RESUMO
Multiple sclerosis (MS) is an autoimmune debilitating disease of the central nervous system caused by a mosaic of interactions between genetic predisposition and environmental factors. The pathological hallmarks of MS are chronic inflammation, demyelination, and neurodegeneration. Oxidative stress, a state of imbalance between the production of reactive species and antioxidant defense mechanisms, is considered one of the key contributors in the pathophysiology of MS. This review is a comprehensive overview of the cellular and molecular mechanisms by which oxidant species contribute to the initiation and progression of MS including mitochondrial dysfunction, disruption of various signaling pathways, and autoimmune response activation. The detrimental effects of oxidative stress on neurons, oligodendrocytes, and astrocytes, as well as the role of oxidants in promoting and perpetuating inflammation, demyelination, and axonal damage, are discussed. Finally, this review also points out the therapeutic potential of various synthetic antioxidants that must be evaluated in clinical trials in patients with MS.
Assuntos
Esclerose Múltipla , Humanos , Esclerose Múltipla/genética , Esclerose Múltipla/tratamento farmacológico , Estresse Oxidativo/fisiologia , Antioxidantes/uso terapêutico , Sistema Nervoso Central/metabolismo , Inflamação/metabolismoRESUMO
Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins released to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen associated with several animal and human diseases, that produces many exotoxins, including the phospholipase C (CpPLC), the main virulence factor in gas gangrene. During this disease, CpPLC generates the formation of neutrophil/platelet aggregates within the vasculature, favoring an anaerobic environment for C. perfringens growth. This work demonstrates that CpPLC induces NETosis in human neutrophils. Antibodies against CpPLC completely abrogate the NETosis-inducing activity of recombinant CpPLC and C. perfringens secretome. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathways, as well as the production of reactive oxygen species (ROS) by the metabolism of arachidonic acid. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that could be relevant in evading NETs. We suggested that in gas gangrene this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis and then escapes and spreads to deeper tissues. Understanding the role of NETs in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.
Assuntos
Armadilhas Extracelulares , Gangrena Gasosa , Animais , Humanos , Armadilhas Extracelulares/metabolismo , Neutrófilos , Clostridium perfringens , Gangrena Gasosa/metabolismo , Gangrena Gasosa/patologia , Proteômica , Fosfolipases Tipo C/metabolismoRESUMO
Bacterial phospholipases and sphingomyelinases are lipolytic esterases that are structurally and evolutionarily heterogeneous. These enzymes play crucial roles as virulence factors in several human and animal infectious diseases. Some bacterial phospholipases C (PLCs) have both phosphatidylcholinesterase and sphingomyelinase C activities. Among them, Listeria monocytogenes PlcB, Clostridium perfringens PLC, and Pseudomonas aeruginosa PlcH are the most deeply understood. In silico predictions of substrates docking with these three bacterial enzymes provide evidence that they interact with different substrates at the same active site. This review discusses structural aspects, substrate specificity, and the mechanism of action of those bacterial enzymes on target cells and animal infection models to shed light on their roles in pathogenesis.
Assuntos
Esfingomielina Fosfodiesterase/metabolismo , Esfingomielina Fosfodiesterase/fisiologia , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/fisiologia , Animais , Clostridium perfringens/enzimologia , Clostridium perfringens/patogenicidade , Humanos , Listeria monocytogenes/enzimologia , Listeria monocytogenes/patogenicidade , Fosfolipases , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Fosfolipases Tipo C/genéticaRESUMO
SARS-CoV-2 variants of concern show reduced neutralization by vaccine-induced and therapeutic monoclonal antibodies; therefore, treatment alternatives are needed. We tested therapeutic equine polyclonal antibodies (pAbs) that are being assessed in clinical trials in Costa Rica against five globally circulating variants of concern: alpha, beta, epsilon, gamma and delta, using plaque reduction neutralization assays. We show that equine pAbs efficiently neutralize the variants of concern, with inhibitory concentrations in the range of 0.146-1.078 µg/mL, which correspond to extremely low concentrations when compared to pAbs doses used in clinical trials. Equine pAbs are an effective, broad coverage, low-cost and a scalable COVID-19 treatment.
