Activation of M1 muscarinic acetylcholine receptors by proline-rich oligopeptide 7a (

Background: The bioactive peptides derived from snake venoms of the Viperidae family species have been promising as therapeutic candidates for neuroprotection due to their ability to prevent neuronal cell loss, injury, and death. Therefore, this study aimed to evaluate the cytoprotective effects of a synthetic proline-rich oligopeptide 7a (PRO-7a; <EDGPIPP) from Bothrops jararaca snake, on oxidative stress-induced toxicity in neuronal PC12 cells and astrocyte-like C6 cells. Methods: Both cells were pre-treated for four hours with different concentrations of PRO-7a, submitted to H2O2-induced damage for 20 h, and then the oxidative stress markers were analyzed. Also, two independent neuroprotective mechanisms were investigated: a) L-arginine metabolite generation via argininosuccinate synthetase (AsS) activity regulation to produce agmatine or polyamines with neuroprotective properties; b) M1 mAChR receptor subtype activation pathway to reduce oxidative stress and neuron injury. Results: PRO-7a was not cytoprotective in C6 cells, but potentiated the H2O2-induced damage to cell integrity at a concentration lower than 0.38 μM. However, PRO-7a at 1.56 µM, on the other hand, modified H2O2-induced toxicity in PC12 cells by restoring cell integrity, mitochondrial metabolism, ROS generation, and arginase indirect activity. The α-Methyl-DL-aspartic acid (MDLA) and L-NΩ-Nitroarginine methyl ester (L-Name), specific inhibitors of AsS and nitric oxide synthase (NOS), which catalyzes the synthesis of polyamines and NO from L-arginine, did not suppress PRO-7a-mediated cytoprotection against oxidative stress. It suggested that its mechanism is independent of the production of L-arginine metabolites with neuroprotective properties by increased AsS activity. On the other hand, the neuroprotective effect of PRO-7a was blocked in the presence of dicyclomine hydrochloride (DCH), an M1 mAChR antagonist. Conclusions: For the first time, this work provides evidence that PRO-7a-induced neuroprotection seems to be mediated through M1 mAChR activation in PC12 cells, which reduces oxidative stress independently of AsS activity and L-arginine bioavailability.(AU)

Conhecimentos sobre o manejo de vítimas do acidente botrópico no serviço de emergência / Knowledge about botropic accident victim management in emergency service / Conocimiento sobre el manejo de víctimas de accidentes botópicos en emergencia

Objetivo: identificar o conhecimento da equipe de enfermagem, no manejo do paciente vítima de acidente botrópico no serviço de emergência. Métodos: estudo descritivo, exploratório e com abordagem qualitativa. Desenvolvido em 2019, com profissionais da equipe de enfermagem do setor de emergência adulto de um Hospital Universitário. Utilizou-se entrevistas semiestruturadas, transcritas e analisadas por meio de estatística descritiva simples e análise de conteúdo. Resultados: participaram do estudo 34 profissionais da equipe de enfermagem, sendo 82% do sexo feminino, com média de idade de 37 anos. A análise de conteúdo revelou três categorias: conhecimento sobre acidentes por animais peçonhentos, trajetória acadêmica e profissional no contexto de animais peçonhentos e manejo da equipe de enfermagem do paciente vítima de acidente botrópico. Conclusão: os resultados demonstram o conhecimento e condutas da equipe de enfermagem frente ao acidente botrópico e evidenciam a necessidade de sensibilizar a equipe de enfermagem quanto ao reconhecimento do acidente ofídico, especialmente com relação às características apresentadas pelo animal peçonhento e as manifestações clínicas apresentadas pelo paciente. (AU)

Objective: to identify the knowledge of the nursing staff in dealing with bothropic accident victims in the emergency service. Methods: descriptive, exploratory study with a qualitative approach. Developed in 2019, with professionals from the nursing staff of the adult emergency sector of a University Hospital. A semi-structured interview was used, transcribed and analyzed using simple descriptive statistics and content analysis. Results: 34 professionals from the nursing team participated in the study, being 82% female, with an average age of 37 years. The content analysis revealed three categories: knowledge about accidents caused by poisonous animals, academic and professional trajectory in the context of poisonous animals and management of the nursing team to the patient victim of bothropic accidents. Conclusão: the results demonstrate the knowledge and behaviors of the nursing staff in face of bothropic accidents and highlight the need to sensitize the nursing staff regarding the recognition of the snakebite accident, especially regarding the characteristics presented by the venomous animal and the clinical manifestations presented by the patient. (AU)

Objetivo: identificar los conocimientos del equipo de enfermería en el manejo del paciente víctima de accidente botópico en el servicio de urgencias. Métodos: estudio descriptivo, exploratorio con abordaje cualitativo. Desarrollado en 2019, con profesionales del personal de enfermería del servicio de urgencias de adultos de un Hospital Universitario. Se utilizó una entrevista semiestructurada, transcrita y analizada mediante estadística descriptiva simple y análisis de contenido. Resultados: participaron en el estudio 34 profesionales del equipo de enfermería, de los cuales el 82% eran mujeres, con una edad promedio de 37 años. El análisis de contenido reveló tres categorías: conocimiento sobre accidentes causados por animales venenosos, trayectoria académica y profesional en el contexto de animales venenosos, y manejo del equipo de enfermería al paciente víctima de accidentes bottrópicos. Conclusión: los resultados demuestran el conocimiento y conducta del equipo de enfermería ante el accidente bottrópico y muestran la necesidad de sensibilizar al personal de enfermería sobre el reconocimiento del accidente de la serpiente, especialmente en lo que respecta a las características que presenta el animal venenoso y las manifestaciones clínicas que presenta el paciente. (AU)

