Pub Date : 2024-08-13DOI: 10.1016/j.toxcx.2024.100202
Andrés Sánchez, Maykel Cerdas, Jairo Gutiérrez, Mariángela Vargas, Álvaro Segura, María Herrera, Stephanie Chaves-Araya, Ronald Sánchez, Mauren Villalta, Gina Durán, Adriana Sánchez, Gabriela Solano, Daniel Cordero, Paola Sánchez, José María Gutiérrez, Guillermo León
The performance of dynamic body-feed filtration (DBF) in the removal of bulky solids produced during the manufacturing of snake antivenoms using the caprylic acid method was evaluated. For this purpose, diatomites with different filterability properties were compared in a bench-scale study to assess their effectiveness in removing the precipitated material formed after the addition of caprylic acid to equine hyperimmune plasma. C1000 diatomite at a concentration of 90 g/L of precipitated plasma showed the best performance. Then, the process was scaled up to three batches of 50 L of hyperimmune horse plasma. At this pilot scale, 108 ± 4% of the immunoglobulins present following plasma precipitation were recovered after DBF. The antivenoms generated using this procedure met quality specifications. When compared to open filtration systems commonly used at an industrial scale by many antivenom manufacturers, DBF has a similar yield and produces filtrates with comparable physicochemical characteristics. However, DBF ensures the microbiological quality of the primary clarification in a way that open systems cannot. This is because: 1) DBF is performed in a single-use closed device of depth filters which prevents microbial contamination, and 2) DBF removes bulky material in few minutes instead of the more than 24 h needed by open filtration systems, thus reducing the risk of contamination. It was concluded that DBF is a cost-effective, easily validated, and GMP-compliant alternative for primary clarification following caprylic acid precipitation of plasma in snake antivenom production.
{"title":"Pilot-scale evaluation of a dynamic body-feed filtration system for primary clarification of snake antivenoms produced by the caprylic acid method","authors":"Andrés Sánchez, Maykel Cerdas, Jairo Gutiérrez, Mariángela Vargas, Álvaro Segura, María Herrera, Stephanie Chaves-Araya, Ronald Sánchez, Mauren Villalta, Gina Durán, Adriana Sánchez, Gabriela Solano, Daniel Cordero, Paola Sánchez, José María Gutiérrez, Guillermo León","doi":"10.1016/j.toxcx.2024.100202","DOIUrl":"10.1016/j.toxcx.2024.100202","url":null,"abstract":"<div><p>The performance of dynamic body-feed filtration (DBF) in the removal of bulky solids produced during the manufacturing of snake antivenoms using the caprylic acid method was evaluated. For this purpose, diatomites with different filterability properties were compared in a bench-scale study to assess their effectiveness in removing the precipitated material formed after the addition of caprylic acid to equine hyperimmune plasma. C1000 diatomite at a concentration of 90 g/L of precipitated plasma showed the best performance. Then, the process was scaled up to three batches of 50 L of hyperimmune horse plasma. At this pilot scale, 108 ± 4% of the immunoglobulins present following plasma precipitation were recovered after DBF. The antivenoms generated using this procedure met quality specifications. When compared to open filtration systems commonly used at an industrial scale by many antivenom manufacturers, DBF has a similar yield and produces filtrates with comparable physicochemical characteristics. However, DBF ensures the microbiological quality of the primary clarification in a way that open systems cannot. This is because: 1) DBF is performed in a single-use closed device of depth filters which prevents microbial contamination, and 2) DBF removes bulky material in few minutes instead of the more than 24 h needed by open filtration systems, thus reducing the risk of contamination. It was concluded that DBF is a cost-effective, easily validated, and GMP-compliant alternative for primary clarification following caprylic acid precipitation of plasma in snake antivenom production.</p></div>","PeriodicalId":37124,"journal":{"name":"Toxicon: X","volume":"23 ","pages":"Article 100202"},"PeriodicalIF":3.6,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590171024000195/pdfft?md5=3dd29b384f64d71414bc636ba033eb5f&pid=1-s2.0-S2590171024000195-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141993675","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-28DOI: 10.1016/j.toxcx.2024.100201
