Diversity of phospholipases A2 from Bothrops atrox snake venom: adaptive advantages for snakes compromising treatments for snakebite patients

The evolution of snake venoms resulted in multigene toxin families that code for structurally similar isoforms eventually harboring distinct functions. PLA2s are dominant toxins in viper venoms, and little is known about the impact of their diversity on human envenomings and neutralization by antivenoms. Here, we show the isolation of three distinct PLA2s from B. atrox venom. FA1 is a Lys-49 homologue, and FA3 and FA4 are catalytic Asp-49 PLA2s. FA1 and FA3 are basic myotoxic proteins, while FA4 is an acid non-myotoxic PLA2. FA3 was the most potent toxin, inducing higher levels of edema, inflammatory nociception, indirect hemolysis, and anticoagulant activity on human, rat, and chicken plasmas. FA4 presented lower anticoagulant activity, and FA1 had only a slight effect on human and rat plasmas. PLA2s presented differential reactivities with antivenoms, with an emphasis on FA3, which was not recognized or neutralized by the antivenoms used in this study. Our findings reveal the functional and antigenic diversity among PLA2s from B. atrox venom, highlighting the importance of assessing venom variability for understanding human envenomations and treatment with antivenoms, particularly evident here as the antivenom fails to recognize FA3, the most active multifunctional toxin described.

Robust immune response induced by Schistosoma mansoni TSP-2 antigen coupled to bacterial outer membrane vesicles

Purpose: The use of adjuvants can significantly strengthen a vaccine’s efficacy. We sought to explore the immunization efficacy of bacterial outer membrane vesicles (OMVs) displaying the Schistosoma mansoni antigen, SmTSP-2, through a biotin-rhizavidin coupling approach. The rationale is to exploit the nanoparticulate structure and the adjuvant properties of OMVs to induce a robust antigen-specific immune response, in light of developing new vaccines against S. mansoni. Materials and Methods: OMVs were obtained from Neisseria lactamica and conjugated with biotin. The recombinant SmTSP-2 in fusion with the biotin-binding protein rhizavidin (rRzvSmTSP-2) was produced in E. coli and coupled to biotinylated OMVs to generate an OMV complex displaying SmTSP-2 on the membrane surface (OMV:rSmTSP-2). Transmission electron microscopy (TEM) and dynamic light scattering analysis were used to determine particle charge and size. The immunogenicity of the vaccine complex was evaluated in C57BL/6 mice. Results: The rRzvSmTSP-2 protein was successfully coupled to biotinylated OMVs and purified by size-exclusion chromatography. The OMV:rSmTSP-2 nanoparticles showed an average size of 200 nm, with zeta potential around – 28 mV. Mouse Bone Marrow Dendritic Cells were activated by the nanoparticles as determined by increased expression of the co-stimulatory molecules CD40 and CD86, and the proinflammatory cytokines (TNF-α, IL-6 and IL-12) or IL-10. Splenocytes of mice immunized with OMV:rSmTSP-2 nanoparticles reacted to an in vitro challenge with SmTSP-2 with an increased production of IL-6, IL-10 and IL-17 and displayed a higher number of CD4+ and CD8+ T lymphocytes expressing IFN-γ, IL-4 and IL-2, compared to mice immunized with the antigen alone. Immunization of mice with OMV:rSmTSP-2 induced a 100-fold increase in specific anti-SmTSP-2 IgG antibody titers, as compared to the group receiving the recombinant rSmTSP-2 protein alone or even co-administered with unconjugated OMV. Conclusion: Our results demonstrate that the SmTSP-2 antigen coupled with OMVs is highly immunogenic in mice, supporting the potential effectiveness of this platform for improved antigen delivery in novel vaccine strategies.

The modulatory effect of crotoxin and its phospholipase A2 subunit from Crotalus durissus terrificus venom on dendritic cells interferes with the generation of effector CD4+ T lymphocytes