Snakebites in Northeastern Brazil: accessing clinical-epidemiological profile as a strategy to deal with Neglected Tropical Diseases

ABSTRACT Background: Brazil ranks first in the number of snakebites in South America. A detailed analysis of these cases is required to improve the public health planning. In this study, we retrospectively examined the clinical and epidemiological profiles of snakebites in Maranhão between January 2009 and December 2019. Methods: Data were obtained from the compulsory notification forms provided by the Health Department of Maranhão. Results: A total of 17,658 cases were recorded during the study period. Most of the bites were from snakes belonging to the genus Bothrops. Medical care was mostly within three hours after the bite. Most cases were classified as mild and most victims recovered; however, 139 deaths were recorded. Most bites occurred among people aged 20-39 years, mainly among rural workers. The most frequent local clinical manifestations were pain, edema, and ecchymosis. The most common systemic clinical manifestations include neuroparalysis, vagal syndrome, and myolysis. Most snakebites occurred between January and March. The municipalities with the highest number of notifications were Buriticupu (936 cases), Arame (705 cases), and Grajaú (627 cases). Conclusions: The clinical profile of snakebites in Maranhão is similar to that observed in other states of Northeast Brazil. However, we found that some systemic manifestations are not compatible with the etiology of snakebites, which leads us to believe that the problem could be the lack of knowledge of the health professionals at the site of envenomation, who may not be ready for attendance, and an important lack of health centers with snake antivenom to treat snakebites.

Compartment Syndrome Secondary to Bothrops spp. Envenomation in Triângulo Mineiro, Region, Minas Gerais, Brazil

ABSTRACT Background: Snakebite is a neglected global health problem with high morbidity. We describe compartment syndrome (CS) cases related to snakebites by Bothrops spp. Methods: The medical records of patients admitted with snakebites envenomation were reviewed. Results: Of 47 patients with Bothrops spp. envenomation (4 male; mean age: 42 years), 7 (15%) developed CS. The mean time to antivenom administration was 9.5 hours. The time to fasciotomy was variable. Seven patients developed infection and four had acute kidney injury. Conclusions: The incidence of CS is higher than that reported previously. This may be due to the clinical severity and long delay before administering antivenom.

Métodos alternativos para determinação da potência de veneno e antiveneno botrópico: aplicações e perspectivas para o controle da qualidade / Alternative methods for potency evaluation for Bothrops venom and antivenom: applications and perspectives for quality control

Introdução: Acidentes com animais peçonhentos são classificados como doenças tropicais negligenciadas e são atualmente a mais frequente causa de intoxicação em humanos no Brasil. O único tratamento disponível é a rápida administração de antivenenos específicos e de qualidade garantida. Para assegurar a eficácia e a segurança desses produtos, são realizados ensaios de determinação da potência in vivo para veneno e antiveneno, desde as etapas de produção até sua liberação final. Apesar dos diversos estudos sobre métodos   alternativos ao ensaio murino, nenhum método foi efetivamente validado. Objetivo: Compilar os métodos alternativos desenvolvidos para os antivenenos botrópicos, avaliando sua disponibilidade, perspectivas e aplicações em laboratórios de produção e controle da qualidade. Método: Foi realizada uma busca nas bases PubMed, BVS e Scopus entre novembro de 2021 e junho de 2022. Foram identificados 89 trabalhos, dos quais 31 foram selecionados de acordo com os critérios de elegibilidade. Resultados: Nos métodos alternativos identificados, observamos a preferência de 42,80% dos estudos por metodologias que utilizem linhagens celulares como método alternativo aos ensaios murinos, sendo que a maioria destes trabalhos 58,30% optou pela linhagem celular Vero. Conclusões: Pela diversidade das toxinas encontradas em cada gênero de serpentes, entende-se que é de extrema importância que o ensaio de potência dos antivenenos tenha como base a avaliação e a quantificação precisa da inibição da atividade biológica dos venenos. Ensaios de citotoxicidade são amplamente utilizados e têm acumulado evidências de sua adequação como importante ferramenta alternativa ao ensaio murino para o controle da qualidade de veneno e antiveneno antibotrópico.

Introduction: Accidents with venomous animals are classified as neglected tropical diseases and are currently the most frequent cause of intoxication in humans in Brazil. The only available treatment is the rapid administration of specific, quality-assured antivenoms. To ensure the efficacy and safety of these products, in vivo potency determination tests for venom and antivenom are performed during the production stages, until final release. Despite several studies on alternative methods to the murine assay, no method has been effectively validated. Objective: To compile alternative methods developed for Bothrops antivenoms, assessing the availability of the methods and the prospects and applications in Bothrops venom and antivenom production and quality control laboratories. Method: A search was conducted in PubMed, BVS, and Scopus databases between November 2021 and June 2022. 89 articles were identified, of which 31 were selected according to the eligibility criteria. Results: We observed in the alternative methods identified a preference of 42.80% of the studies for methodologies that use cell lines as an alternative method to the murine assays, and most of these works (58.30%) opted for a VERO cell line. Conclusions: Due to the diversity of toxins found in each genus of snakes, it is understood that the potency assay for antivenoms should be based on the evaluation and precise quantification of the inhibition of biological activity of venoms. Cytotoxicity assays are widely used and have been accumulating evidence of their suitability as an important alternative tool to the murine assay for quality control for Bothrops venom and antivenom.