Stephen Wilson Kpordze , Victor Atunga Mobegi , Gideon Mutie Kikuvi , Joseph Kangangi Gikunju , Courage Kosi Setsoafia Saba , Jackan Moshe , James Hungo Kimotho
The Black mamba, D. polylepis, is one of the many venomous snakes found in Kenya, and known to account for some snakebite incidents. The Kenyan Ministry of Health data reveals annual 15,000 snakebites occurrences. Also, 1 in 15 people in Kenya gets bitten by a snake, and tragically, 1 in 147 of these individuals die of snakebite yearly. Traditionally, antivenoms for treatment are produced from horse or sheep but have complicated and expensive production issues. Alternative production approaches, such as using IgY antibodies derived from chicken egg yolks, may overcome disadvantages with traditional antivenom manufacturing techniques. In this current study, D. polylepis specific IgY polyclonal antibodies were purified from the egg yolks of chickens immunized with D. polylepis venom. These antibodies were subsequently assessed for their in-vivo neutralizing capacity vis-à-vis commercial antivenoms, PANAF-Premium and VINS. The IgY antibodies were purified by ammonium sulfate precipitation and affinity-chromatography, with quality and specificity determined by SDS-PAGE and ELISA. The LD50 of D. polylepis was found to be 0.54 mg/kg in chicks, and 0.34 mg/kg in mice, respectively. Pool of extracted IgY yielded 2.8 mg/mL concentration. Purified IgY under non-reducing and reducing conditions on SDS-PAGE exhibited a single-protein band of about 183 kDa and two bands (67 kDa and 25 kDa), respectively. The minimum-edematogenic dose was 0.05 μg. Anti-D. polylepis IgY antibodies and two antivenoms demonstrated the capacity to neutralize the toxic activities of D. polylepis venom. This study confirms a successful IgY generation against Black mamba venom for the first time, and observed toxic effects of the venom as well as neutralizing capacity of antivenoms.
{"title":"Generation of chicken-based IgY polyclonal antibodies against Dendroaspis polylepis and preclinical evaluation of envenomation-neutralizing efficacy vis-à-vis selected commercial antivenoms","authors":"Stephen Wilson Kpordze , Victor Atunga Mobegi , Gideon Mutie Kikuvi , Joseph Kangangi Gikunju , Courage Kosi Setsoafia Saba , Jackan Moshe , James Hungo Kimotho","doi":"10.1016/j.toxcx.2024.100201","DOIUrl":"https://doi.org/10.1016/j.toxcx.2024.100201","url":null,"abstract":"<div><p>The Black mamba, <em>D. polylepis</em>, is one of the many venomous snakes found in Kenya, and known to account for some snakebite incidents. The Kenyan Ministry of Health data reveals annual 15,000 snakebites occurrences. Also, 1 in 15 people in Kenya gets bitten by a snake, and tragically, 1 in 147 of these individuals die of snakebite yearly. Traditionally, antivenoms for treatment are produced from horse or sheep but have complicated and expensive production issues. Alternative production approaches, such as using IgY antibodies derived from chicken egg yolks, may overcome disadvantages with traditional antivenom manufacturing techniques. In this current study, <em>D. polylepis</em> specific IgY polyclonal antibodies were purified from the egg yolks of chickens immunized with <em>D. polylepis</em> venom. These antibodies were subsequently assessed for their <em>in-vivo</em> neutralizing capacity vis-à-vis commercial antivenoms, PANAF-Premium and VINS. The IgY antibodies were purified by ammonium sulfate precipitation and affinity-chromatography, with quality and specificity determined by SDS-PAGE and ELISA. The LD<sub>50</sub> of <em>D. polylepis</em> was