RESUMO
Despite vaccines are the main strategy to control the ongoing global COVID-19 pandemic, their effectiveness could not be enough for individuals with immunosuppression. In these cases, as well as in patients with moderate/severe COVID-19, passive immunization with anti-SARS-CoV-2 immunoglobulins could be a therapeutic alternative. We used caprylic acid precipitation to prepare a pilot-scale batch of anti-SARS-CoV-2 intravenous immunoglobulins (IVIg) from plasma of donors immunized with the BNT162b2 (Pfizer-BioNTech) anti-COVID-19 vaccine (VP-IVIg) and compared their in vitro efficacy and safety with those of a similar formulation produced from plasma of COVID-19 convalescent donors (CP-IVIg). Both formulations showed immunological, physicochemical, biochemical, and microbiological characteristics that meet the specifications of IVIg formulations. Moreover, the concentration of anti-RBD and ACE2-RBD neutralizing antibodies was higher in VP-IVIg than in CP-IVIg. In concordance, plaque reduction neutralization tests showed inhibitory concentrations of 0.03-0.09 g/L in VP-IVIg and of 0.06-0.13 in CP-IVIg. Thus, VP-IVIg has in vitro efficacy and safety profiles that justify their evaluation as therapeutic alternative for clinical cases of COVID-19. Precipitation with caprylic acid could be a simple, feasible, and affordable alternative to produce formulations of anti-SARS-CoV-2 IVIg to be used therapeutically or prophylactically to confront the COVID-19 pandemic in middle and low-income countries.
RESUMO
Resumen Objetivo: Efectuar un análisis de los logros en Costa Rica en cuanto al problema de los envenenamientos por mordeduras de serpientes, y señalar tareas pendientes para reducir aún más el impacto de esta patología. Metodología: Se efectuó una revisión de bibliografía relacionada con el estudio del envenenamiento ofídico en Costa Rica y con los avances efectuados en el tema, en el país. Paralelamente, se identificaron aspectos que requieren atención en el manejo de esta enfermedad tropical desatendida. Conclusiones: Desde las primeras décadas del siglo XX, se han realizado avances significativos en la comprensión y manejo del problema de los envenenamientos por mordedura de serpiente en Costa Rica. Se ha trabajado desde una visión integral que incluye: investigación científico-tecnológica, producción y distribución de antivenenos, esfuerzos en prevención, capacitación de las personas profesionales de la salud en el diagnóstico y tratamiento, y docencia de grado y posgrado en el tema. El país ha asumido un papel de liderazgo a nivel internacional, tanto en el plano académico como en la provisión de antivenenos a muchos países. No obstante, aún quedan aspectos del problema que requieren nuevos esfuerzos en nuestro medio, sobre todo en lo referente a la atención de las consecuencias biomédicas, psicológicas, sociales y económicas que sufren las personas afectadas.
Abstract Aim: To analyze the achievements made in Costa Rica in confronting the problem of snakebite envenomings, and to identify pending tasks to further reduce the impact of this pathology. Methods: A review of the literature on snakebite envenomings in Costa Rica was carried out, identifying the main achievements reached in the country. In parallel, issues that require renewed attention in the management of this neglected tropical disease were identified. Conclusions: Since the first decades of the 20th century, significant advances have been made in Costa Rica for understanding and confronting the problem of snakebite envenomings. An integrative perspective has been implemented, which includes scientific and technological research, production and distribution of antivenoms, prevention campaigns, training of health professionals in the diagnosis and treatment of envenomings, and teaching at graduate and undergraduate levels. Costa Rica has had a leading international role in this topic at the academic level, and by providing antivenoms to many countries. Nevertheless, there are issues that require further efforts, especially regarding the attention to the biomedical, psychological, social and economic consequences suffered by people affected by snakebites.