Dendritic Cells (DCs) direct either cellular immune response or tolerance. The crotoxin (CTX) and its CB subunit (phospholipase A2) isolated from Crotalus durissus terrificus rattlesnake venom modulate the DCs maturation induced by a TLR4 agonist. Here, we analyzed the potential effect of CTX and CB subunit on the functional ability of DCs to induce anti-ovalbumin (OVA) immune response. Thus, CTX and CB inhibited the maturation of OVA/LPS-stimulated BM-DCs from BALB/c mice, which means inhibition of costimulatory and MHC-II molecules expression and proinflammatory cytokines secretion, accompanied by high expression of ICOSL, PD-L1/2, IL-10 and TGF-β mRNA expression. The addition of CTX and CB in cultures of BM-DCs incubated with ConA or OVA/LPS inhibited the proliferation of CD3+ or CD4+T cells from OVA-immunized mice. In in vitro experiment of co-cultures of purified CD4+T cells of DO11.10 mice with OVA/LPS-stimulated BM-DCs, the CTX or CB induced lowest percentage of Th1 and Th2 and CTX induced increase of Treg cells. In in vivo, CTX and CB induced lower percentage of CD4+IFNγ+ and CD4+IL-4+ cells, as well as promoted CD4+CD25+IL-10+ population in OVA/LPS-immunized mice. CTX in vivo also inhibited the maturation of DCs. Our findings demonstrate that the modulatory action of CTX and CB on DCs interferes with the generation of adaptive immunity and, therefore contribute for the understanding of the mechanisms involved in the generation of cellular immunity, which can be useful for new therapeutic approaches for immune disorders.

Crotoxin isolated from Crotalus durissus terrificus venom modulates the functional activity of dendritic cells via formyl peptide receptors

The Crotalus durissus terrificus rattlesnake venom, its main toxin, crotoxin (CTX), and its crotapotin (CA) and phospholipase A(2) (CB) subunits modulate the immune system. Formyl peptide receptors (FPRs) and lipoxin A(4) (LXA(4)) are involved in CTX's effect on macrophages and neutrophils. Dendritic cells (DCs) are plasticity cells involved in the induction of adaptive immunity and tolerance maintenance. Therefore, we evaluated the effect of CTX, CA or CB on the maturation of DCs derived from murine bone marrow (BM). According to data, CTX and CB-but not CA-induced an increase of MHC-II, but not costimulatory molecules on DCs. Furthermore, CTX and CB inhibited the expression of costimulatory and MHC-II molecules, secretion of proinflammatory cytokines and NF-kappa Bp65 and p38/ERK1/2-MAPK signaling pathways by LPS-incubated DCs. Differently, CTX and CB induced IL-10, PGE(2) and LXA(4) secretion in LPS-incubated DCs. Lower proliferation and IL-2 secretion were verified in coculture of CD3(+) cells and DCs incubated with LPS plus CTX or CB compared with LPS-incubated DCs. The effect of CTX and CB on DCs was abolished in cultures incubated with a FPRs antagonist. Hence, CTX and CB exert a modulation on functional activity of DCs; we also checked the involvement the FPR family on cell activities.

Crotoxin isolated from Crotalus durissus terrificus venom modulates the functional activity of dendritic cells via formyl peptide receptors

The Crotalus durissus terrificus rattlesnake venom, its main toxin, crotoxin (CTX), and its crotapotin (CA) and phospholipase A(2) (CB) subunits modulate the immune system. Formyl peptide receptors (FPRs) and lipoxin A(4) (LXA(4)) are involved in CTX's effect on macrophages and neutrophils. Dendritic cells (DCs) are plasticity cells involved in the induction of adaptive immunity and tolerance maintenance. Therefore, we evaluated the effect of CTX, CA or CB on the maturation of DCs derived from murine bone marrow (BM). According to data, CTX and CB-but not CA-induced an increase of MHC-II, but not costimulatory molecules on DCs. Furthermore, CTX and CB inhibited the expression of costimulatory and MHC-II molecules, secretion of proinflammatory cytokines and NF-kappa Bp65 and p38/ERK1/2-MAPK signaling pathways by LPS-incubated DCs. Differently, CTX and CB induced IL-10, PGE(2) and LXA(4) secretion in LPS-incubated DCs. Lower proliferation and IL-2 secretion were verified in coculture of CD3(+) cells and DCs incubated with LPS plus CTX or CB compared with LPS-incubated DCs. The effect of CTX and CB on DCs was abolished in cultures incubated with a FPRs antagonist. Hence, CTX and CB exert a modulation on functional activity of DCs; we also checked the involvement the FPR family on cell activities.

