Clinical outcomes in a murine model after envenoming by the Amazonian scorpions Tityus strandi and Tityus dinizi.

The Brazilian Amazon is home to a rich fauna of scorpion species of medical importance, some of them still poorly characterized regarding their biological actions and range of clinical symptoms after envenoming. The Amazonian scorpion species Tityus strandi and Tityus dinizi constitute some of the scorpions in this group, with few studies in the literature regarding their systemic repercussions. In the present study, we characterized the clinical, inflammatory, and histopathological manifestations of T. strandi and T. dinizi envenoming in a murine model using Balb/c mice. The results show a robust clinical response based on clinical score, hyperglycemia, leukocytosis, increased cytokines, and histopathological changes in the kidneys and lungs. Tityus strandi envenomed mice presented more prominent clinical manifestations when compared to Tityus dinizi, pointing to the relevance of this species in the medical scenario, with both species inducing hyperglycemia, leukocytosis, increased cytokine production in the peritoneal lavage, increased inflammatory infiltrate in the lungs, and acute tubular necrosis after T. strandi envenoming. The results presented in this research can help to understand the systemic manifestations of scorpion accidents in humans caused by the target species of the study and point out therapeutic strategies in cases of scorpionism in remote regions of the Amazon.

The complex repertoire of Tityus spp. venoms: Advances on their composition and pharmacological potential of their toxins.

Animal venoms are a rich and complex source of components, including peptides (such as neurotoxins, anionic peptides and hypotensins), lipids, proteins (such as proteases, hyaluronidases and phospholipases) and inorganic compounds, which affect all biological systems of the envenoming victim. Their action may result in a wide range of clinical manifestations, including tachy/bradycardia, hyper/hypotension, disorders in blood coagulation, pain, edema, inflammation, fever, muscle paralysis, coma and even death. Scorpions are one of the most studied venomous animals in the world and interesting bioactive molecules have been isolated and identified from their venoms over the years. Tityus spp. are among the scorpions with high number of accidents reported in the Americas, especially in Brazil. Their venoms have demonstrated interesting results in the search for novel agents with antimicrobial, anti-viral, anti-parasitic, hypotensive, immunomodulation, anti-insect, antitumor and/or antinociceptive activities. Furthermore, other recent activities still under investigation include drug delivery action, design of anti-epileptic drugs, investigation of sodium channel function, treatment of erectile disfunction and priapism, improvement of scorpion antivenom and chelating molecules activity. In this scenario, this paper focuses on reviewing advances on Tityus venom components mainly through the modern omics technologies as well as addressing potential therapeutic agents from their venoms and highlighting this abundant source of pharmacologically active molecules with biotechnological application.

Mitochondrial swelling in cardiomyocytes: Insights from a murine model of Tityus serrulatus scorpion envenomation.

Immune system hyperactivation is involved with clinical severity and pathological alterations during scorpion envenomation. In a murine model, mice inoculated with a lethal dose of Tityus serrulatus scorpion venom presented mitochondrial swelling in cardiomyocytes, with other structures such as sarcomeres and intercalated disks preserved. Treatment with dexamethasone or knockout animals to the interleukin-1ß receptor do not undergo mitochondrial changes in cardiomyocytes during envenomation.

Beyond Angiogenesis: The Multitasking Approach of the First PEGylated Vascular Endothelial Growth Factor (CdtVEGF) from Brazilian Rattlesnake Venom.

A pioneering study regarding the isolation, biochemical evaluation, functional assays and first PEGylation report of a novel vascular endothelial growth factor from Crotalus durissus terrificus venom (CdtVEGF and PEG-CdtVEGF). CdtVEGF was isolated from crude venom using two different chromatographic steps, representing 2% of soluble venom proteins. Its primary sequence was determined using mass spectrometry analysis, and the molecule demonstrated no affinity to heparin. The Brazilian crotalid antivenom recognized CdtVEGF. Both native and PEGylated CdtVEGF were able to induce new vessel formation and migration, and to increase the metabolic activity of human umbilical endothelial vascular cells (HUVEC), resulting in better wound closure (~50% within 12 h) using the native form. CdtVEGF induced leukocyte recruitment to the peritoneal cavity in mice, with a predominance of neutrophil influx followed by lymphocytes, demonstrating the ability to activate the immune system. The molecule also induced a dose-dependent increase in vascular permeability, and PEG-CdtVEGF showed less in vivo inflammatory activity than CdtVEGF. By unraveling the intricate properties of minor components of snake venom like svVEGF, this study illuminates the indispensable significance of exploring these molecular tools to unveil physiological and pathological processes, elucidates the mechanisms of snakebite envenomings, and could possibly be used to design a therapeutic drug.

