Journal of Venomous Animals and Toxins Including Tropical Diseases最新文献

Background: Cancer is one of the leading causes of death worldwide, with incidence rates continuously increasing, thereby posing a major healthcare challenge. Although many oncological drugs fulfill therapeutic requirements, they often show high toxicity due to their limited specificity. To address this problem, there has been a search for natural therapies, including animal venoms that harbor bioactive molecules with therapeutic potential, as well as biological models that facilitate their study. Consequently, three-dimensional culture models, such as spheroids, play a pivotal role in evaluating anticancer molecules, as they can effectively mimic in vivo tumor microenvironments.

Methods: This study aimed to establish the significance of spheroids in identifying venom-derived molecules as potential therapeutic alternatives against cancer, based on a systematic review conducted from 2010 to 2024. Following PRISMA guidelines, a systematic search was conducted in four databases using the terms "Spheroid" and "Venom". Of the 93 articles identified, 16 satisfied the inclusion criteria for this review.

Results: Notably, several bioactive molecules derived from snake, spider, scorpion, and bee venoms were evaluated using various spheroid formation methods. These molecules demonstrated cytotoxic effects that impaired spheroid formation and disrupted invasion and migration processes.

Conclusion: Overall, the findings indicate that the integration of three-dimensional culture models with venom-derived compounds constitutes a promising preclinical strategy for the development of innovative, venom-based therapeutic strategies for cancer treatment.

Background: In the biomedical field, translational science is the process of applying basic scientific knowledge to advance clinical research through the creation of new drugs, devices, medical procedures, preventive measures, and diagnostic kits. The Covid-19 pandemic exposed a shortage of professionals trained in translational research, essential for responding to global demands. To drive advancements, researchers must overcome the 'valley of death', a critical phase in clinical investigation. In response, CEVAP at São Paulo State University (UNESP), Botucatu, Brazil, has developed a strong 'knowledge industry' centered on Translational Science. As part of its research and innovation efforts, CEVAP has developed two biopharmaceuticals, the fibrin sealant and the apilic antivenom, which are currently in the final stage of development. In 2024, CEVAP began the first Brazilian Contract Development and Manufacturing Organization (CDMO) for developing and producing validated and qualified pilot-scale batches to generate clinical trial material.

Case presentation: The implementation of the optional undergraduate course in Translational Science marks a crucial step in strengthening the 'knowledge industry'. The program, developed in collaboration with São Paulo's three public universities (USP, UNESP, and UNICAMP), also involves an international partnership with the University of Oxford's Department of Pediatrics and the Oxford Research Group LATAM. The successful launch of this course underscores its importance in interdisciplinary education and institutional collaboration. By bridging gaps between research and application, the program equips professionals to meet the growing demand for expertise in translational science. Given the project's success, it will transition into a one-year 'Qualification in Translational Science', available to students enrolled in São Paulo state universities.

Conclusion: The preparation of these professionals will be strategic for transforming basic research into products for health, saving lives, and combating future pandemics that will emerge around the world.

Background: Brazilian waters are home to various venomous fish species, each with its unique venom composition. Although common, envenomation cases are largely underreported, leading to a lack of public health policies for prevention and treatment. Some of the most clinically relevant fish in Brazil include the stingray Potamotrygon orbignyi, the toadfish Thalassophryne nattereri, the scorpionfish Scorpaena plumieri, and the catfish Pseudoplatystoma fasciatum and Cathorops spixii.

Methods: We comprehensively searched reports about accidents involving venomous fish in Brazil and compared the toxic activities of some medically relevant species.

Results: From the biochemical and toxicological evaluation, we found that venoms show a hierarchy in the ability to induce local toxic effects in mice, probably related to the venom compound diversity with species-specific toxins. T. nattereri venom presents greater toxicity, causing more severe local responses than that of P. orbignyi, C. spixii, and P. fasciatum, which cause moderate reactions. The S. plumieri venom induced only a moderate level of edema and could not cause nociception or necrosis. These results highlight that envenomation by P. orbigny, C. spixii, and S. plumieri is marked by proteins with intense hemolytic/proteolytic and phospholipase activity. On the other hand, T. nattereri and P. fasciatum offered a broader panel of new toxin families.

Conclusion: Knowledge of fish venom biochemical and toxicological activities is crucial to antivenom therapy development and helps endorse the study of venomous fish and their impact on the public health system.

Background: Bufadienolides are the main secondary metabolites found in the paratoid gland secretions (PGS) of toads of the Bufonidae family. These compounds are considered the main bioactive components of PGS. The aim of this study was to develop and validate the first method for the quantification of total bufadienolides (free and esterified) in samples of paratoid secretions from toads, using the UV-Vis absorption spectrophotometry technique.

Methods: The proposed method was based on the bathochromic shift induced by the reaction of the α-pyrone group of bufadienolides (296 nm) with a 5% (w:v) aqueous solution of sodium hydroxide and detection at 356 nm, after 60 min (time defined based on the evaluation of kinetic assays).

