BackgroundBitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP).MethodsSolubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically.ResultsA serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity.ConclusionsThe in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO’s recommendation of immunotherapy in cases of human accidents with venomous snakes.Electronic supplementary materialThe online version of this article (10.1186/s40409-018-0176-5) contains supplementary material, which is available to authorized users.

Bitis arietans is a snake of medical importance, as it is responsible for more accidents in humans and domestic animals than all other African snakes put together. The accidents are characterized by local and systemic alterations, such as inflammation, cardiovascular and hemostatic disturbances, which can lead victims to death or permanent disability. However, little is known about the envenomation mechanism, especially regarding the inflammatory response, which is related to severe clinical conditions triggered by the venom. Therefore, the aim of the present study was to evaluate the inflammatory response related to the B. arietans envenomation using a peritonitis mice model. By pharmacological interventions and use of mice genetically deficient of the 5-lipoxygenase enzyme (5-LO−/−) or platelet-activating factor (PAF) receptor (PAFR−/− the participation of eicosanoids and PAF in this response was also investigated. The obtained results demonstrated that the venom induces an in vivo inflammatory response, characterized by an early increased vascular permeability, followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity, accompanied by the production of the eicosanoids LTB4, LTC4, TXB2 and PGE2, as well as the local and systemic production of IL-6 and MCP-1. These inflammatory events were attenuated by the pre-treatment with anti-inflammatory drugs that interfere in lipid mediators’ functions. However, 5-LO−/− mice did not show a reduction of inflammatory response induced by the venom, while PAFR−/− mice showed a reduction in both the PMN leukocytes number and the local and systemic production of IL-6 and MCP-1. This study demonstrated that the Bitis arietans venom contains toxins that trigger an inflammatory process, which is partially dependent on lipid mediators, and may contribute to the envenomation pathology.

NaPi-IIb is a multiple passage protein membrane which is primarily responsible for phosphate uptake in the kidney and in the small intestine. Beyond its physiological functions, their involvement with carcinogenesis was initially described in mid-2003, due to its distinct level of expression in normal and tumor cells of the ovary. Although less common than cervical cancer, epithelial ovarian cancer is considered the most lethal gynecologic malignancy, which is mainly due to diagnosis in the advanced stages as a result of the absence of symptoms during the onset of the disease and the lack of tools for early detection. Here, we produce antibodies that are anti-synthetic peptides that are derived from the regions of second extracellular loop of NaPi-IIb, which is a non-overlapping portion of MX35 epitope. These two 15 distinct amino acid residue peptides, designated as Let#1 and Let#2, are engineered in a very thorough way to detect specific sites only in this isoform, thus excluding cross-reactivity with other carriers of the same family. The lack of immunogenicity of small peptides is surpassed by the conjugation with carrier proteins. Using immunochemical methods, we demonstrate that polyclonal antibodies that are mono-specific for * Corresponding author. Â. A. A. Megale et al. 130the Let#1 and Let#2 peptides recognize proteins that express similar amino acid sequences during blood circulation. Additionally, using flow cytometry, we identify NaPi-IIb in NIH:OVCAR-3 cells. The clear identification of two shorter peptides on the extracellular loop of NaPi-IIb, which are far from the monoclonal antibody MX35-recognizing epitopes, adds new promising tools for ovarian cancer follow-up and staging.

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims’ death or permanent disability. To better characterize the inflammatory process induced by this snake’s venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1β in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Snakebite envenomation is considered a neglected tropical disease, affecting tens of thousands of people each year. The recommended treatment is the use of antivenom, which is composed of immunoglobulins or immunoglobulin fragments obtained from the plasma of animals hyperimmunized with one (monospecific) or several (polyspecific) venoms. In this review, the efforts made in the improvement of the already available antivenoms and the development of new antivenoms, focusing on snakes of medical importance from sub-Saharan Africa and Latin America, are described. Some antivenoms currently used are composed of whole IgGs, whereas others use F(ab’)2 fragments. The classic methods of attaining snake antivenoms are presented, in addition to new strategies to improve their effectiveness. Punctual changes in immunization protocols, in addition to the use of cross-reactivity between venoms from different snakes for the manufacture of more potent and widely used antivenoms, are presented. It is known that venoms are a complex mixture of components; however, advances in the field of antivenoms have shown that there are key toxins that, if effectively blocked, are capable of reversing the condition of in vivo envenomation. These studies provide an opportunity for the use of monoclonal antibodies in the development of new-generation antivenoms. Thus, monoclonal antibodies and their fragments are described as a possible alternative for the production of antivenoms, regardless of the venom. This review also highlights the challenges associated with their development.