Estudo do potencial regulador da crotoxina e suas subunidades isoladas do veneno de Crotalus durissus terrificus sobre a atividade funcional de células dendríticas / Modulatory potential of crotoxin and its subunits isolated from Crotalus durissus terrificus venom on dendritic cells

Pathogens as well as pathogenic molecules are able to activate or modulate the immune system. The Crotalus durissus terrificus rattlesnake venom, its main toxin, crotoxin (CTX) and the CA (crotapotin) or CB (phospholipase A2) subunits have suppressive effect on the immune system. Dendritic cells (DCs) are potent cells from innate immunity responsible by antigen presentation to T lymphocytes and, thus are directly involved in the generation of the adaptive immune response or tolerance. Therefore, we evaluated the modulatory effect of CTX as well as its subunits on functional activity of DCs in vitro. The CTX, CA and CB were purified, analyzed by SDS-PAGE and the endotoxin (LPS) content removed. The cytometric analysis of CD11c expression by cells showed that the protocol using GM- CSF/IL-4 was more efficient in generating DCs in vitro than GM-CSF. Furthermore, these immature CD11c+ cells expressed Toll like receptors 1, 2, 4 and C-type lectin receptors (DCSIGN and MR). DCs were incubated with CTX, CA, CB with or without LPS for 7 days and then analyzed for the expression of class II MHC (MHC-II), CD40, CD80 e CD86 molecules as well as the secretion of proinflammatory cytokines (IL-1β, IL-12, IL-6, TNF-α). CTX, CA or CB did not induce increased expression of costimulatory and MHC-II molecules by DCs, whereas LPS induced high expression of these molecules on DCs. Furthermore, CTX and CB inhibited the expression of CD40, CD80, CD86 and MHC-II molecules by DCs incubated with LPS. Both CTX and CB inhibited the secretion of proinflammatory cytokines by DC stimulated with LPS. However, CA was not able to inhibit the DC maturation induced by LPS. It was also verified that formyl peptide receptor is involved in this effect of CTX and CB on DC maturation induced by LPS. Analysis of IL-10 secretion and gene expression of TGFβ and IL-10 showed that CTX and, at lower intensity the CB, induced high amounts of these anti-inflammatory cytokines by DCs incubated only with the toxins or with LPS. In addition, it was verified increased secretion of prostaglandin E2 and lipoxin A4 by DCs incubated with CTX and CB and, at lower intensity with CA, in the presence or not of LPS. Proliferation assay of CD3+ lymphocytes co-cultured with DCs previoulsy incubated with CTX or CB with or without LPS showed that CTX and CB inhibited the cellular proliferation induced by LPS. These results demonstrate that CTX and CB subunit, but not CA, exert modulatory effect on functional activity of DCs and suggest the involviment of anti-inflammatory mediators in this process.

Agentes patogênicos, assim como moléculas com potencial patogênico são capazes de ativar/modular o sistema imune. O veneno da cascavel Crotalus durissus terrificus, crotoxina (CTX-principal toxina) e suas subunidades CA (crotapotina) ou CB (fosfolipase A2) apresentam efeito supressor sobre o sistema imune. Células dendríticas (DCs) são potentes células da imunidade inata responsáveis pela apresentação dos antígenos para os linfócitos T e, portanto estão envolvidas diretamente na geração da resposta imune adaptativa ou tolerância imunológica. Portanto, no presente estudo, foi investigado o efeito modulador da CTX, bem como da CA e CB sobre a atividade funcional das DCs in vitro. CTX, CA e CB foram purificadas, analisadas por SDS-PAGE e o conteúdo de endotoxina (LPS) eliminado. DCs imaturas (iDCs) foram diferenciadas a partir da medula óssea de camundongos BALB/c com GM-CSF ou GM-CSF/IL-4 por 7 dias. A análise por citometria de fluxo da expressão de CD11c revelou que o protocolo de utilização do GM-CSF/IL-4 foi mais eficiente em gerar DCs in vitro. Além disso, estas células imaturas (CD11c+ ) expressam receptores do tipo Toll 1, 2, 4 e de lectina tipo C (DC-SIGN e MR). DCs imaturas foram incubadas com CTX, CA ou CB na presença ou não de LPS e analisadas quanto à expressão das moléculas de MHC de classe II (MHC-II), CD40, CD80 e CD86, bem como a secreção de citocinas pró-inflamatórias (IL-1β, IL-12, IL6, TNF-α). A CTX, CA e CB não induziram aumento da expressão das moléculas MHC-II e coestimuladoras nas DCs in vitro, enquanto que o LPS induziu alta expressão dessas moléculas nas DCs. Além disso, CTX e CB inibiram a expressão de CD40, CD80, CD86 e MHC-II em DCs incubadas com LPS, sendo esse efeito mais acentuado com a CTX. Tanto a CTX como CB inibiram a secreção das citocinas pró-inflamatórias pelas DCs estimuladas com LPS. Por outro lado, a subunidade CA não foi capaz de modular a maturação de DCs induzida pelo LPS. Pôde ser observado ainda, que receptor formil peptídeo está envolvido nesse efeito da CTX e CB sobre a maturação das DCs induzida pelo LPS. A análise da secreção IL-10 e expressão gênica de TGF-β e IL-10 pelas DCs incubadas com CTX, CA, CB na presença ou não de LPS mostraram que a CTX e, em menor intensidade, a CB induzem aumento dessas citocinas pelas DCs incubadas somente com as toxinas ou juntamente com LPS. Além disso, foi observada maior produção de prostaglandina E2 e lipoxina A4 pelas DCs incubadas com CTX ou CB, e em menor intensidade com CA, na presença ou não de LPS. O ensaio de proliferação de linfócitos CD3+ cocultivados com DCs previamente incubadas com CTX ou CB na presença ou não de LPS mostrou que a CTX e CB em associação com LPS inibiram a proliferação celular quando comparada à induzida somente pelo LPS. Estes resultados demonstraram que a CTX e CB, mas não CA, exercem efeito modulador sobre a atividade funcional das DCs e sugerem a participação dos mediadores antiinflamatórios nesse processo.