Pioneering in vitro characterization of macrophage response induced by scorpion venoms from the Brazilian Amazon.

There are several scorpion species of medical relevance around the world. Some of them are well characterized by their toxins and clinical outcomes. Brazilian Amazon has a great amount of these arthropods that have an impact in the scorpionism events specifically in this region of Brazil. Recently, several studies pointed out the immune system activation during scorpion envenouming as an important facet of scorpionism, inducing a sepsis-like state that culminates in clinical severity and death. In this work, we characterized the macrophage response of three species of clinical relevance in Brazilian Amazon: Tityus silvestris, T. metuendus and T. obscurus and one specie with no toxic effects to humans, Brotheas amazonicus. All the four species analyzed were able to induce pro- and anti-inflammatory cytokine production in a J774.1 murine macrophage model. This activation was dependent on TLR2/TLR4/MyD88 activation and abolished by TLRs antagonists. These results suggest that the venoms of the four species analyzed were able to induce macrophage response in agreement to the well-established immune activation by T. serrulatus venom. Our findings provide new insights into the clinical repercussions of scorpionism of uncharacterized species and point to new biotechnological applications of these venoms and possible supportive therapies in scorpionism.

Baccharin and p-coumaric acid from green propolis mitigate inflammation by modulating the production of cytokines and eicosanoids.

ETHNOPHARMACOLOGICAL RELEVANCE: Green propolis is produced by Apis mellifera honeybees using Baccharis dracunculifolia D.C. (Asteraceae) as substrate. This Southern Brazilian native plant and green propolis have been used in traditional medicine to treat gastric diseases, inflammation and liver disorders. AIM OF THE STUDY: Investigate the effects of baccharin (Bac) or p-coumaric acid (pCA) isolated from B. dracunculifolia D.C. (Asteraceae) over the inflammation induced by lipopolysaccharide (LPS) in vivo. MATERIALS AND METHODS: Inflammation was induced by LPS injection into air-pouches in mice, which were subsequently treated with Bac or pCA. Lavage fluid was collected from air pouches for the quantification of cellular influx via microscopy, and quantification of inflammatory mediators via colorimetric methods, ELISA and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: LPS-induced inflammation increased cellular influx and increased the levels of parameters related to vascular permeability and edema formation, such as nitric oxide (NO) and protein extravasation. Moreover, LPS increased the levels of cytokines and eicosanoids in the air-pouches. Importantly, both Bac and pCA suppressed the infiltration of neutrophils, production of NO and protein extravasation. Notably, the compounds promote differential regulation of cytokine and eicosanoid production. CONCLUSIONS: Our results suggest that Bac from green propolis directly affects inflammation by inhibiting the production of cytokines and eicosanoids, while pCA may exert direct, but also indirect effects on inflammation by stimulating the production of regulatory effectors such as interkeukin-10 in vivo.

Interleukin-1 receptor-induced PGE2 production controls acetylcholine-mediated cardiac dysfunction and mortality during scorpion envenomation.

Scorpion envenomation is a leading cause of morbidity and mortality among accidents caused by venomous animals. Major clinical manifestations that precede death after scorpion envenomation include heart failure and pulmonary edema. Here, we demonstrate that cardiac dysfunction and fatal outcomes caused by lethal scorpion envenomation in mice are mediated by a neuro-immune interaction linking IL-1 receptor signaling, prostaglandin E2, and acetylcholine release. IL-1R deficiency, the treatment with a high dose of dexamethasone or blockage of parasympathetic signaling using atropine or vagotomy, abolished heart failure and mortality of envenomed mice. Therefore, we propose the use of dexamethasone administration very early after envenomation, even before antiserum, to inhibit the production of inflammatory mediators and acetylcholine release, and to reduce the risk of death.

Pioneering Study on Rhopalurus crassicauda Scorpion Venom: Isolation and Characterization of the Major Toxin and Hyaluronidase.

Scorpionism is responsible for most accidents involving venomous animals in Brazil, which leads to severe symptoms that can evolve to death. Scorpion venoms consist of complexes cocktails, including peptides, proteins, and non-protein compounds, making separation and purification procedures extremely difficult and time-consuming. Scorpion toxins target different biological systems and can be used in basic science, for clinical, and biotechnological applications. This study is the first to explore the venom content of the unexplored scorpion species Rhopalurus crassicauda, which inhabits exclusively the northernmost state of Brazil, named Roraima, and southern region of Guyana. Here, we pioneer the fractionation of the R. crassicauda venom and isolated and characterized a novel scorpion beta-neurotoxin, designated Rc1, and a monomeric hyaluronidase. R. crassicauda venom and Rc1 (6,882 Da) demonstrated pro-inflammatory activities in vitro and a nociceptive response in vivo. Moreover, Rc1 toxin showed specificity for activating Nav1.4, Nav1.6, and BgNav1 voltage-gated ion channels. This study also represents a new perspective for the treatment of envenomings in Roraima, since the Brazilian scorpion and arachnid antivenoms were not able to recognize R. crassicauda venom and its fractions (with exception of hyaluronidase). Our work provides useful insights for the first understanding of the painful sting and pro-inflammatory effects associated with R. crassicauda envenomings.