Lesión renal aguda secundaria a mordedura de serpiente del género bothrops: a propósito de un caso

Introducción: el accidente ofídico es una intoxicación ocasionada por la inoculación de veneno a través de la mordedura de una serpiente, la cual genera alteraciones locales y sistémicas. Dicho evento representa un importante problema de salud pública en los países en vía de desarrollo debido a su alta frecuencia y gravedad. Las serpientes del género Bothrops son responsables de la mayoría de los accidentes y esto se debe, en parte, a su amplia distribución geográfica y comportamiento agresivo cuando se sienten amenazadas. La lesión renal aguda es una causa importante de mortalidad en pacientes con mordedura de serpientes de este género. Objetivo: describir el caso de un paciente femenino con lesión renal aguda inducida por mordedura de serpiente del género Bothrops. Simultáneamente se detallan los datos epidemiológicos más relevantes de dicha entidad, sus factores de riesgo, los posibles mecanismos fisiopatológicos que explican la génesis de la misma, su diagnóstico y manejo clínico. Se resalta la importancia de su identificación precoz y tratamiento oportuno. Presentación del caso: se presenta el caso de una paciente de 40 años que ingresa a una institución de cuarto nivel de complejidad por presentar lesión renal aguda y coagulopatía por consumo secundario a mordedura de serpiente del género Bothrops, con presencia de sangrado vaginal y hematuria que progresa a anuria y hematemesis, requiriendo transfusión de hemoderivados y terapia de reemplazo renal, medidas con las cuales presentó una evolución clínica favorable. Discusión y conclusión: la lesión renal aguda asociada a la mordedura de serpientes del género Bothrops es una complicación clínica común y potencialmente mortal que no debe ser olvidada. Los eventos fisiopatológicos responsables de esta complicación incluyen mecanismos inmunológicos, trastornos de la coagulación, pigmenturia, nefrotoxicidad directa y la respuesta inflamatoria con efectos hemodinámicos sistémicos y renales. Se hace imprescindible el conocimiento de dicha entidad en aras de reconocerla de forma precoz e instaurar su tratamiento oportuno con el fin de reducir sus complicaciones potencialmente fatales.

Introduction: The ophidian accident is an intoxication caused by the inoculation of venom through the bite of a snake, which generates local and systemic alterations. This event represents an important public health problem in developing countries due to its high frequency and severity. Snakes of the genus Bothrops are responsible for the majority of accidents, and this is partly due to their wide geographic distribution and aggressive behavior when threatened. Acute kidney injury is an important cause of mortality in patients with snake bites of this genus. Purpose: To describe the case of a female patient with bothrops snakebite-induced acute kidney injury. Simultaneously, the most relevant epidemiological data of said entity, its risk factors, the possible pathophysiological mechanisms that explain its genesis, its diagnosis and clinical management are detailed. The importance of its early identification and timely treatment is highlighted. Case presentation: We present the case of a 40-year-old female patient who was admitted to a fourth level of complexity institution due to acute kidney injury and consumption coagulopathy secondary to bothrops snakebite, with the presence of vaginal bleeding and hematuria that progressed to anuria. and hematemesis; requiring transfusion of blood products and renal replacement therapy, measures with which she presented a favorable clinical evolution. Conclusion and discussion: Acute kidney injury associated with the bite of bothrops snakes is a common and life-threatening clinical complication that should not be overlooked. The pathophysiological events responsible for this complication include immunological mechanisms, coagulation disorders, pigmenturia, direct nephrotoxicity, and the inflammatory response with systemic and renal hemodynamic effects. Knowledge of this entity is essential in order to recognize it early and establish its timely treatment in order to reduce its potentially fatal complications.

Massive acute ischemic stroke after Bothrops spp. envenomation in southwestern Colombia: Case report and literature review / Accidente cerebrovascular isquémico agudo masivo por veneno de Bothrops spp. en el suroccidente de Colombia: reporte de caso y revisión de la literatura

Bothrops spp. envenomation and its relationship with ischemic stroke has complex pathogenesis. Local effects such as edema, pain, redness, necrosis, and systemic manifestations like coagulation disorders, thrombosis, renal failure, and hemorrhage have been reported. Hemorrhagic stroke is a common neurological complication but ischemic stroke is poorly understood. We present here the case of a 50-year-old male with no comorbidities referred from a rural area in southwest Colombia with a Bothrops spp. snakebite on the left hand. On admission, the patient presented with a deterioration of consciousness and required mechanical ventilation assistance. The MRI showed multiple ischemic areas in the bilateral frontal- temporal and occipital regions. Two months later, the patient had a favorable resolution, although central paresis in the III and VI cranial nerves and positive Babinski's sign persisted. As already mentioned, the pathophysiology of ischemic stroke due to snakebite is complex but the procoagulant activity of the venom components, the hypovolemic shock, the endothelial damage, and the thromboinflammation can explain it, and although it rarely occurs, it should be considered as a complication of ophidian accidents caused by Bothrops spp.

La mordedura de serpientes Bothrops spp. y el ataque cerebrovascular isquémico tienen una patogenia compleja. Se reconocen efectos locales como edema, dolor, enrojecimiento y necrosis, así como manifestaciones sistémicas como trastornos de la coagulación, trombosis, insuficiencia renal y hemorragia, por lo que el accidente cerebrovascular hemorrágico es una complicación neurológica común, pero, en cambio, el accidente cerebrovascular isquémico es poco conocido. Se presenta el caso de un paciente de 50 años, sin comorbilidades, remitido de una zona rural del suroccidente de Colombia debido a la mordedura de una serpiente Bothrops spp. en su mano izquierda. En el momento del ingreso, el paciente presentaba deterioro de la conciencia y requirió asistencia respiratoria mecánica. Mediante resonancia magnética, se observaron múltiples áreas isquémicas bilaterales en la región fronto-temporal y en la occipital. Dos meses después, el paciente había evolucionado favorablemente, pero persistían la paresia en los pares craneales III y VI y el signo de Babinski. La fisiopatología del accidente cerebrovascular isquémico por mordedura de serpiente es compleja. La actividad procoagulante de los componentes del veneno, el choque hipovolémico, el daño endotelial y la tromboinflamación pueden explicar el accidente cerebrovascular isquémico que, aunque raro, debe considerarse como una complicación del accidente ofídico causado por serpientes Bothrops spp.

