Editorial: Novel immunotherapies against envenomings by snakes and other venomous animals

Editorial on the Research TopicUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Genetic and toxinological divergence among populations of Tityus trivittatus Kraepelin, 1898 (Scorpiones: Buthidae) inhabiting Paraguay and Argentina

Autor de correspondencia: [email protected] by scorpions in genus Tityus is a public health problem in Tropical America. One of the most medically significant species is Tityus trivittatus, which is known to occur from southwest Brazil to central-northern and eastern Argentina. In this work, we studied the lethality, composition, antigenicity, and enzymatic activity of venom from a T. trivittatus population found further north in urban areas of eastern Paraguay, where it has caused serious envenomation of children. Our results indicate that the population is of medical importance as it produces a potently toxic venom with an LD50 around 1.19 mg/kg. Venom neutralization in preliminary mouse bioassays was complete when using Brazilian anti-T. serrulatus antivenom but only partial when using Argentinean anti-T. trivittatus antivenom. Venom competitive solid-phase enzyme immunoassays and immunoblotting from Argentinean and Paraguayan T. trivittatus populations indicated that antigenic differences exist across the species range. SDS-PAGE showed variations in type and relative amounts of venom proteins between T. trivitattus samples from Argentina and Paraguay. MALDI-TOF mass spectrometry indicated that while some sodium channel toxins are shared, including β-toxin Tt1g, others are population-specific. Proteolytic activity by zymography and peptide identification through nESI-MS/MS also point out that population-specific proteases may exist in T. trivitattus, which are postulated to be involved in the envenoming process. A time-calibrated molecular phylogeny of mitochondrial COI sequences revealed a significant (8.14%) genetic differentiation between the Argentinean and Paraguayan populations, which appeared to have diverged between the mid Miocene and early Pliocene. Altogether, toxinological and genetic evidence indicate that T. trivitattus populations from Paraguay and Argentina correspond to distinct, unique cryptic species, and suggest that further venom and taxonomic diversity exists in synanthropic southern South American Tityus than previously thought.Consejo Nacional de Ciencia y TecnologíaPrograma Paraguayo para el Desarrollo de la Ciencia y Tecnología. Programa de repatriación y radicación de investigadores del exteriorPrograma Paraguayo para el Desarrollo de la Ciencia y Tecnología. Programa Nacional de Incentivo a los Investigadore

Mesenchymal Stromal Cell-Based Therapies as Promising Treatments for Muscle Regeneration After Snakebite Envenoming

Snakebite envenoming is a global neglected disease with an incidence of up to 2.7 million new cases every year. Although antivenoms are so-far the most effective treatment to reverse the acute systemic effects induced by snakebite envenoming, they have a limited therapeutic potential, being unable to completely neutralize the local venom effects. Local damage, such as dermonecrosis and myonecrosis, can lead to permanent sequelae with physical, social, and psychological implications. The strong inflammatory process induced by snake venoms is associated with poor tissue regeneration, in particular the lack of or reduced skeletal muscle regeneration. Mesenchymal stromal cells (MSCs)-based therapies have shown both anti-inflammatory and pro-regenerative properties. We postulate that using allogeneic MSCs or their cell-free products can induce skeletal muscle regeneration in snakebite victims, improving all the three steps of the skeletal muscle regeneration process, mainly by anti-inflammatory activity, paracrine effects, neovascularization induction, and inhibition of tissue damage, instrumental for microenvironment remodeling and regeneration. Since snakebite envenoming occurs mainly in areas with poor healthcare, we enlist the principles and potential of MSCs-based therapies and discuss regulatory issues, good manufacturing practices, transportation, storage, and related-procedures that could allow the administration of these therapies, looking forward to a safe and cost-effective treatment for a so far unsolved and neglected health problem.The authors are supported by the University Pablo de Olavide (Sevilla), the University Miguel Hernández (Elche, Alicante), National University Toribio Rodriguez de Mendoza (Chachapoyas, Peru) Grants: Contrato N° 09-2019-FONDECYT-BM-INC.INV to JRT, JDRF 2-SRA-2019-837-S-B and AVI-GVA COVID-19-68 to BS, Fundación Andaluza de I+D and Al-Andalus Biopharma Project (FAID-2018-1). The authors CC-O, CG-D, and TCSA were supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq) (Process: 406163/2018-9), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil - CAPES (Program COFECUB Process: 88881.191812/2018-01) and by Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil (FAPEMIG)

Evaluation of mesenchymal cells and dapsone for the treatment of dermonecrotic wounds caused by Loxosceles laeta venom in rabbits