Interleukin-1 Receptor-Induced Nitric Oxide Production in the Pancreas Controls Hyperglycemia Caused by Scorpion Envenomation.

Tityus serrulatus causes numerous scorpion envenomation accidents and deaths worldwide. The symptoms vary from local to systemic manifestations, culminating in pulmonary edema and cardiogenic shock. Among these events, transitory hyperglycemia is a severe manifestation that influences pulmonary edema, hemodynamic alterations, and cardiac disturbances. However, the molecular mechanism that leads to increased glucose levels after T. serrulatus envenomation remains unknown. This study aimed to investigate our hypothesis that hyperglycemia due to scorpion envenomation involves inflammatory signaling in the pancreas. The present study showed that T. serrulatus venom induces the production of IL-1α and IL-1ß in the pancreas, which signal via IL-1R and provoke nitric oxide (NO) production as well as edema in ß-cells in islets. Il1r1-/- mice were protected from transitory hyperglycemia and did not present disturbances in insulin levels in the serum. These results suggest that the pathway driven by IL-1α/IL-1ß-IL-1R-NO inhibits insulin release by ß-cells, which increases systemic glucose concentration during severe scorpion envenomation. A supportive therapy that inhibits NO production, combined with antiserum, may help to prevent fatal outcomes of scorpion envenomation. Our findings provide novel insights into the design of supportive therapy with NO inhibitors combined with antiscorpion venom serum to overcome fatal outcomes of scorpion envenomation.

Immunomodulatory activity of hyaluronidase is associated with metabolic adaptations during acute inflammation.

OBJECTIVE AND DESIGN: Investigate survival outcomes, and immunological and metabolomic effects of hyaluronidase (Hz) treatment during mouse models of acute inflammation and sepsis. METHODS: Survival of C57Bl/6 mice was monitored after lethal challenge with lipopolysaccharide (LPS) or cecal and ligation puncture (CLP)-induced sepsis and treated with Hz or saline. Mice were also challenged with LPS and treated with Hz for leukocyte counting, cytokine quantification and determination of metabolomic profiles in the peritoneal fluid. RESULTS: Hz treatment improved survival outcomes after lethal challenge with LPS or CLP-induced sepsis. LPS challenge promoted acute neutrophil accumulation and production of interleukin-1ß (IL-1ß) and IL-6 in the peritoneum, whereas Hz treatment suppressed neutrophil infiltration and cytokine production. We further characterized the metabolomic alterations caused by LPS challenge, which predicted activity of metabolic pathways related to fatty acids and eicosanoids. Hz treatment had a profound effect over the metabolic response, reflected by reductions of the relative levels of fatty acids. CONCLUSION: Collectively, these data demonstrate that Hz treatment is associated with metabolic reprogramming of pathways that sustain the inflammatory response.

Scorpion envenomation and inflammation: Beyond neurotoxic effects.

Scorpion envenomation results in a wide range of clinical manifestations that are mostly attributed to the activation of the autonomic nervous system by venom toxins. In fact, sympathetic and parasympathetic disturbances play important roles during poisoning. However, scorpion venom also induces a complex hyperinflammatory state that occurs parallel to systemic inflammatory response syndrome and acute sepsis. After a scorpion sting, innate immune cells are exposed to the venom molecules, which bind to pattern recognition receptors and activate pro-inflammatory pathways that contribute toward the promotion of severe symptoms, such as pulmonary edema, and eventually lead to death. In this review, we highlight studies that pointed out inflammation as a major pathological facet of scorpion envenomation, so as to provide novel targets to improve therapeutics for scorpionism.

Lipoxin A4 encapsulated in PLGA microparticles accelerates wound healing of skin ulcers.