Synthesis, characterization and utilization of a new series of 1,2,3-triazole derivatives to neutralize some toxic activities of Bothrops jararaca snake venom

Abstract Snake envenomation is a public health problem, and while serum therapy prevents death, the local effects of venoms can lead to amputations or morbidities. Thus, alternative treatments deserve attention. In this study, we tested eight derivatives of 1,2,3-triazole against some toxic activities of Bothrops jararaca venom. The derivatives were synthesized, and their structures analyzed by infrared and nuclear magnetic resonance. After that, the ability of compounds to inhibit hemolysis, coagulation, proteolysis, hemorrhaging, edema, and lethal activities of B. jararaca venom was investigated. The derivatives were incubated with B. jararaca venom (incubation protocol), administered before (prevention protocol) or after (treatment protocol) injecting venom into the mice. Then, hemorrhaging assay occurred. As a result, most of the derivatives inhibited the activities, even if they were incubated, injected before or after B. jararaca venom. However, the derivatives TRI 07 and TRI 18 seemed to be the most efficient in impairing hemorrhaging. The derivatives showed a low drug score of toxicity based on an in silico technique. Therefore, the derivatives fulfilled physicochemical and biological requirements to become drugs, and they may be a brand new initiative for designing antivenom molecules to complement antivenom therapy to efficiently block tissue necrosis or any other local effects.

Secondary infection profile after snakebite treated at a tertiary referral center in the Brazilian Amazon

ABSTRACT Background: Bothrops envenomations can often lead to complications, such as secondary infections. Methods: This cross-sectional study analyzed the medical records of all patients diagnosed with snakebite. Results: A total of 127 patients were included. Clindamycin was the most commonly prescribed antibiotic, with 105 patients (82.7%) receiving it as the primary antibiotic regimen. In 31 (24.4%) individuals, the first-choice antibiotic did not cease the infection. Conclusions: Secondary infection is an important complication resulting from snakebites, and evidence-based management of this complication can contribute to better clinical outcomes.

Venenos de serpentes do gênero Bothrops: impacto da glicosilação na complexidade dos proteomas e função de toxinas / Bothrops snake venoms: impact of glycosylation on the complexity of proteomes and toxins function

A variabilidade estrutural é uma característica das proteínas de venenos de serpentes, e a glicosilação é uma das principais modificações pós-traducionais que contribui para a diversificação de seus proteomas. Recentes estudos de nosso grupo demonstraram que venenos do gênero Bothrops são marcadamente definidos pelo seu conteúdo de glicoproteínas, e que a maioria das estruturas de N-glicanos dos tipos híbrido e complexo identificados em oito venenos deste gênero contêm unidades de ácido siálico. Em paralelo, em glicoproteínas do veneno de B. cotiara foi identificada a presença de uma estrutura de N-acetilglicosamina bissecada. Assim, com o objetivo de investigar a variação do conteúdo de glicoproteínas, assim como os mecanismos envolvidos na geração dos diferentes venenos de Bothrops, neste estudo foram analisados comparativamente os glicoproteomas de nove venenos do gênero Bothrops (B. atrox, B. cotiara, B. erythromelas, B. fonsecai, B. insularis, B. jararaca, B. jararacussu, B. moojeni e B. neuwiedi). As abordagens glicoproteômicas envolveram cromatografia de afinidade e ensaio de pull-down utilizando, respectivamente, as lectinas SNA (aglutinina de Sambucus nigra) e MAL I (lectina de Maackia amurensis), que mostram afinidade por unidades de ácido siálico nas posições, respectivamente, α2,6 e α2,3; e cromatografia de afinidade com a lectina PHA-E (eritroaglutinina de Phaseolus vulgaris), que reconhece N-acetilglicosamina bissecada. Ainda, eletroforese de proteínas, blot de lectina, e identificação de proteínas por espectrometria de massas foram empregadas para caracterizar os glicoproteomas. As lectinas geraram frações dos venenos enriquecidas de diferentes componentes, onde as principais classes de glicoproteínas identificadas foram metaloprotease, serinoprotease, e L-amino ácido oxidase, além de outras enzimas pouco abundantes nos venenos. Os diferentes conteúdos de proteínas reconhecidas por essas lectinas, com especificidades distintas, ressaltaram novos aspectos da variabilidade dos subproteomas de glicoproteínas desses venenos, dependendo da espécie. Ainda, considerando que metaloproteases e serinoproteases são componentes abundantes nesses venenos e fundamentais no quadro de envenenamento botrópico, e que estas enzimas contêm diversos sítios de glicosilação, o papel das unidades de ácido siálico na atividade proteolítica das mesmas foi avaliado. Assim, a remoção enzimática de ácido siálico (i) alterou o padrão de gelatinólise em zimografia da maioria dos venenos, (ii) diminuiu a atividade proteolítica de alguns venenos sobre o fibrinogênio e a atividade coagulante do plasma humano de todos os venenos, e (iii) alterou o perfil de hidrólise de proteínas plasmáticas pelo veneno de B. jararaca, indicando que este carboidrato pode desempenhar um papel na interação das proteases com seus substratos proteicos. Em contraste, o perfil da atividade amidolítica dos venenos não se alterou após a remoção de ácido siálico e incubação com o substrato Bz-Arg-pNA, indicando que ácido siálico não é essencial em N-glicanos de serinoproteases atuando sobre substratos não proteicos. Em conjunto, esses resultados expandem o conhecimento sobre a variabilidade de proteomas de venenos do gênero Bothrops e apontam a importância das cadeias de carboidratos contendo ácido siálico nas atividades enzimáticas das proteases desses venenos