Assuntos
Mordeduras de Serpentes/história , Antivenenos , Costa RicaRESUMO
This study describes the basic epidemiological features of snakebites in El Salvador for the period 2014-2019 on the basis of data provided by the national system of information on morbidity and mortality (Sistema de Morbi-Mortalidad via Web, SIMMOW) of the Ministry of Health of El Salvador. The total number of cases per year ranged from 161 (2017) to 215 (2016). Incidences ranged from 2.52 cases to 3.38 cases per 100,000 population per year, corresponding to the years 2017 and 2016, respectively. Five deaths were recorded in the six-year period, four in 2015 and one in 2016, for a case fatality rate of 0.44%. Snakebites peaked during the rainy season (May to November) and mostly affected people in the age groups of 10-30 years. The male/female ratio was 1.59. The Departments (local political units) showing the highest number of cases were Santa Ana, Libertad, Chalatenango, Sonsonate, and La Unión. Most cases were attended at departmental and regional hospitals (second level of attention). The incidence and mortality due to snakebite envenoming in El Salvador are the lowest reported for Central America. This may be related to the fact that Bothrops asper, the medically most important snake species in the region, is not distributed in El Salvador, where the rattlesnake Crotalus simus predominates.
Assuntos
Mordeduras de Serpentes/epidemiologia , Animais , Bothrops , Crotalus , El Salvador/epidemiologia , Feminino , Humanos , Incidência , MasculinoRESUMO
Considering the etiology of multiple sclerosis (MS) is still unknown, experimental models resembling specific aspects of this immune-mediated demyelinating human disease have been developed to increase the understanding of processes related to pathogenesis, disease evolution, evaluation of therapeutic interventions, and demyelination and remyelination mechanisms. Based on the nature of the investigation, biological models may include in vitro, in vivo, and ex vivo assessments. Even though these approaches have disclosed valuable information, every disease animal model has limitations and can only replicate specific features of MS. In vitro and ex vivo models generally do not reflect what occurs in the organism, and in vivo animal models are more likely used; nevertheless, they are able to reproduce only certain stages of the disease. In vivo MS disease animal models in mammals include: experimental autoimmune encephalomyelitis, viral encephalomyelitis, and induced demyelination. This review examines and describes the most common biological disease animal models for the study of MS, their specific characteristics and limitations.
Assuntos
Modelos Animais de Doenças , Esclerose Múltipla , Animais , Células Cultivadas , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/fisiopatologia , Humanos , Esclerose Múltipla/patologia , Esclerose Múltipla/fisiopatologiaRESUMO
Gas gangrene, or clostridial myonecrosis, is usually caused by Clostridium perfringens and may occur spontaneously in association with diabetes mellitus, peripheral vascular disease, or some malignancies but more often after contamination of a deep surgical or traumatic lesion. If not controlled, clostridial myonecrosis results in multiorgan failure, shock, and death, but very little is known about the muscle regeneration process that follows myonecrosis when the infection is controlled. In this study, we characterized the muscle regeneration process after myonecrosis caused in a murine experimental infection with a sublethal inoculum of C. perfringens vegetative cells. The results show that myonecrosis occurs concomitantly with significant vascular injury, which limits the migration of inflammatory cells. A significant increase in cytokines that promote inflammation explains the presence of an inflammatory infiltrate; however, impaired interferon gamma (IFN-γ) expression, a reduced number of M1 macrophages, deficient phagocytic activity, and a prolongation of the permanence of inflammatory cells lead to deficient muscle regeneration. The expression of transforming growth factor ß1 (TGF-ß1) agrees with the consequent accumulation of collagen in the muscle, i.e., fibrosis observed 30 days after infection. These results provide new information on the pathogenesis of gas gangrene caused by C. perfringens, shed light on the basis of the deficient muscle regenerative activity, and may open new perspectives for the development of novel therapies for patients suffering from this disease.