Results: The proposed method showed wide linearity (r = 0.9999), low LOD (1.3 × 10^-4 µg/mL) and LOQ (3.9 × 10^-4 µg/mL), recovery (84%-99%), repeatability (%RSD ≤ 5), reproducibility and robustness (p > 0.05). The total bufadienolide content in PGS extracts from 12 samples of R. diptycha ranged from 478 to 801 mg of EqMB/g of extract, while the R. granulosa sample presented 661 mg of EqMB/g of extract.

Conclusion: The new developed method is innovative, simple, fast, accurate, robust, low cost, and can contribute to future research focused on the quantification of total bufadienolides in samples of toad glandular secretions. In addition to serving as a strategic tool in the selection of work matrices, optimizing time, and minimizing costs.

Background: Deinagkistrodon acutus, or the hundred-pace snake, poses severe health risks due to its venom. Envenomation by this snake leads to complications such as hemorrhage, edema, and coagulopathy. Traditional antivenoms are limited by venom variability and often contain non-neutralizing antibodies, highlighting the need for more precise and effective immunogens.

Methods: This study utilized epitope-based antibody technology to develop a targeted sera against venom metalloproteinases (MPs) and phospholipases A₂ (PLA₂s). Twelve antigenic epitopes were identified via bioinformatics, leading to the design of a composite antigen peptide, EpiMPLA. It was engineered to be expressed via two expression systems, resulting in the recombinant immunogens, ProMPLA and p2AMPLA.

Results: Immunization with ProMPLA and p2AMPLA produced robust antibody responses in mice, effectively inhibiting MPs and PLA₂s. In vitro assays demonstrated that sera from immunized mice reduced the activity of these venom enzymes, minimized venom-induced hemorrhage and edema, and restored blood coagulation. At a venom dose of 2×LD₅₀, all mice in the control group died, while survival rates were 90% for anti-ProMPLA and 70% for anti-p2AMPLA.

Conclusion: The EpiMPLA epitope represents a promising candidate for generating neutralizing antibodies against D. acutusvenom, demonstrating its potential to address critical gaps in current antivenom therapy. These findings not only validate the feasibility of epitope-based antivenom development but also pave the way for further research to optimize this strategy.

Background: Osteosarcoma is the most common primary bone tumor in humans. It is a locally aggressive tumor at the primary site, with metastasis being the main cause of death in patients. Studies on dogs have gained prominence in oncology, as they are valuable spontaneous models of osteosarcoma. In the context of natural compounds, biotoxins are attracting increasing research interest as new therapeutic agents against cancer, such as melittin, that represents 40 to 50% of the dry weight of bee venom, and studies have already shown its antitumor effects.

Methods: We analyzed the anti-migratory and anti-invasive potential of melittin, with the wound healing and Transwell tests, apoptosis with Annexin V/IP and cell viability with the MTT test in 2D and 3D models.

Results: Melittin had a cytotoxic effect on osteosarcoma cell lines, with an IC50 between 1.5 and 2.5 µg/mL. In the wound healing test and Transwell test, melittin prevented cell migration and invasion, resulting in cell death due to iodide propidium marking in canine, murine and human cell lines. Melittin exhibited cytotoxicity in a 3D model of osteospheres, with a significantly higher IC50 in this type of culture, with values between 3.5 and 4.0 µg/mL.

Conclusion: We conclude that melittin has antitumor and antimetastatic properties in canine, murine and human osteosarcoma cell lines. Consequently, we believe that further research on this promising compound will facilitate its application in the development of therapeutic agents for osteosarcoma, ultimately contributing to improved survival outcomes for cancer patients.

Background: Spiders of medical importance in the Amazon region belong to the genera Phoneutria, Loxosceles and Latrodectus. Natural history data show that Phoneutria spp. occur in both periodically flooded forest areas (igapós) and non-flooded areas (terra firme), as well as in commercial plantations in the Amazon. Negative interactions with wandering spiders (Phoneutria spp.) can occur along forest trails, leading to homes, schools and workplaces. Harmful species, such as Loxosceles amazonica and Latrodectus aff. curacaviensis, are mainly associated with accidents in rural settings.

Methods: To understand the dynamics of spider accidents in the Brazilian Amazon, we conducted a search for scientific articles in five databases (Google Scholar, PubMed/MEDLINE, Scopus by Elsevier and SciELO). In addition, we analyzed the content of four reference books on the ecological aspects of Amazonian spiders. All told, we identified 64 eligible studies, including six regional surveys published between 1996 and 2016.

Results: From 2015 to 2022, a total of 25 human lives were lost to spider envenomation in the Brazilian Amazon. An analysis of the data revealed that many riverside families engage in agricultural practices that expose them to venomous animals. Hospital data reveal that most patients bitten by spiders come from impoverished rural communities, which rely on public hospitals of Brazil's Unified Health System (SUS) for medical treatment. The results indicate that spider bites in the Amazon represent a neglected public health problem, especially in locations far from capital cities.

Conclusion: Amerindian and non-Amerindian communities living in areas at high risk of venomous animal attacks do not receive adequate attention in health policies. Given the wide dispersion of rural populations vulnerable to venomous animal incidents in the Amazon, the establishment of new referral medical centers is an essential strategy, especially for riverside communities with limited access to health services.