Structural variability is a feature of snake venom proteins, and glycosylation is one of the main post-translational modifications that contributes to the diversification of venom proteomes. Recent studies by our group have shown that Bothrops venoms are markedly defined by their glycoprotein content, and that most hybrid and complex N-glycan structures identified in eight venoms of this genus contain sialic acid units. In parallel, the presence of a bisected N-acetylglucosamine structure was identified in B. cotiara venom glycoproteins. Thus, with the aim of investigating the variation in the content of glycoproteins, as well as the mechanisms involved in the generation of different Bothrops venoms, in this study the glycoproteomes of nine Bothrops venoms (B. atrox, B. cotiara, B. erythromelas, B. fonsecai, B. insularis, B. jararaca, B. jararacussu, B. moojeni e B. neuwiedi) were comparatively analyzed. The glycoproteomic approaches involved affinity chromatography and pulldown using, respectively, the lectins SNA (Sambucus nigra agglutinin) and MAL I (Maackia amurensis lectin), which show affinity for sialic acid units at positions, respectively, α2,6 and α2,3, and affinity chromatography with PHA-E (Phaseolus vulgaris erythroagglutinin), which recognizes bisected N-acetylglucosamine. In addition, protein electrophoresis, lectin blot, and protein identification by mass spectrometry were employed for glycoproteome characterization. The lectins generated venom fractions enriched with different components, where the main classes of glycoproteins identified were metalloprotease, serine protease, and L-amino acid oxidase, in addition to other low abundant enzymes. The different contents of proteins recognized by these lectins of distinct specificities highlighted new aspects of the variability of the glycoprotein subproteomes of these venoms, depending on the species. Furthermore, considering that metalloproteases and serine proteases are abundant components of these venoms and essential in Bothrops envenomation, and that these enzymes contain several glycosylation sites, the role of sialic acid units in their proteolytic activities was evaluated. Thus, enzymatic removal of sialic acid (i) altered the pattern of gelatinolysis in zymography of most venoms, (ii) decreased the proteolytic activity of some venoms on fibrinogen and the clotting activity of human plasma of all venoms, and (iii) altered the hydrolysis profile of plasma proteins by B. jararaca venom, indicating that this carbohydrate may play a role in the interaction of proteases with their protein substrates. In contrast, the profile of amidolytic activity of the venoms did not change after removal of sialic acid and incubation with the substrate Bz-Arg-pNA, indicating that sialic acid is not essential in N-glycans of serine proteases acting on small substrates. Together, these results expand the knowledge about the variability of proteomes of Bothrops venoms and point to the importance of carbohydrate chains containing sialic acid in the enzymatic activities of venom proteases

Analysis of the protein profile of the venoms of snakes Bothrops asper, Bothrocophias myersi and Crotalus durissus from the Colombian Andean Region obtained by RP-HPLC / Análisis del perfil proteico de los venenos de Bothrops asper, Bothrocophias myersi y Crotalus durissus serpientes de la Región Andina en Colombia obtenidos por RP-HPLC

ABSTRACT Snake venoms comprise a highly complex mixture of proteins, and there is also a high interspecific and intraspecific variability in their composition, even in the same region. Our aim was to compare the composition of the venoms of Bothrocophias myersi, Crotalus durissus, and Bothrops asper, snakes from the Colombian Andean region by Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). The venoms were given to the research group under an agreement with Fundación Zoológica de Cali. The venoms pool was obtained by manual extraction, lyophilized and frozen. The venom protein was quantified by direct measurement with Nanodrop® 280 nm. The protein composition was established by RP-HPLC, using a Lichosper 100 RP, C18 column (250X4 mm) with a pore size of 5-m, as well as by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The highest quantity of protein was found in the venom of B. myersi (108.6 mg/ mL) followed by C. durissus (78.1 mg/mL) and B. asper (74.1 mg/mL). All venoms showed bands of 15 and 50 KDa by using SDS-PAGE. B. myersi venom chromatogram exhibited 16 peaks by RP-HPLC. We conclude that the composition of the three venoms is quite similar, being phospholipase A2 the common protein therein, and together with metalloproteinases they were the most abundant protein families in the venom of B. myersi. SDS-PAGE and RP-HPLC techniques allow a first approach to the profile of the venoms, which in turn could clarify the clinical syndrome produced.

RESUMEN Los venenos de las serpientes comprenden una mezcla compleja de proteínas, y existe una alta variabilidad interespecífica e intra-específica en su composición, incluso en la misma región. Nuestro objetivo fue comparar la composición de los venenos de Bothrocophias myersi, Crotalus durissus y Bothrops asper de la región andina de Colombia, mediante cromatografía líquida de alta eficiencia en fase reversa (RP-HPLC). Los venenos fueron entregados al grupo de investigación mediante un convenio con la Fundación Zoológica de Cali. El pool de venenos fue obtenido por extracción manual, liofilizado y congelado. La proteína de los venenos fue cuantificada por Absorbancia 280nm por medición directa con Nanodrop®. La composición proteica se estableció por RP-HPLC, utilizando una columna Lichosper 100 RP, C18 (250X4 mm) con un tamaño de poro de 5-jm, así como por electroforesis en gel dodecil sulfato de sodio-poliacrilamida (SDS-PAGE). La mayor cantidad de proteínas se encontró en el veneno de B. myersi (108.6 mg/mL), seguido de C. durissus (78.1 mg/mL) y B. asper (74.1 mg/mL). Todos los venenos mostraron bandas de 15 y 50 KDa por SDS-PAGE. El cromatograma de B. myersi exhibió 16 picos por RP-HPLC. Concluimos que la composición de los tres venenos es bastante similar, siendo la fosfolipasa A2 la proteína común en estos y junto con las metaloproteinasas fueron las familias de proteínas más abundantes en el veneno de B. myersi. Las técnicas de SDS-PAGE y el RP-HPLC permiten un primer acercamiento al perfil de los venenos, lo que a su vez podría contribuir a esclarecer el síndrome clínico producido.