This study aimed to evaluate the efficacy of mesenchymal stem cells (MSC), alone or associated with dapsone (DAP), in treating dermonecrotic wounds caused by Loxosceles laeta venom. Twenty-five male rabbits were distributed into five groups. Negative control received ultrapure water (C-), whilst all other groups were injected with 20 μg of L. laeta venom. After 4 hours, each group received one of the following treatments: PBS (C+), DAP, MSC, and DAP+MSC. Animals were evaluated daily and photographic records made for analysis of wound area. Twelve days after, animals were euthanized and skin samples removed for histological analysis. We observed that DAP showed the best percentage of wound contraction at day 3. In the treatments using MSCs, a negative value of wound contraction was observed for the isolated MSCs, as well as a lower contraction value for the association of the MSC + DAP when compared to PBS, probably, by the increase in initial inflammation after the application of stem cells, due to the fact that MSCs secrete a broad spectrum of bioactive molecules such as cytokines and growth factors that favor regeneration. Histologically, it was observed that animals of C+ showed extensive areas of necrosis, ulcers, neutrophilic infiltrate, and mineralization. Collagen deposition showed increase in MSC+DAP treatment, however vascularization remained unchanged. This is the first report using MSC and MSC+DAP as a treatment for cutaneous loxoscelism and more studies are needed to determine its use as an alternative therapy for dermonecrotic lesions caused by Loxosceles spider. Key words: loxoscelism; MSC; stem cells; spider venom; wound repair

Screening of expression libraries using ELISA : identification of immunogenic proteins from Tityus bahiensis and Tityus serrulatus venom.

The present report describes the use of ELISA with cDNA expression libraries in the identification of immunogenic proteins. The methodology described was applied using libraries constructed with mRNA isolated from Tityus serrulatus and Tityus bahiensis venom glands. In addition we describe for the first time the sequence of a neurotoxin from Tityus bahiensis venom gland named TbTx5 whose amino acid sequencing showed 93% similarity with the Tityus bahiensis TbTx IV-5 neurotoxin. The methodology described can be used for the generation of an immunogenic bank in order to contribute to genome and proteome projects

Acidic Phospholipase A2-Peptide Derivative Modulates Oxidative Status and Microstructural Reorganization of Scar Tissue after Cutaneous Injury

From in vitro and in vivo models, the proliferative and healing potential of an acidic phospholipase A2 (LAPLA2) from Lachesis muta venom was investigated. The LAPLA2 proliferative activity was evaluated on fibroblasts and keratinocytes cultured, and the antioxidant and regenerative potential of LAPLA2 was analyzed in a murine model. The animal study consisted of four groups: C (negative control): 0.9% NaCl; SS (positive control): 1% silver sulfadiazine; L1 group: 0.5% LAPLA2; and L2 group: 0.25% LAPLA2. Wounds were topically treated daily for 12 days, and scar tissue samples were collected every 4 days. In vitro, LAPLA2 stimulated marked time-dependent cell proliferation. In vivo, it increased the antioxidant activity of superoxide dismutase (SOD) and catalase (CAT) and decreased malondialdehyde (MDA) and carbonyl protein (CP) levels in scar tissue treated with LAPLA2 at 0.5%. This peptide was effective in stimulating cellular proliferation, neoangiogenesis, type I and III collagen deposition, and maturation in a time-dependent-way, reducing the time required for wound closure. Our results indicated that LAPLA2 presented a remarkable potential in improving the oxidative status and microstructural reorganization of the scar tissue by stimulation of cellularity, angiogenesis, colagenogenesis, and wound contraction, suggesting that the peptide could be a potential candidate for a new healing drug

In vitro assessment of cytotoxic activities of Lachesis muta muta snake venom.

Envenomation by the bushmaster snake Lachesis muta muta is considered severe, characterized by local effects including necrosis, the main cause of permanent disability. However, cellular mechanisms related to cell death and tissue destruction, triggered by snake venoms, are poorly explored. The purpose of this study was to investigate the cytotoxic effect caused by L. m. muta venom in normal human keratinocytes and to identify the cellular processes involved in in cellulo envenomation. In order to investigate venom effect on different cell types, Alamar Blue assay was performed to quantify levels of cellular metabolism as a readout of cell viability. Apoptosis, necrosis and changes in mitochondrial membrane potential were evaluated by flow cytometry, while induction of autophagy was assessed by expression of GFP-LC3 and analyzed using fluorescence microscopy. The cytotoxic potential of the venom is shown by reduced cell viability in a concentration-dependent manner. It was also observed the sequential appearance of cells undergoing autophagy (by 6 hours), apoptosis and necrosis (12 and 24 hours). Morphologically, incubation with L. m. muta venom led to a significant cellular retraction and formation of cellular aggregates. These results indicate that L. m. muta venom is cytotoxic to normal human keratinocytes and other cell lines, and this toxicity involves the integration of distinct modes of cell death. Autophagy as a cell death mechanism, in addition to apoptosis and necrosis, can help to unravel cellular pathways and mechanisms triggered by the venom. Understanding the mechanisms that underlie cellular damage and tissue destruction will be useful in the development of alternative therapies against snakebites

Immunodetection of the "brown" spider (Loxosceles intermedia) dermonecrotoxin with an scFv-alkaline phosphatase fusion protein

International audienceBites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32-35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39-20000ng/mL and a detection limit of 39ng/mL without any cross-reaction

Use of Phage Display technology in development of canine visceral leishmaniasis vaccine using synthetic peptide trapped in sphingomyelin/cholesterol liposomes

International audienc