Assuntos
Clostridium perfringens/patogenicidade , Gangrena Gasosa/fisiopatologia , Músculo Esquelético/fisiologia , Regeneração , Animais , Citocinas/metabolismo , Fibrose , Gangrena Gasosa/etiologia , Gangrena Gasosa/imunologia , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Necrose , Infiltração de NeutrófilosRESUMO
BACKGROUND: Bothrops, Crotalus and Lachesis represent the most medically relevant genera of pitvipers in Central and South America. Similarity in venom phenotype and physiopathological profile of envenomings caused by the four nominal Lachesis species led us to hypothesize that an antivenom prepared against venom from any of them may exhibit paraspecificity against all the other congeneric taxa. METHODS: To assess this hypothesis, in this work we have applied antivenomics and immunochemical methods to investigate the immunoreactivity of three monovalent antivenoms and two polyvalent antivenoms towards the venoms from different geographic populations of three different Lachesis species. The ability of the antivenoms to neutralize the proteolytic, hemorrhagic, coagulant, and lethal activities of the seven Lachesis venoms was also investigated. RESULTS: A conspicuous pattern of immunorecognition and cross-neutralization for all effects was evident by the polyspecific antivenoms, indicating large immunoreactive epitope conservation across the genus during more than 10 million years since the Central and South American bushmasters diverged. CONCLUSIONS: Despite the broad geographic distribution of Lachesis, antivenoms against venoms of different species are effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that they can be used equivalently for the clinical treatment of any lachesic envenoming. GENERAL SIGNIFICANCE: This study demonstrates that antivenoms raised against venom of different Lachesis species are indistinctly effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that antivenoms against conspecific venoms may be used equivalently for the clinical treatment of envenomings caused by any bushmaster species.
Assuntos
Antivenenos/farmacologia , Reações Cruzadas , Venenos de Crotalídeos/antagonistas & inibidores , Fatores Imunológicos/farmacologia , Testes de NeutralizaçãoRESUMO
EchiTAb-plus-ICP is an antivenom prepared from plasma of horses hyperimmunized with the venoms of the carpet viper (Echis ocellatus), the puff adder (Bitis arietans) and the black-necked spitting cobra (Naja nigricollis). Therefore, the use of this antivenom has been limited to Western Africa. In order to expand the neutralization scope of EchiTAb-plus-ICP, we supplemented the immunogenic mixture with the venoms of B. arietans, the black mamba (Dendroaspis polylepis), the Mozambique spitting cobra (Naja mossambica), the snouted cobra (N. annulifera), and the rinkhals (Hemachatus haemachatus) from Swaziland. The ability of the expanded-scope antivenom, hereby named EchiTAb + ICP, to neutralize the venoms of B. arietans, D. polylepis, N. mossambica and H. haemachatus was similar to those of FAV Afrique and the SVA African antivenoms. In comparison to the SAIMR antivenom, the expanded-scope EchiTAb + ICP had lower ability to neutralize the venom of B. arietans, but similar ability to neutralize the venoms of D. polylepis, N. mossambica and H. haemachatus. Owing to its low protein concentration, the expanded-scope EchiTAb + ICP had lower ability to neutralize the venom of N. annulifera than FAV Afrique and the SAIMR antivenoms. However, when formulated at a protein concentration as high as FAV Afrique and SAIMR antivenoms, the expanded-scope EchiTAb + ICP showed similar capacity to neutralize this poorly immunogenic venom. Our results encourage the transition to the new EchiTAb + ICP antivenom, with an expanded neutralization scope that includes venoms of some of the most medically important elapids from Southern Africa. Clinical trials are required to determine the minimum effective-safe dose of the new EchiTAb + ICP for each type of envenomation.