Aspectos biomédicos y epidemiológicos del accidente ofídico en el departamento del Cauca, Colombia, 2009-2018 / Snakebite biomedical and epidemiological aspects in the department of Cauca, Colombia, 2009-2018

Resumen | Introducción. El ofidismo en Colombia es un problema de salud pública, lo cual se hace evidente al examinar los datos epidemiológicos a nivel latinoamericano, pues el país ocupa el tercer lugar en número de accidentes ofídicos después de Brasil y México. Objetivo. Hacer un análisis retrospectivo de los casos de accidente ofídico ocurridos entre 2009 y 2018 en el departamento de Cauca, según los datos del Sistema de Vigilancia en Salud Pública (Sivigila) registrados en el Instituto Departamental de Salud del Cauca. Materiales y métodos. Se recopiló e interpretó la información consignada en las fichas de notificación obligatoria de accidente ofídico en los 10 años de estudio. Se determinaron la incidencia y la frecuencia de accidentes según la distribución geográfica y los agentes causales, y se analizaron las variables socioeconómicas relacionadas. Resultados. Se registraron 1.653 casos y una baja mortalidad. Los géneros Bothrops y Bothriechis causaron la mayoría (77,43 %) de los accidentes, seguidos por el género Micrurus (2,9 %). La mayoría de las notificaciones procedían del sur del departamento; las personas de sexo masculino y los agricultores fueron los más afectados, con mordeduras en las extremidades superiores principalmente. Las principales manifestaciones del envenenamiento fueron las hemorrágicas, más frecuentes que la necrosis y la infección. Aunque el criterio para utilizar la seroterapia no siempre fue el mejor, las complicaciones iatrogénicas no fueron frecuentes. Conclusiones. Los municipios de El Tambo y Piamonte, las personas de sexo masculino y las áreas rurales, fueron las variables más afectadas por el ofidismo, principalmente el ofidismo botrópico. Las mayores incidencias se presentaron en la zona sur del Cauca, en las cuencas de los ríos Patía y Caquetá.

Abstract | Introduction: Snakebite is a relevant public health problem in Colombia, which is evident in epidemiological data at the Latin American level, where it ranks third place in the number of snakebites, after México and Brazil. Objective: To perform a retrospective analysis of snakebite accidents in the department of Cauca between 2009 and 2018 based on the data from the Public Health Surveillance System of Colombia reported to the Health Institute of Cauca. Materials and methods: We compiled and interpreted the information from the compulsory snakebite notification forms for the study's 10 years period. We determined snakebite incidence and frequency according to the geographical distribution and the causal agents, and we analyzed related socioeconomic variables as well. Results: The records accounted for 1,653 cases and low mortality. Bothrops and Bothriechis species (77.43%) caused most of the accidents followed by Micrurus (2.9%). Most of the notifications came from the south of the department, and males and farmers were the most affected with bites mainly in the upper extremities. During poisoning, hemorrhagic manifestations predominated over necrosis and infections; serotherapy criteria were not always adequately applied, however, iatrogenic complications were not frequent. Conclusions: El Tambo and Piamonte municipalities, being male, and rural areas were the variables most affected by ophidism, mainly bothropic ophidism. The highest incidences occurred in the southern part of Cauca, in the Patía and Caquetá river basins.

Variaciones de caracteres morfológicos dentro de la especie Bothrops alternatus / Morphological characters variations within the Bothrops alternatus species

Resumen El género Bothrops (yarará) es responsable de más del 95% de los accidentes por ofidios venenosos en Argentina. La mayoría de éstos son notificados en provincias del Nordeste y Noroeste. Bothrops alternatus, es la especie más frecuentementehallada, siendo el vipérido de gran talla más meridional del mundo y una de las especies de mayor importancia sanitaria en Argentina, pudiendo hallarse en relación a núcleos urbanos. Presentan en el dorso figuras arriñonadas características, que remedan atubos de teléfono o porotos unidos, completos o incompletos, hasta la forma fusionada. Presenta foseta loreal, órgano termore-ceptor ubicado entre el ojo y la fosa nasal. Su dentición es solenoglifa, dientes inoculadores huecos, con conducto cerrado, fijosa un hueso maxilar móvil. Si bien se describen variaciones entre animales, se considera una especie monotípica. Su identificaciónes importante dada la abundancia de especímenes y de hallazgos en regiones periurbanas.

Abstract The Bothrops genus is responsible for more than 95% of ophidian accidents in Argentina, most of these are reported in the Northeast and Northwest provinces. Bothrops alternatus, is the most frequent species of the genus Bothrops (yarará), themost southern large-scale viperid in the world and one of the most important species in Argentina, being able to be found in rela-tion to urban nucleus. Their characteristic features are kidney-like figures in the skin, that mimic phone tubes or beans, completeor incomplete, until the merged form. It presents a loreal pit, thermoreceptor organ located between the eye and the nostril. Itsdentition is solenoglyphous, hollow inoculating teeth, with closed duct, fixed to a mobile maxillary bone. Although variationsbetween animals are described, it is considered a monotypic species. The identification of this snake is important due their abun-dance in around urban regions and its frequency of finding.