Lipoxin A4 (LXA4) is involved in the resolution of inflammation and wound healing; however, it is extremely unstable. Thus, to preserve its biological activities and confer stability, we encapsulated LXA4 in poly-lactic-co-glycolic acid (PLGA) microparticles (LXA4-MS) and assessed its application in treating dorsal rat skin lesions. Ulcers were sealed with fibrin adhesive and treated with either LXA4-MS, unloaded microparticles (Un-MS), soluble LXA4, or PBS/glue (vehicle). All groups were compared at 0, 2, 7, and 14 days post-lesions. Our results revealed that LXA4-MS accelerated wound healing from day 7 and reduced initial ulcer diameters by 80%. Soluble LXA4, Un-MS, or PBS closed wounds by 60%, 45%, and 39%, respectively. LXA4-MS reduced IL-1ß and TNF-α, but increased TGF-ß, collagen deposition, and the number of blood vessels. Compared to other treatments, LXA4-MS reduced inflammatory cell numbers, myeloperoxidase (MPO) concentration, and metalloproteinase-8 (MMP8) mRNA in scar tissue, indicating decreased neutrophil chemotaxis. In addition, LXA4-MS treatment increased macrophages and IL-4, suggesting a positive impact on wound healing. Finally, we demonstrated that WRW4, a selective LXA4 receptor (ALX) antagonist, reversed healing by 50%, indicating that LXA4 must interact with ALX to induce wound healing. Our results show that LXA4-MS could be used as a pharmaceutical formulation for the treatment of skin ulcers.

Ocorrência de enteroparasitoses em manipuladores de alimentos de restaurantes em Parnaíba, Piauí-Brasil / Occurrence of intestinal parasites in food handlers working in restaurants of the city of Parnaíba, Piauí-Brazil

As enteroparasitoses têm se mostrado um sério problema de saúde pública no Brasil e no mundo.O processo de transmissão fecal-oral dessas doenças facilita sua disseminação por meio dealimentos. Este trabalho teve como objetivos avaliar a ocorrência de contaminação parasitária emmanipuladores de alimentos de restaurantes de médio e grande porte na cidade de Parnaíba, Piauí, ediscutir a influência destes na transmissão de parasitoses. Foram analisadas amostras fecais de 251indivíduos na faixa etária entre 20 e 59 anos, segundo os métodos de Hoffman, Pons e Janer e de Willis. Foram aplicados questionários estruturados sobre os aspectos socioeconômicos e sanitáriosdos indivíduos. A análise estatística dos resultados foi realizada pelos testes exato de Fischer equi-quadrado para identificar associações entre as variáveis com nível de significância de 5 por cento. Oíndice de positividade para parasitos nas amostras foi de 51 por cento (129 amostras), sendo identificadoscasos de poliparasitismo. Entre os protozoários foram encontrados: Entamoeba coli (38 por cento),Endolimax nana (26 por cento), Entamoeba histolytica/E. dispar (17 por cento), Iodamoeba bustchlii (8 por cento). Entre os helmintos: Ascaris lumbricoides (48 por cento), ancilostomídeos (19 por cento), Enterobius vermicularis (13 por cento),Strongyloides stercolaris (10 por cento), Hymenolepis nana (6 por cento) e Taenia spp (4 por cento). Verificou-se elevada prevalência (51 por cento) de enteroparasitoses em manipuladores de alimentos, com predominânciasignificativa (p<0,05) de protozooses (74 por cento) sobre helmintíases (26 por cento).

Introduction: Intestinal parasites are a serious public health problem in Brazil and worldwide, affectingindividuals from several social classes. The fecal-oral route of transmission of these infectionsfacilitates their spread by food. This study aimed to evaluate the occurrence of parasitic contaminationsamong restaurant food handlers in the city of Parnaíba, Piauí, also discussing their possible influenceson the transmission of enteroparasites. Methodology: Fecal samples from 251 individuals agedbetween 20 and 59 years were analyzed. Hoffman, Pons and Janer and Willis methods were performedon samples. Structured questionnaires about socio-economic and health aspects of individuals wereapplied. Fisher’s exact and chi-square tests with significance level of 5 percent were used to identifyassociations between categorical variables. Results: Positive samples for parasites were found in 51 percent (129 samples). Cases of polyparasitism were also found. Protozoa detected were Entamoeba coli(38 percent ), Endolimax nana (26 percent ), Entamoeba histolytica / E. dispar (17 percent ), Iodamoeba bustchlii (8 percent ).Helminths detected were Ascaris lumbricoides (48 percent ), ancylostomides (19 percent ), Enterobius vermicularis(13 percent ), Strongyloides stercoralis (10 percent ), Hymenolepis nana (6 percent ) and Taenia spp. (4 percent ). Conclusion:A high prevalence (51 percent ) of intestinal parasites was found in restaurant food handlers. Prevalence ofprotozoan infections (74 percent ) was significantly (p<0.05) higher than helminth infections (26 percent ).