Assuntos
Antivenenos/farmacologia , Venenos Elapídicos/antagonistas & inibidores , África Austral , Animais , Antivenenos/química , CavalosRESUMO
A new whole IgG, freeze-dried, polyspecific antivenom was prepared from the plasma of horses immunized with the venoms of the snakes Daboia russelii, Echis carinatus, Hypnale hypnale, and Naja naja from Sri Lanka. The preclinical neutralizing ability of this antivenom against several toxic and enzymatic activities of these four venoms was analyzed, and compared with that of a batch of VINS antivenom (India) being currently used in Sri Lanka. The activities tested were: lethality, hemorrhagic, in vitro coagulant, proteinase and phospholipase A2. Both antivenoms neutralized, to a different extent, these activities of the venom of D. russelii, E. carinatus, and N. naja. In general, the polyspecific Sri Lankan antivenom was more effective than the Indian antivenom in the neutralization of the venoms of D. russelii and E. carinatus, whereas the Indian antivenom showed a higher efficacy against the venom of N. naja. Regarding H. hypnale, the new Sri Lankan antivenom was effective in the neutralization of all activities tested, whereas the Indian antivenom neutralized lethality but not hemorrhagic, coagulant, proteinase and PLA2 activities, in agreement with the fact that this venom is not included in the immunization mixture for this antivenom. Results suggest that the new polyspecific Sri Lankan antivenom has a satisfactory preclinical neutralizing profile and compares favorably with the Indian antivenom. This is ready to be tested in a clinical trial to evaluate its efficacy and safety in human victims of snakebite envenomings by D. russelii, E. carinatus and H. hypnale in Sri Lanka.
Assuntos
Antivenenos/uso terapêutico , Animais , Antivenenos/imunologia , Antivenenos/farmacologia , Humanos , Sri LankaRESUMO
The application of proteomic tools to the study of snake venoms has led to an impressive growth in the knowledge about their composition (venomics), immunogenicity (antivenomics), and toxicity (toxicovenomics). About one-third of all venomic studies have focused on elapid species, especially those of the Old World. The New World elapids, represented by coral snakes, have been less studied. In recent years, however, a number of venomic studies on Micrurus species from North, Central, and South America have been conducted. An overview of these studies is presented, highlighting the emergence of some patterns and trends concerning their compositional, functional, and immunological characteristics. Results gathered to date, encompassing 18 out of the approximately 85 species of Micrurus, reveal a dichotomy of venom phenotypes regarding the relative abundance of the omnipresent phospholipases A2 (PLA2) and 'three-finger' toxins (3FTx): a group of species express a PLA2-predominant venom composition, while others display a 3FTx-predominant compositional pattern. These two divergent toxin expression phenotypes appear to be related to phylogenetic positions and geographical distributions along a North-South axis in the Americas, but further studies encompassing a higher number of species are needed to assess these hypotheses. The two contrasting phenotypes also show correlations with some toxic functionalities, complexity in the diversity of proteoforms, and immunological cross-recognition patterns. The biological significance for the emergence of a dichotomy of venom compositions within Micrurus, in some cases observed even among sympatric species that inhabit relatively small geographic areas, represents a puzzling and challenging area of research which warrants further studies.
Assuntos
Venenos Elapídicos/química , Elapidae , Evolução Química , Proteômica , Animais , Venenos Elapídicos/metabolismo , Eletroforese em Gel de PoliacrilamidaRESUMO
Bacterial sphingomyelinases and phospholipases are a heterogeneous group of esterases which are usually surface associated or secreted by a wide variety of Gram-positive and Gram-negative bacteria. These enzymes hydrolyze sphingomyelin and glycerophospholipids, respectively, generating products identical to the ones produced by eukaryotic enzymes which play crucial roles in distinct physiological processes, including membrane dynamics, cellular signaling, migration, growth, and death. Several bacterial sphingomyelinases and phospholipases are essential for virulence of extracellular, facultative, or obligate intracellular pathogens, as these enzymes contribute to phagosomal escape or phagosomal maturation avoidance, favoring tissue colonization, infection establishment and progression, or immune response evasion. This work presents a classification proposal for bacterial sphingomyelinases and phospholipases that considers not only their enzymatic activities but also their structural aspects. An overview of the main physiopathological activities is provided for each enzyme type, as are examples in which inactivation of a sphingomyelinase- or a phospholipase-encoding gene impairs the virulence of a pathogen. The identification of sphingomyelinases and phospholipases important for bacterial pathogenesis and the development of inhibitors for these enzymes could generate candidate vaccines and therapeutic agents, which will diminish the impacts of the associated human and animal diseases.
