundación Oswaldo Cruz; Brasi

Thermal, Structural, Morphological and Electrical Characterization of Cerium-Containing 45S5 for Metal Implant Coatings

Biomaterial coatings on dental implants are increasingly being used as a solution to the problems of rejection and implant loss. Bioglass® has been seen as a promising material for coating metal implants, increasing the integration rate and improving the bond between the bone and the implant. Multifunctional bioactive glasses can exhibit antibacterial, antitumor and antioxidant effects with the addition of therapeutic ions. The cerium ion has shown an antioxidant effect through mimicking mechanisms and by acting as a scavenger of reactive oxygen species (ROS), which is important for avoiding oxidative stress post-surgery. Furthermore, it is possible to store electrical charge on the bioglass surface, which potentiates osseointegration. In this work, glasses with various percentages of cerium oxide (0.25, 0.5, 1 and 2 mol%) were developed and structurally and electrically analyzed. It was verified that the cerium insertion did not modify the amorphous phase characteristic of the glass but showed an increase in the number of non-bridging oxygens (NBOs). This increase in NBOs did not modify the electrical conductivity in either the ac or dc regime. The similar permittivity values of the glasses also suggest that their storage ability is unchanged with the addition of CeO2. Concerning the impedance spectroscopy (IS) data, a decrease in resistance is visible with the addition of cerium oxide, suggesting a favorable behavior for applications as an antioxidant through the electro-Fenton reaction

In Situ Synthesis of AZO-Np in Guar Gum/PVOH Composite Fiber Mats for Potential Bactericidal Release

Since the number of antibiotic-resistant bacterial infections is growing and cases are getting worse every year, the search for new alternative bactericidal wound dressing treatments is becoming crucial. Within this context, the use of polysaccharides from plants and seeds in innovative biopolymer technologies is of key importance. In this work, bio-nano-composite guar gum/polyvinyl alcohol (PVOH) membranes loaded with aluminum-doped zinc oxide nanoparticles were produced via electrospinning. Citric acid was added to the mixture to increase spinnability. However, depending on the pH, zinc oxide nanoparticles are partially dissociated, decreasing their bactericidal efficiency. Thus, a second successful alkaline thermo-chemical regrowth step was added to the process to treat the obtained fibers. This alkaline thermo-chemical treatment reconstituted both the nanoparticles and their bactericidal properties. The Staphylococcus aureus antibacterial assay results show that the membranes obtained after the alkaline thermo-chemical treatment presented a 57% increase in growth inhibition

Effects of Decavanadate Salts with Organic and Inorganic Cations on Escherichia coli, Giardia intestinalis, and Vero Cells

Decavanadate salts with nicotinamide (3-pyridinecarboxamide, 3-pca) and isonicotinamide (4-pyridinecarboxamide, 4-pca) in both neutral and protonated forms, (3-Hpca) 4 [H 2 V 10 O 28 ]·2H 2 O·2(3-pca) (complex I) and (4-Hpca) 4 [H 2 V 10 O 28 ]·2(4-pca) (complex II), have been synthesized and characterized by vibrational spectroscopy (infrared and Raman), thermogravimetric analysis (TGA), 51 V NMR, and single-crystal X-ray diffraction analysis. The effects of sodium decavanadate (henceforth called NaV 10 ) and compounds I and II on Escherichia coli, Giardia intestinalis, and Vero (African green monkey epithelial kidney) cells were evaluated. Enhanced growth inhibitory activity against E. coli cultures was observed upon treatment with products I and II when compared to that with NaV 10 (GI 50 values of 2.8, 4.0, and 11 mmol L -1 , respectively), as well as lower cell viability as measured by the intake of propidium iodide (PI). Exposure of Giardia trophozoites to NaV 10 and II revealed reduction in trophozoite viability (GI 50 values of ca. 10 μmol L -1 ) and affected the parasite adherence to both polystyrene culture tubes and a monolayer of Vero cells, even at low concentrations. A lesser effect on Giardia was shown for I. Furthermore, all three compounds were significantly less toxic to Vero cells than the reference drug, albendazole, employed in the treatment of giardiasis. Toxicity reports of oxidovanadium compounds toward Giardia are unprecedented and open a path to the development of new therapeutic agents