The African snake Bitis arietans is of great medical importance and is found in sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. Contributes complacently in the epidemiology of snakebites in humans and animals. The lack of specific antivenoms aggravates this situation. Identifying toxins, knowing their toxic properties and developing antitoxins are the goals of emerging projects. This project aims at monoclonal anti-metalloprotease (mAbs) from the toxin of Bitis arietans venom. mAbs will serve as sources of complementarity determining regions (CDR). The main applied methodologies will not develop the project fit mainly in the purification of metalloprotease and immunization of mice to obtain lymphocytes and replicate it. So far, we have been able to highlight a metalloprotease of interest, which will be properly identified for the production of antibodies, a finding that was confirmed by proteomic and transcriptomic analyzes. The next step will be to immunize mice and validate the antibodies produced.

MEGALE, Ângela Alice Amadeu. Inflammatory properties of the Bitis arietans snake venom: contribution of lipid mediators in vivo envenomation and the action of toxins purified from the venom upon human macrophages. 2019. 203 p. Thesis (Ph.

Ao meu orientador, professor Wilmar Dias da SilvaPrimeiramente, por ter confiado em minha capacidade, e pela paciência que me ensina uma Imunologia que não encontro nos livros e, sem dúvida, por ser para mim uma constante inspiração. À minha querida mãe, Josefa Alice AmadeuPor me ensinar, com muito amor e paciência, a ser quem sou e por estar sempre ao meu lado. Ao meu amado esposo, Joel José Megale GabriliPor me apoiar em tudo que preciso, por me dar forças quando não as tenho mais, por me amar da maneira que sou e me ajudar a cada dia ser uma pessoa melhor. AGRADECIMENTOSAgradeço a Deus pela proteção, força, companhia e pelos anjos que coloca em minha vida, tornando minha caminhada mais fácil; Ao meu orientador, Dr. Wilmar Dias da Silva que acreditou em mim desde o primeiro dia.Nunca esquecerei do dia que conheci o senhor e fui apresentada ao projeto peptídeos, quando eu disse que achava o projeto e suas ideias maravilhosas, mas que sabia muito pouco de Imunologia, e o senhor me disse que isso não era problema, pois me ensinaria tudo que fosse preciso. E de fato, foi o que aconteceu. Obrigada por toda a paciência, por suas aulas e todos os ensinamentos. Muito Obrigada! É um privilégio conviver com alguém como o senhor, que além de um excelente cientista, é um ser humano admirável; Agradeço à professora Dra. Denise Tambourgi, por gentilmente ter aberto as portas do laboratório e por seus pertinentes conselhos; Ao Dr. Osvaldo Sant'anna, pela ajuda para obtenção dos camundongos da linhagem High III e da sílica SBA-15, e, principalmente por me ajudar no protocolo de imunização e por me passar um pouco de sua ampla experiência; À Dra. Leticia Batista Azevedo Rangel, por colaborar de maneira fundamental com este projeto, cedendo as sequências de aminoácidos dos peptídeos Let#1 e Let#2; Ao Instituto do Câncer do Estado de São Paulo (ICESP), pela colaboração com a coleta, armazenamento e envio das amostras de soro humano para análise, principalmente a toda a equipe do setor de pesquisa clínica coordenado pelo Dr. Roberto Jun Arai; À banca de qualificação, Dr. José Alexandre Marzagão Barbuto, Dra. Maristela Martins de Camargo e Dr. Enéas de Carvalho, pela contribuição dada a este projeto. Sem dúvida, foram de grande importância a avaliação, as correções e os conselhos passados. Em especial, agradeço o Dr. Enéas pela indispensável contribuição para a conclusão desta etapa do trabalho; À minha amada e insubstituível mãe, Josefa Alice Amadeu, por toda paciência, carinho, educação, força e amor incondicional. Que Deus te proteja sempre e que eu possa te dar muito orgulho para tentar retribuir tudo que a senhora fez e faz por mim. Te amo imensuravelmente;Ao meu melhor amigo e esposo, Joel José Megale Gabrili, que me mostra o melhor lado da vida e divide comigo todos os momentos, os tornando únicos. Te amo por tudo que você é e por estar sempre do meu lado. Sem dúvida, te conhecer e conviver com você me torna uma pessoa melhor a cada dia. Obrigada por existir e por me dar mais uma linda família para amar;Às minhas lindas e a...