Assuntos
Bactérias/patogenicidade , Fungos/patogenicidade , Fosfolipase D/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Fosfolipases Tipo C/metabolismo , Bactérias/enzimologia , Firmicutes/enzimologia , Firmicutes/patogenicidade , Fungos/enzimologia , Proteobactérias/enzimologia , Proteobactérias/patogenicidade , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Fatores de Virulência/metabolismoRESUMO
The venom of Micrurus lemniscatus, a coral snake of wide geographical distribution in South America, was fractionated by reverse-phase HPLC and the fractions screened for phospholipase A2 (PLA2) activity. The major component of the venom, a PLA2, here referred to as 'Lemnitoxin', was isolated and characterized biochemically and toxicologically. It induces myotoxicity upon intramuscular or intravenous injection into mice. The amino acid residues Arg15, Ala100, Asn108, and a hydrophobic residue at position 109, which are characteristic of myotoxic class I phospholipases A2, are present in Lemnitoxin. This PLA2 is antigenically related to M. nigrocinctus nigroxin, Notechis scutatus notexin, Pseudechis australis mulgotoxin, and Pseudonaja textilis textilotoxin, as demonstrated with monoclonal and polyclonal antibodies. Lemnitoxin is highly selective in its targeting of cells, being cytotoxic for differentiated myotubes in vitro and muscle fibers in vivo, but not for undifferentiated myoblasts or endothelial cells. Lemnitoxin is not lethal after intravenous injection at doses up to 2µg/g in mice, evidencing its lack of significant neurotoxicity. Lemnitoxin displays anticoagulant effect on human plasma and proinflammatory activity also, as it induces paw edema and mast cell degranulation. Thus, the results of this work demonstrate that Lemnitoxin is a potent myotoxic and proinflammatory class I PLA2.
Assuntos
Edema/induzido quimicamente , Venenos Elapídicos/enzimologia , Venenos Elapídicos/toxicidade , Elapidae/metabolismo , Mediadores da Inflamação/toxicidade , Doenças Musculares/induzido quimicamente , Fosfolipases A2/toxicidade , Animais , Coagulação Sanguínea/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Creatina Quinase/sangue , Relação Dose-Resposta a Droga , Edema/enzimologia , Venenos Elapídicos/isolamento & purificação , Venenos Elapídicos/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Mediadores da Inflamação/isolamento & purificação , Mediadores da Inflamação/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Doenças Musculares/enzimologia , Doenças Musculares/patologia , Fosfolipases A2/isolamento & purificação , Fosfolipases A2/metabolismo , Ratos Wistar , Análise de Sequência de Proteína , Fatores de TempoRESUMO
Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis.
Assuntos
Toxinas Bacterianas/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , MAP Quinase Quinase 1/metabolismo , Melanoma/patologia , Músculo Esquelético/patologia , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fosfolipases Tipo C/farmacologia , Animais , Western Blotting , Células CHO , Proliferação de Células/efeitos dos fármacos , Cricetulus , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, plays a key role in the pathogenesis of gas gangrene. CpPLC may lead to cell lysis at concentrations that cause extensive degradation of plasma membrane phospholipids. However, at sublytic concentrations it induces cytotoxicity without inducing evident membrane damage. The results of this work demonstrate that CpPLC becomes internalized in cells by a dynamin-dependent mechanism and in a time progressive process: first, CpPLC colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. Lysosomal damage in the target cells is evident 9 h after CpPLC exposure. Our previous work demonstrated that CpPLCinduces ERK1/2 activation, which is involved in its cytotoxic effect. In this work we found that cholesterol sequestration, dynamin inhibition, as well as inhibition of actin polymerization, prevent CpPLC internalization and ERK1/2 activation, involving endocytosis in the signalling events required for CpPLC cytotoxic effect at sublytic concentrations. These results provide new insights about the mode of action of this bacterial phospholipase C, previously considered to act only locally on cell membrane.