found to be 0.54 mg/kg in chicks, and 0.34 mg/kg in mice, respectively. Pool of extracted IgY yielded 2.8 mg/mL concentration. Purified IgY under non-reducing and reducing conditions on SDS-PAGE exhibited a single-protein band of about 183 kDa and two bands (67 kDa and 25 kDa), respectively. The minimum-edematogenic dose was 0.05 μg. Anti-<em>D. polylepis</em> IgY antibodies and two antivenoms demonstrated the capacity to neutralize the toxic activities of <em>D. polylepis</em> venom. This study confirms a successful IgY generation against Black mamba venom for the first time, and observed toxic effects of the venom as well as neutralizing capacity of antivenoms.</p></div>","PeriodicalId":37124,"journal":{"name":"Toxicon: X","volume":"23 ","pages":"Article 100201"},"PeriodicalIF":3.6,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590171024000183/pdfft?md5=bbf40e6654e30620cd2d2ab2d4cacd6e&pid=1-s2.0-S2590171024000183-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141482434","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-06DOI: 10.1016/j.toxcx.2024.100199
Aspassia D. Chatziefthimiou , James S. Metcalf , William B. Glover , James T. Powell , Sandra A. Banack , Paul A. Cox , Moncef Ladjimi , Ali A. Sultan , Hiam Chemaitelly , Renee A. Richer
Biocrusts dominate the soil surface in deserts and are composed of diverse microbial communities that provide important ecosystem services. Cyanobacteria in biocrusts produce many secondary metabolites, including the neurotoxins BMAA, AEG, DAB, anatoxin-a(S) (guanitoxin), and the microcystin hepatotoxins, all known or suspected to cause disease or illness in humans and other animals. We examined cyanobacterial growth and prevalence of these toxins in biocrusts at millimeter-scales, under a desert-relevant illumination gradient. In contrast to previous work, we showed that hydration had an overall positive effect on growth and toxin accumulation, that nitrogen was not correlated with growth or toxin production, and that phosphorus enrichment negatively affected AEG and BMAA concentrations. Excess illumination positively correlated with AEG, and negatively correlated with all other toxins and growth. Basic pH negatively affected only the accumulation of BMAA. Anatoxin-a(S) (guanitoxin) was not correlated with any tested variables, while microcystins were not detected in any of the samples. Concerning toxin pools, AEG and BMAA were good predictors of the presence of one another. In a newly conceptualized scheme, we integrate aspects of biocrust growth and toxin pool accumulations with arid-relevant desertification drivers.
{"title":"Cyanotoxin accumulation and growth patterns of biocrust communities under variable environmental conditions","authors":"Aspassia D. Chatziefthimiou , James S. Metcalf , William B. Glover , James T. Powell , Sandra A. Banack , Paul A. Cox , Moncef Ladjimi , Ali A. Sultan , Hiam Chemaitelly , Renee A. Richer","doi":"10.1016/j.toxcx.2024.100199","DOIUrl":"https://doi.org/10.1016/j.toxcx.2024.100199","url":null,"abstract":"<div><p>Biocrusts dominate the soil surface in deserts and are composed of diverse microbial communities that provide important ecosystem services. Cyanobacteria in biocrusts produce many secondary metabolites, including the neurotoxins BMAA, AEG, DAB, anatoxin-a(<em>S</em>) (guanitoxin), and the microcystin hepatotoxins, all known or suspected to cause disease or illness in humans and other animals. We examined cyanobacterial growth and prevalence of these toxins in biocrusts at millimeter-scales, under a desert-relevant illumination gradient. In contrast to previous work, we showed that hydration had an overall positive effect on growth and toxin accumulation, that nitrogen was not correlated with growth or toxin production, and that phosphorus enrichment negatively affected AEG and BMAA concentrations. Excess illumination positively correlated with AEG, and negatively correlated with all other toxins and growth. Basic pH negatively affected only the accumulation of BMAA. Anatoxin-a(<em>S</em>) (guanitoxin) was not correlated with any tested variables, while microcystins were not detected in any of the samples. Concerning toxin pools, AEG and BMAA were good predictors of the presence of one another. In a newly conceptualized scheme, we integrate aspects of biocrust growth and toxin pool accumulations with arid-relevant desertification drivers.