Filogenia da sensibilidade da acetilcolinesterase cerebral de peixes ao metil-paraoxon como um possível marcador ambiental / Phylogeny of the sensitivity of fish brain acetylcholinesterase to methyl-paraoxon as a possible marker environmental

Todos os animais com células nervosas e musculares possuem a enzima acetilcolinesterase (EC 3.1.1.7, AChE) e essa seqüência de aminoácidos está presente em muitas outras proteínas, com ou sem atividade catalítica, fato que permite que essa proteína seja utilizada em estudos de filogenia e de evolução. A grande diferença de afinidade entre substratos e inibidores é utilizada como um biomarcador para estudos genéticos de vários grupos de animais, especialmente insetos. Peixes possuem uma grande diferença na sensibilidade da AChE ao metil-paraoxon (MP) em relação aos animais terrestres e essa diferença pode estar relacionada à evolução. As constantes cinéticas de inibição (CCI) para o mecanismo de inibição progressivamente irreversível da AChE cerebral ao MP foram determinadas em duas fontes de AChE como um modelo para avaliação do potencial de uso das CCI como biomarcador para estudos evolucionários e filogenéticos. As CCI da AChE cerebral de seis exemplares de tainha (Mugil liza), coletados em duas lagoas da costa do Estado do Rio de Janeiro (Araruama e Saquarema), em tempos diferentes (2005 e 2007, respectivamente), foram determinadas em dois laboratórios distintos (CESTEH - FIOCRUZ e Dept. Bioquímica - UERJ). As CCI, medidas separadamente em cada exemplar, indicaram que essas constantes são preservadas em todos os exemplares de uma mesma espécie e que a metodologia empregada pode ser conduzida em laboratórios distintos sem grandes variações. A AChE cerebral de tainha foi tomada como um exemplo de enzima menos sensível (IC50 = 2118nM) e a de galinha comercial (Gallus gallus domesticus) como um exemplo de uma enzima muito mais sensível ao MP (IC50 = 26nM)...

The enzyme acetylcholinesterase (EC 3.1.1.7, AChE) is present in all animalswith neurons and muscle cells, and many other proteins have the same sequence, with or without catalytic activity, allowing them to be used for phylogenetic and evolutionary studies. The great difference between substrates and inhibitor affinities makes it useful as a biomarker for genetic studies of various animals groups, especially insects. There are great differences among fish in AChE sensitivity to methyl-paraoxon (MP) in relation to terrestrial animals, and these differences can be related to evolution. The inhibition kinetic constants (IKC) for progressive irreversible inhibition of brain AChE with MP were determined in these two AChE sources as models for evaluating IKC as potential biomarkers in evolutionary and phylogenetic studies, especially among fish. IKC of brain AChE from six specimens of Mugil liza, a very common coastal fish, was collected from two of Brazil's lagoons in Rio de Janeiro State during 2005 at Araruama and 2007 at Saquarema. First samples were assayed at CESTEH - Fundação Oswaldo Cruz and the latter at Dept. Bioquímica - Universidade do Estado do Rio de Janeiro. The IKC was measured separately for each fish showing that these constants weremaintained for all animals of the same species and that this methodology can beused in different laboratories without variations. The cerebral AChE of tainha was used as an example of a less sensitive enzyme (Concentration which inhibits 50% of enzyme activity after 30 minutes of incubation, or IC50, = 2118nM). The commercial hen (Gallus gallus domesticus) was used an example of a very highly sensitiveenzyme to MP (IC50 = 26nM)...