Assuntos
Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/toxicidade , Endocitose , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/toxicidade , Linhagem Celular , HumanosRESUMO
We report the proteomic analysis of the Atlantic bushmaster, Lachesis muta rhombeata, from Brazil. Along with previous characterization of the venom proteomes of L. stenophrys (Costa Rica), L. melanocephala (Costa Rica), L. acrochorda (Colombia), and L. muta muta (Bolivia), the present study provides the first overview of the composition and distribution of venom proteins across this wide-ranging genus, and highlights the remarkable similar compositional and pharmacological profiles across Lachesis venoms. The paraspecificity of two antivenoms, produced at Instituto Vital Brazil (Brazil) and Instituto Clodomiro Picado (Costa Rica) using different conspecific taxa in the immunization mixtures, was assessed using genus-wide comparative antivenomics. This study confirms that the proteomic similarity among Lachesis sp. venoms is mirrored in their high immunological conservation across the genus. The clinical and therapeutic consequences of genus-wide venomics and antivenomics investigations of Lachesis venoms are discussed. BIOLOGICAL SIGNIFICANCE: The proteomics characterization of L. m. rhombeata venom completes the overview of Lachesis venom proteomes and confirms the remarkable toxin profile conservation across the five clades of this wide-ranging genus. Genus-wide antivenomics showed that two antivenoms, produced against L. stenophrys or L. m. rhombeata, exhibit paraspecificity towards all other congeneric venoms. Our venomics study shows that, despite the broad geographic distribution of the genus, monospecific antivenoms may achieve clinical coverage for any Lachesis sp. envenoming.
Assuntos
Antivenenos , Venenos de Crotalídeos , Proteoma , Viperidae , Animais , Antivenenos/química , Antivenenos/genética , Antivenenos/imunologia , Venenos de Crotalídeos/química , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/imunologia , Cavalos , Proteoma/química , Proteoma/genética , Proteoma/imunologia , Especificidade da Espécie , Viperidae/genética , Viperidae/imunologiaRESUMO
We report the proteomic analysis of ontogenetic changes in venom composition of the Central American bushmaster, Lachesis stenophrys, and the characterization of the venom proteomes of two congeneric pitvipers, Lachesis melanocephala (black-headed bushmaster) and Lachesis acrochorda (Chochoan bushmaster). Along with the previous characterization of the venom proteome of Lachesis muta muta (from Bolivia), our present outcome enables a comparative overview of the composition and distribution of the toxic proteins across genus Lachesis. Comparative venomics revealed the close kinship of Central American L. stenophrys and L. melanocephala and support the elevation of L. acrochorda to species status. Major ontogenetic changes in the toxin composition of L. stenophrys venom involves quantitative changes in the concentration of vasoactive peptides and serine proteinases, which steadily decrease from birth to adulthood, and age-dependent de novo biosynthesis of Gal-lectin and snake venom metalloproteinases (SVMPs). The net result is a shift from a bradykinin-potentiating and C-type natriuretic peptide (BPP/C-NP)-rich and serine proteinase-rich venom in newborns and 2-years-old juveniles to a (PI>PIII) SVMP-rich venom in adults. Notwithstanding minor qualitative and quantitative differences, the venom arsenals of L. melanocephala and L. acrochorda are broadly similar between themselves and also closely mirror those of adult L. stenophrys and L. muta venoms. The high conservation of the overall composition of Central and South American bushmaster venoms provides the ground for rationalizing the "Lachesis syndrome", characterized by vagal syntomatology, sensorial disorders, hematologic, and cardiovascular manifestations, documented in envenomings by different species of this wide-ranging genus. This finding let us predict that monospecific Lachesic antivenoms may exhibit paraspecificity against all congeneric species.