</p></div>","PeriodicalId":37124,"journal":{"name":"Toxicon: X","volume":"23 ","pages":"Article 100199"},"PeriodicalIF":0.0,"publicationDate":"2024-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S259017102400016X/pdfft?md5=f0941ac9f6ce1df1faba6aa715d3d57a&pid=1-s2.0-S259017102400016X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141325915","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nowadays, more than two billion inhabitants of underdeveloped tropical and subtropical countries are at risk of being stung by scorpions. Scorpion stings annually cause 2000–3000 deaths as they can lead to the respiratory and/or cardiovascular complications. Pathogenesis of lung damage under scorpion envenomation is often comprehensive. Respiratory failure can have a cardiogenic origin, associated with venom neurotoxin action. However, some venom components can stimulate pro-inflammatory signaling cascades followed by cytokines synthesis, recruit and activate immune cells, participating in the inflammatory response in lung injury. Scorpions of the Leiurus genus ("deathstalker") are one of the most dangerous Arthropoda. To date, 22 species of this genus have been described, but the venom composition and the mechanisms of tissues damage under envenomation have been studied to some extent only for L. quinquestriatus, L. hebraeus, and L. abdullahbayrami. Scorpions of L. macroctenus species are expected to be very hazardous, but the possibility of their venom cause inflammation in the lung tissue has not been investigated to date. Therefore, in this study, we focused on evaluating the levels of cytokines and their regulators – transcription factors (HIF-1α and NF-κB) and growth factors (FGF-2, VEGF, and EGF) – in rat lung homogenates after L. macroctenus envenomation. The results revealed a decrease in the levels of most pro-inflammatory cytokines (IL-6, IL-8, IL-1β and TNF-α) with simultaneous rise in the content of both anti-inflammatory cytokines (IL-4 and IL-10) and interferon-γ. Furthermore, the levels of all researched transcription factors and growth factors were shown to be increased too. The detected changes peak occurred at 24 h, whereas a tendency towards all indicators values normalization was observed in 72 h after venom injection. Thus, our results did not reveal signs of a classic inflammatory process in the lungs of rats injected with L. macroctenus venom. However, the obtained data indicate venom influence both on cytokine profile and on their regulators content in the rat lungs, which is a feature of certain alterations in the innate immune response, caused by studied venom components. But, the mechanisms of the changes we found require additional researches.
{"title":"Cytokines and their regulators in rat lung following scorpion envenomation","authors":"Valery Gunas , Oleksandr Maievskyi , Tatyana Synelnyk , Nataliia Raksha , Tetiana Vovk , Tetiana Halenova , Olexiy Savchuk , Igor Gunas","doi":"10.1016/j.toxcx.2024.100198","DOIUrl":"https://doi.org/10.1016/j.toxcx.2024.100198","url":null,"abstract":"<div><p>Nowadays, more than two billion inhabitants of underdeveloped tropical and subtropical countries are at risk of being stung by scorpions. Scorpion stings annually cause 2000–3000 deaths as they can lead to the respiratory and/or cardiovascular complications. Pathogenesis of lung damage under scorpion envenomation is often comprehensive. Respiratory failure can have a cardiogenic origin, associated with venom neurotoxin action. However, some venom components can stimulate pro-inflammatory signaling cascades followed by cytokines synthesis, recruit and activate immune cells, participating