Características clínico - epidemiológicas de los accidentes ofídicos en un hospital de la Amazonía del Perú / Clinico-epidemiologic characteristics of ophidic accidents in a hospital of the amazonia of Peru

Introducción: En el Perú, los accidentes ofídicos son un importante problema de salud pública, debido a una amplia distribución territorial de diferentes tipos de serpientes. Objetivo: Describir las características clínico - epidemiológicas y el manejo de los accidentes ofídicos registrados en un hospital de Perú. Métodos: Estudio de serie de casos, realizado entre enero del 2011 a diciembre 2015 en el Hospital Santa Rosa de Puerto Maldonado. Se revisaron 256 historias clínicas de pacientes con diagnóstico definitivo de ofidismo. Se analizaron variables sociodemográficas, clínicas y evolución de los pacientes. Resultados: El 78,5 por ciento de pacientes fueron varones, el grupo de edad más afectado fueron los adultos. La mayoría de los accidentes ocurrieron en el mes de marzo (12,5 por ciento). Un 39 por ciento de los pacientes se dedicaba al trabajo agrícola. Entre las 16:00 y 19:59 horas se produjeron 35,2 por ciento de los accidentes, 71,1 por ciento reportó como causante del accidente a la serpiente Bothrops atrox. El 71,5 por ciento de las mordeduras se presentaron en miembros inferiores. La mayoría de pacientes recibió tratamiento específico con antibotrópico polivalente (90,6 por ciento). Conclusiones: En la población estudiada, el sexo más afectado es el masculino con una edad entre 27 a 59 años. La serpiente más frecuente causante de los accidentes fue la Bothrops atrox, más conocida entre los pobladores como "jergón". En la mayoría de los casos se administró como tratamiento específico el suero antiofídico(AU)

Introduction: In Peru, ophidian accidents are an important public health problem due to a wide territorial distribution of different types of snakes. Objective: To describe the clinical-epidemiological characteristics and the management of ophidian accidents registered in a hospital in Peru. Methods: Case series study carried out between January 2011 to December 2015 at the Santa Rosa Hospital in Puerto Maldonado, 256 medical records of patients with a definitive diagnosis of ophidism were reviewed. Sociodemographic and clinical variables and the evolution of the patients were analyzed. Results: A total of 78.5 percent of patients were men, the age group most affected were adults. Most of the accidents occurred in March (12.5 percent). 39 percent of the patients were engaged in agricultural work. Between 4:00 p.m. and 7:59 p.m., 35.2 percent of the accidents occurred. 71.1 percent reported the Bothrops atrox snake as the cause of the accident. 71.5 percent of the bites occurred on the lower limbs. Most patients received specific treatment with polyvalent anti-botropic (90.6 percent). Conclusions: In the studied population, the most affected sex is the male with an age between 27 and 59 years. The most frequent snake causing the accidents was the Bothrops atrox, better known among the inhabitants as "jergón". In most cases, antivenom was administered as a specific treatment(AU)

Hemostatic evaluation of rabbits envenomed with Bothrops alternatus treated with anti-bothropic serum, desmopressin and tranexamin acid / Avaliação hemostática de coelhos envenenados com Bothrops alternatus e tratados com soro antibotrópico, desmopressina e ácido tranexâmico

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.(AU)

No Brasil, as serpentes do gênero Bothrops são responsáveis por milhares de acidentes, e seus venenos são compostos principalmente de enzimas proteolíticas. Embora o soro antiofídico produzido pelos institutos brasileiros seja notavelmente eficiente, mais estudos são necessários, especialmente na medicina veterinária. O veneno contem enzimas e proteínas não-enzimáticas que interferem com a hemostasia levando a hemorragias ou trombose. A associação de outros tratamentos ao soro antibotrópico foi a razão para o desenvolvimento do presente estudo. O objetivo deste estudo foi avaliar as alterações da hemostasia causadas pelo veneno de Bothrops alternatus em coelhos, após tratamento com soro antibotrópico, ácido tranexâmico e desmopressina. Vinte coelhos da Nova Zelândia foram distribuídos em cinco grupos (n = 4) que foram submetidos a experimentos com 150mcg/kg de veneno de B. alternatus por injeção intramuscular. O Grupo 1 (G1) foi o controle positivo e recebeu veneno e PBS / BSA, enquanto o Grupo 2 (G2) foi tratado com ácido tranexâmico, o Grupo 3 (G3) com desmopressina, o Grupo 4 (G4) com ácido tranexâmico e soro antibotrópico, e o Grupo 5 (G5) com soro antibotrópico e desmopressina. As amostras de sangue foram coletadas antes da administração do veneno, e uma, quatro, oito e 12 horas após os tratamentos para realização de tempo de tromboplastina parcial ativada parcial (TTPa), tempo de protrombina (TP), tempo de trombina (TT) e mensuração de fibrinogênio. Para o ensaio de geração de trombina (TG) foi realizado com um pool de amostras nos tempos finais (8 e 12h). Ao final das 12h, todos os animais foram sacrificados e a necropsia foi realizada. Amostras de tecido muscular, coração, pulmões e rins foram analisadas. Os testes TTPa, TP, TT e fibrinogênio não mostraram diferenças significativas entre os grupos e os tempos. No entanto, o TG indicou que o veneno causa um estado de hipocoagulabilidade, que não foi revertido pelos tratamentos propostos. Na histologia, foram observadas inflamação muscular, hemorragia e necrose, além de hemorragia em outros tecidos, sem diferenças entre os grupos. O envenenamento por B. alternatus causa hipocoagulabilidade detectada mais precocemente pelo teste de geração de trombina. O uso de ácido tranexâmico e desmopressina para estabilização da hemostasia após a inoculação do veneno não mostrou vantagem na restauração da coagulação.(AU)