Background: Leishmaniasis is a neglected disease that mainly affects impoverished populations and receives limited attention from governments and research institutions. Current treatments are based on antimonial therapies, which present high toxicity and cause significant side effects, such as cardiotoxicity and hepatotoxicity. This study proposes using crotapotin, isolated from Crotalus durissus terrificus venom, as a potential inhibitor of the enzyme trypanothione reductase from Leishmania braziliensis (LbTR).

Methods: In silico assays were conducted to evaluate the interaction of crotapotin with LbTR using molecular docking and molecular dynamics techniques. Recombinant LbTR was expressed in E. coli, and its enzymatic activity was confirmed. The inhibitory action of crotapotin on LbTR was then tested in enzymatic assays.

Results: The stability of these interactions was confirmed over 200 ns molecular dynamics simulations, with a clustering analysis using the GROMACS method revealing a total of 12 distinct clusters. The five most representative clusters showed low RMSD values, indicating high structural stability of the LbTR-crotapotin complex. In particular, cluster 1, with 3,398 frames and an average RMSD of 0.189 nm from the centroid, suggests a dominant stable conformation of the complex. Additional clusters maintained average RMSD values between 0.173 nm and 0.193 nm, further reinforcing the robustness of the complex under physiological conditions. Recombinant LbTR expression was successful, yielding 4.8 mg/L with high purity, as verified by SDS-PAGE. In the enzymatic assays, crotapotin partially inhibited LbTR activity, with an IC₅₀ of 223.4 μM.

Conclusion: The in silico findings suggest a stable and structured interaction between crotapotin and LbTR, with low structural fluctuation, although the inhibition observed in in vitro assays was moderate. These results indicate the potential of crotapotin as a promising basis for developing specific LbTR inhibitors, contributing to the bioprospecting of new antiparasitic agents.

Background: Scorpion envenomation poses a significant health threat in endemic regions, eliciting complex immune responses in affected individuals. Recent research suggests that the timing of envenomation - whether it occurs during the day or night - may influence the host inflammatory response and subsequent organ damage. This study investigates the impact of envenomation timing on host inflammatory and oxidative responses using an experimental scorpion envenomation model.

Methods: Mice were divided into two groups, corresponding to their resting phase (day) and activity phase (night), and were monitored for twenty-four hours post-envenomation. We analyzed systemic inflammatory markers, hormonal changes within the hypothalamic-pituitary-adrenal (HPA) axis, and assessed liver toxicity.

Results: Our findings reveal that the release of the myeloperoxidase enzyme, along with the pro-inflammatory cytokines IL-6 and IL-17, varied significantly based on the timing of envenomation. Notably, envenomation occurring during the nighttime resulted in elevated levels of these mediators. We also observed a pronounced imbalance in oxidative stress, characterized by a higher presence of prooxidant species during the daytime and enhanced antioxidant activities during the nighttime. This diurnal variation highlights the dynamic nature of the inflammatory and oxidative processes. Importantly, our analysis points to the probable involvement of corticosterone, the final effector of the HPA axis, in modulating these variations in the inflammatory response. By influencing both the intensity of the immune response and the degree of oxidative stress, corticosterone appears to play a pivotal role in the overall pathophysiology of scorpion envenomation.

Conclusion: This study provides valuable insights into how the timing of scorpion envenomation influences inflammatory responses and organ-specific toxicity, offering potential implications for the treatment and management of envenomation cases.

Background: Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately seven million individuals worldwide, with the highest number of cases in Latin America. CD has two phases, of which the chronic phase is characterized by reduced efficacy in drug therapies. This and other factors make developing new strategies that aim to identify molecules capable of becoming alternatives to or complement current chemotherapy vitally important.

Methods: Cruzain and AsCystatin were obtained recombinantly through expression in E. coli. Bioinformatic assays were conducted with both molecules, followed by in vitro enzyme inhibition assays. Subsequently, in silico studies allowed for the design of peptides, which were then assessed for molecular interactions with cruzain. The designed peptides were synthesized, and their inhibitory potential on cruzain and their trypanocidal and cytotoxic effects in vitro were finally assessed.

Results: AsCystatin, a potential inhibitor of cysteine proteases, was identified from previously published scientific literature. In silico assays suggested that AsCystatin interacts with key regions of cruzain, and was subsequently produced through heterologous expression, obtaining a protein with a high degree of purity. Next, the inhibition of AsCystatin on the activity of cruzain was assessed, observing that approximately 20 µM of cystatin could inhibit 50% of the catalytic activity of the recombinant enzyme. Based on the in-silico analysis performed previously, original, and modified peptides were designed and tested, which allowed for identifying four peptides with inhibitory capacity on the enzymatic activity of cruzain. Finally, three of these peptides showed trypanocidal activity on epimastigote forms of T. cruzi in in vitro models.

Conclusion: It was possible to identify AsCystatin and four peptides derived from this protein with inhibitory activity on cruzain, highlighting the trypanocidal effect of these peptides observed in in vitro assays.