in the inflammatory response in lung injury. Scorpions of the <em>Leiurus</em> genus (\"deathstalker\") are one of the most dangerous <em>Arthropoda</em>. To date, 22 species of this genus have been described, but the venom composition and the mechanisms of tissues damage under envenomation have been studied to some extent only for <em>L. quinquestriatus</em>, <em>L. hebraeus</em>, and <em>L. abdullahbayrami</em>. Scorpions of L. <em>macroctenus</em> species are expected to be very hazardous, but the possibility of their venom cause inflammation in the lung tissue has not been investigated to date. Therefore, in this study, we focused on evaluating the levels of cytokines and their regulators – transcription factors (HIF-1α and NF-κB) and growth factors (FGF-2, VEGF, and EGF) – in rat lung homogenates after <em>L. macroctenus</em> envenomation. The results revealed a decrease in the levels of most pro-inflammatory cytokines (IL-6, IL-8, IL-1β and TNF-α) with simultaneous rise in the content of both anti-inflammatory cytokines (IL-4 and IL-10) and interferon-γ. Furthermore, the levels of all researched transcription factors and growth factors were shown to be increased too. The detected changes peak occurred at 24 h, whereas a tendency towards all indicators values normalization was observed in 72 h after venom injection. Thus, our results did not reveal signs of a classic inflammatory process in the lungs of rats injected with <em>L. macroctenus</em> venom. However, the obtained data indicate venom influence both on cytokine profile and on their regulators content in the rat lungs, which is a feature of certain alterations in the innate immune response, caused by studied venom components. But, the mechanisms of the changes we found require additional researches.</p></div>","PeriodicalId":37124,"journal":{"name":"Toxicon: X","volume":"22 ","pages":"Article 100198"},"PeriodicalIF":0.0,"publicationDate":"2024-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590171024000158/pdfft?md5=8ddc657f0aa88232fe56ede6c867b4d7&pid=1-s2.0-S2590171024000158-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140540487","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-07DOI: 10.1016/j.toxcx.2024.100197
Mátyás A. Bittenbinder , Eric Wachtel , Daniel Da Costa Pereira , Julien Slagboom , Nicholas R. Casewell , Paul Jennings , Jeroen Kool , Freek J. Vonk
Snakebite envenoming is a global health issue that affects millions of people worldwide, and that causes morbidity rates surpassing 450,000 individuals annually. Patients suffering from snakebite morbidities may experience permanent disabilities such as pain, blindness and amputations. The (local) tissue damage that causes these life-long morbidities is the result of cell- and tissue-damaging toxins present in the venoms. These compounds belong to a variety of toxin classes and may affect cells in various ways, for example, by affecting the cell membrane. In this study, we have developed a high-throughput in vitro assay that can be used to study membrane disruption caused by snake venoms using phospholipid vesicles from egg yolk as a substrate. Resuspended chicken egg yolk was used to form these vesicles, which were fluorescently stained to allow monitoring of the degradation of egg yolk vesicles on a plate reader. The assay proved to be suitable for studying phospholipid vesicle degradation of crude venoms and was also tested for its applicability for neutralisation studies of varespladib, which is a PLA2 inhibitor. We additionally made an effort to identify the responsible toxins using liquid chromatography, followed by post-column bioassaying and protein identification using high-throughput venomics. We successfully identified various toxins in the venoms of C. rhodostoma and N. mossambica, which are likely to be involved in the observed vesicle-degrading effect. This indicates that the assay can be used for screening the membrane degrading activity of both crude and fractionated venoms as well as for neutralisation studies.