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.(AU)

Efficacy of the 20-minute whole blood clotting test (WBCT20) in the diagnosis of coagulation alteration related to snakebites in a Western Brazilian Amazon hospital

Abstract Introduction: The efficacy of 20-minute whole blood clotting (WBCT20) and the Lee-White clotting time (LWCT) tests in diagnosing coagulation alterations from snakebites were compared. Methods: We evaluated 89 snakebite cases treated at the Hospital Regional do Juruá em Cruzeiro do Sul, Acre, Brazil. Results: WBCT20 results were normal in 33.7% and unclottable in 66.3% of cases, while LWCT results were normal in 23.6% and altered (prolonged or unclottable) in 76.4% of cases, with no significant differences. Conclusions: The WBCT20 is important for rapidly diagnosing coagulation alterations from snakebites. Furthermore, it is efficient, inexpensive, and can be deployed in isolated hospitals.

Severe tissue complications in patients of Bothrops snakebite at a tertiary health unit in the Brazilian Amazon: clinical characteristics and associated factors

Abstract INTRODUCTION: Snakebites in the Brazilian Amazon are caused mostly by snakes from the Bothrops genus and envenomated patients may suffer from tissue complications. METHODS: This study aimed to identify risk factors for severe tissue complications (STC) in patients with Bothrops snakebite in the Amazonas state, Brazil. RESULTS: Snakebites that were classified as severe and affected female patients with comorbidities presented greater risks of developing STCs. In addition, hospitalizations of patients with STC exceeded 5 days. CONCLUSIONS: Clinical and epidemiological characteristics can prove essential for assessing the evolution of STC and clinical prognosis of patients with Bothrops snakebites.

Síndrome hemolítico urémico por ingesta de veneno de una serpiente del género Bothrops: Reporte de un caso / Hemolytic uremic syndrome due to ingestion of venom from a snake of the genus Bothrops: Report of a case

Resumen El síndrome hemolítico urémico (SHU) es una enfermedad que se caracteriza por la tríada anemia hemolítica no inmune, trombocitopenia e insuficiencia renal aguda, en la que las lesiones subyacentes están mediadas por un proceso de microangiopatía trombótica (MAT) sistêmica. Existen distintas causas que pueden desencadenar el proceso de la MAT que caracteriza el SHU. Se describe el caso de una mujer, con antecedentes de diabetes mellitus e hipertensión arterial, quién ingirió veneno de serpiente del género Bothrops con fines medicinales y 72 horas después debió ser hospitalizada por edema de miembros inferiores, dolor abdominal, equimosis e ictericia. Al ingreso, se le realizó analítica sanguínea que constató trombocitopenia, elevación de azoados y anemia; de igual forma se documentaron datos de falla renal aguda, por lo que se diagnosticó con SHU secundario a la ingesta de veneno de serpiente. En el artículo se describen aspectos del diagnóstico y el manejo clínico que se realizó y se estable que el compromiso renal por veneno de serpiente Bothrops es una entidad poco frecuente que si no se maneja de manera adecuada puede ser letal, y más aún si existen comorbilidades predisponentes.

Abstract Hemolytic uremic syndrome (HUS) is a disease characterized by the triad of non-immune hemolytic anemia, thrombocytopenia, and acute renal failure, in which the underlying lesions are mediated by a process of systemic thrombotic microangiopathy (TMA). There are different causes that can trigger the MAT process that characterizes HUS. The case of a woman with a history of diabetes mellitus and arterial hypertension is described, who ingested snake venom of the genus Bothrops for medicinal purposes, and 72 hours later she had to be hospitalized for edema of the lower limbs, abdominal pain, ecchymosis and jaundice. Upon admission, he underwent blood tests that found thrombocytopenia, elevated nitrogen levels and anemia; Similarly, data on acute kidney failure were documented, for which HUS was diagnosed secondary to the ingestion of snake venom. The article describes aspects of the diagnosis, and the clinical management that was carried out and it was established that renal involvement by Bothrops snake venom is a rare entity, which if not managed properly can be lethal, and even more so if there are predisposing comorbidities.

Hemorragia cerebral fatal después de una mordedura de serpiente Bothrops asper en la región del Catatumbo, Colombia / Fatal brain hemorrhage after Bothrops asper bite in the Catatumbo region of Colombia

Resumen: Las mordeduras de serpientes son un problema de salud pública en regiones tropicales y subtropicales del mundo. Ocurren, especialmente, en trabajadores rurales, y son una importante fuente de discapacidad y mortalidad. Se presenta el caso de un hombre de 59 años, agricultor de la región del Catatumbo (Colombia), quien sufrió la mordedura de una serpiente Bothrops asper, la cual le produjo una hemorragia cerebral fatal. Se llama la atención sobre el grave trastorno hemorrágico en contraste con los leves cambios en el sitio de la mordedura, así como sobre la necesidad del tratamiento temprano de la intoxicación con el suero antiofídico, incluso, en ausencia de manifestaciones cutáneas significativas.

Abstract: Snake bites are a public health problem in tropical and subtropical regions of the world. They occur especially in rural workers and are an important source of disability and mortality. We present the case of a 59-year-old farmer from the Catatumbo region of Colombia who was bitten by a B. asper snake and suffered a fatal brain hemorrhage after the event. We draw attention to the severe bleeding disorder in contrast with the slight changes at the site of the bite, as well as on the importance of the early treatment of poisoning with antivenom even in the absence of significant skin manifestations.