毒蛇咬伤是一个全球性的健康问题,影响着全球数百万人,每年的发病率超过 45 万人。被蛇咬伤的患者可能会终身残疾,如疼痛、失明和截肢。造成这些终身疾病的(局部)组织损伤是毒液中的细胞和组织损伤毒素造成的。这些化合物属于多种毒素类别,可通过各种方式影响细胞,例如影响细胞膜。在这项研究中,我们开发了一种高通量体外检测方法,可以利用蛋黄中的磷脂囊泡作为底物来研究蛇毒造成的细胞膜破坏。我们使用重悬的鸡卵黄来形成这些囊泡,并对其进行荧光染色,以便在平板阅读器上监测卵黄囊泡的降解情况。事实证明,这种检测方法适用于研究粗制毒液的磷脂囊泡降解,同时还测试了它是否适用于varespladib(一种PLA2抑制剂)的中和研究。此外,我们还努力利用液相色谱法鉴定毒素,然后进行柱后生物测定,并利用高通量毒液组学鉴定蛋白质。我们成功鉴定了 C. rhodostoma 和 N. mossambica 毒液中的多种毒素,这些毒素很可能参与了观察到的囊泡降解效应。这表明该检测方法可用于筛选粗制和分馏毒液的膜降解活性以及中和研究。
{"title":"Development of a membrane-disruption assay using phospholipid vesicles as a proxy for the detection of cellular membrane degradation","authors":"Mátyás A. Bittenbinder , Eric Wachtel , Daniel Da Costa Pereira , Julien Slagboom , Nicholas R. Casewell , Paul Jennings , Jeroen Kool , Freek J. Vonk","doi":"10.1016/j.toxcx.2024.100197","DOIUrl":"https://doi.org/10.1016/j.toxcx.2024.100197","url":null,"abstract":"<div><p>Snakebite envenoming is a global health issue that affects millions of people worldwide, and that causes morbidity rates surpassing 450,000 individuals annually. Patients suffering from snakebite morbidities may experience permanent disabilities such as pain, blindness and amputations. The (local) tissue damage that causes these life-long morbidities is the result of cell- and tissue-damaging toxins present in the venoms. These compounds belong to a variety of toxin classes and may affect cells in various ways, for example, by affecting the cell membrane. In this study, we have developed a high-throughput <em>in vitro</em> assay that can be used to study membrane disruption caused by snake venoms using phospholipid vesicles from egg yolk as a substrate. Resuspended chicken egg yolk was used to form these vesicles, which were fluorescently stained to allow monitoring of the degradation of egg yolk vesicles on a plate reader. The assay proved to be suitable for studying phospholipid vesicle degradation of crude venoms and was also tested for its applicability for neutralisation studies of varespladib, which is a PLA<sub>2</sub> inhibitor. We additionally made an effort to identify the responsible toxins using liquid chromatography, followed by post-column bioassaying and protein identification using high-throughput venomics. We successfully identified various toxins in the venoms of <em>C. rhodostoma</em> and <em>N. mossambica,</em> which are likely to be involved in the observed vesicle-degrading effect. This indicates that the assay can be used for screening the membrane degrading activity of both crude and fractionated venoms as well as for neutralisation studies.</p></div>","PeriodicalId":37124,"journal":{"name":"Toxicon: X","volume":"22 ","pages":"Article 100197"},"PeriodicalIF":0.0,"publicationDate":"2024-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590171024000146/pdfft?md5=f3c6b065aae885ef6b6ad2c21df4387c&pid=1-s2.0-S2590171024000146-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140546317","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-03DOI: 10.1016/j.toxcx.2024.100195
Xavier Araya , Mitchel Okumu , Gina Durán , Aarón Gómez , José María Gutiérrez , Guillermo León
Mice are routinely used in snake venom research but are costly and subject to pain and suffering. The crustacean Artemia salina could be an alternative to mice, but data to support its adoption in snake venom research is limited. The aim of the present study was to evaluate the suitability of A. salina as a surrogate of mice in assessing the toxicity of venoms and the preclinical efficacy of antivenoms. The toxicity of venoms from 22 snakes of medical importance in sub–Saharan Africa was evaluated in mice (intraperitoneally; i.p. and intravenously; i.v.) and in A. salina. Subsequently, the capacity of a commercial antivenom to neutralize the toxicity of these venoms in mice and A. salina was investigated. There was a positive correlation between the i.v. median lethal doses (LD50s) and the i.p. LD50s in mice (r = 0.804; p < 0.0001), a moderate correlation between the i.v. LD50s in mice and the median lethal concentrations (LC50s) in A. salina (r = 0.606; p = 0.003), and a moderate correlation between the i.p. LD50s in mice and the LC50s in A. salina (r = 0.426; p = 0.048). Moreover, there was a strong correlation between the i.p. median effective doses (ED50s) and the i.v. ED50s in mice (r = 0.941, p < 0.0001), between the i.p. ED50s in mice and the ED50s in A. salina (r = 0.818, p < 0.0001), and between the i.v. ED50s in mice and the ED50s in A. salina (r = 0.972, p < 0.0001). These findings present A. salina as a promising candidate for reducing reliance on mice in snake venom research. Future investigations should build upon these findings, addressing potential limitations and expanding the scope of A. salina in venom research and antivenom development.
{"title":"Assessment of the Artemia salina toxicity assay as a substitute of the mouse lethality assay in the determination of venom-induced toxicity and preclinical efficacy of antivenom","authors":"Xavier Araya , Mitchel Okumu , Gina Durán , Aarón Gómez , José María Gutiérrez , Guillermo León","doi":"10.1016/j.toxcx.2024.100195","DOIUrl":"https://doi.org/10.1016/j.toxcx.2024.100195","url":null,"abstract":"<div><p>Mice are routinely used in snake venom research but are costly and subject to pain and suffering. The crustacean <em>Artemia salina</em> could be an alternative to mice, but data to support its adoption in snake venom research is limited. The aim of the present study was to evaluate the suitability of <em>A. salina</em> as a surrogate of mice in assessing the toxicity of venoms and the preclinical efficacy of antivenoms. The toxicity of venoms from 22 snakes of medical importance in sub–Saharan Africa was evaluated in mice (intraperitoneally; i.p. and intravenously; i.v.) and in <em>A. salina</em>. Subsequently, the capacity of a commercial antivenom to neutralize the toxicity of these venoms in mice and <em>A. salina</em> was investigated. There was a positive correlation between the i.v. median lethal doses (LD<sub>50s</sub>) and the i.p. LD<sub>50s</sub> in mice (r = 0.804; p < 0.0001)<em>,</em> a moderate correlation between the i.v. LD<sub>50s</sub> in mice and the median lethal concentrations (LC<sub>50s</sub>) in <em>A. salina</em> (r = 0.606; p = 0.003)<em>,</em> and a moderate correlation between the i.p. LD<sub>50s</sub> in mice and the LC<sub>50s</sub> in <em>A. salina (</em>r = 0.426; p = 0.048). Moreover, there was a strong correlation between the i.p. median effective doses (ED<sub>50s</sub>) and the i.v. ED<sub>50s</sub> in mice (r = 0.941, p < 0.0001)<em>,</em> between the i.p. ED<sub>50s</sub> in mice and the ED<sub>50s</sub> in <em>A. salina</em> (r = 0.818, p < 0.0001)<em>,</em> and between the i.v. ED<sub>50s</sub> in mice and the ED<sub>50s</sub> in <em>A. salina</em> (r = 0.972, p < 0.0001). These findings present <em>A. salina</em> as a promising candidate for reducing reliance on mice in snake venom research. Future investigations should build upon these findings, addressing potential limitations and expanding the scope of <em>A. salina</em> in venom research and antivenom development.</p></div>","PeriodicalId":37124,"journal":{"name":"Toxicon: X","volume":"22 ","pages":"Article 100195"},"PeriodicalIF":0.0,"publicationDate":"2024-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590171024000122/pdfft?md5=aa23aafcc27eb0fb3fdb905f3edcbc73&pid=1-s2.0-S2590171024000122-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140535666","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-03DOI: 10.1016/j.toxcx.2024.100196
Mya Nila Win , Khin Than Yee , Kyae Mhon Htwe , Ei Ei Thin , Su Mon Win , Aung Myat Kyaw , Myo Myo Aye , Kyaw Kyaw Khaing , Wai Myat Thwe , Khin Khin Htwe , Aung Zaw
Snakebite is a neglected public health issue, with many scientific and medical issues to be solved. Cobras are among the most common venomous snakes in Myanmar and are responsible for a considerable number of severe snakebite envenoming. There are three species of cobra (Naja kaouthia, Naja mandalayensis and Ophiophagus hannah) in Myanmar. The study aims to characterize the N. kaouthia and N. mandalayensis venoms and to investigate the efficacy of anti-cobra antivenom (BPI) against the two venoms. Protein components and fibrinogenolytic activity were determined by SDS-PAGE. Enzymatic activities for PLA2, protease and acetylcholinesterase were determined by spectrophotometric method. Anticoagulant activity was determined by recalcification time of citrated human plasma. Myotoxicity, necrotizing activity, median lethal dose (LD50) and median effective dose (ED50) were determined by WHO recommended methods. The SDS-PAGE displayed the proteins and enzymes containing in two venoms were different. N. kaouthia venom exhibited more in PLA2, acetylcholinesterase, anticoagulant, fibrinogenolytic and necrotizing activities than N. mandalayensis venom. N. mandalayensis venom had more protease activity and myotoxicity than N. kaouthia venom. The median lethal dose (LD50) of N. kaouthia and N. mandalayensis venom was 4.33 μg/mouse and 5.04 μg/mouse respectively. Both venoms induced fibrinogen Aα chain degradation in 30 min (N. kaouthia) and in 6 h (N. mandalayensis). The same median effective dose (ED50) (19.56 μg/mouse) showed that anti-NK antivenom can neutralize against lethal effect of N. mandalayensis venom. It can also neutralize the protease activity, anticoagulant activity and fibrinogenolytic activity of both venoms. Immunodiffusion and immunoblotting studies showed that the antivenom recognized its homologous venom (N. kaouthia) and cross-reacted against the heterologous venom (N. mandalayensis). The anti-NK antivenom is suitable to use for N. mandalayensis bite if monospecific antivenom is not available.
蛇咬伤是一个被忽视的公共卫生问题,有许多科学和医学问题有待解决。眼镜蛇是缅甸最常见的毒蛇之一,也是造成大量严重蛇咬伤的罪魁祸首。缅甸有三种眼镜蛇(Naja kaouthia、Naja mandalayensis 和 Ophiophagus hannah)。本研究旨在描述 N. kaouthia 和 N. mandalayensis 毒液的特征,并调查抗眼镜蛇毒液(BPI)对这两种毒液的疗效。蛋白质成分和纤维蛋白原溶解活性通过 SDS-PAGE 进行测定。用分光光度法测定了 PLA2、蛋白酶和乙酰胆碱酯酶的酶活性。抗凝活性通过柠檬酸化人血浆的再凝时间来测定。肌毒性、坏死活性、中位致死剂量(LD50)和中位有效剂量(ED50)采用世界卫生组织推荐的方法测定。SDS-PAGE 显示两种毒液所含的蛋白质和酶不同。N.kaouthia毒液的PLA2、乙酰胆碱酯酶、抗凝血、纤维蛋白原溶解和坏死活性均高于N.mandalayensis毒液。曼德勒毒液的蛋白酶活性和肌毒性高于卡乌瑟氏毒液。N.kaouthia和N.mandalayensis毒液的中位致死剂量(LD50)分别为4.33微克/小鼠和5.04微克/小鼠。两种毒液都能在 30 分钟内(N. kaouthia)和 6 小时内(N. mandalayensis)诱导纤维蛋白原 Aα 链降解。相同的中位有效剂量(ED50)(19.56 μg/只小鼠)表明,抗 NK 毒液可以中和曼德勒虫毒液的致死效应。它还能中和两种毒液的蛋白酶活性、抗凝血活性和纤维蛋白原溶解活性。免疫扩散和免疫印迹研究表明,抗蛇毒血清能识别同源毒液(N. kaouthia),并与异源毒液(N. mandalayensis)发生交叉反应。如果没有单特异性抗蛇毒血清,抗 NK 抗蛇毒血清适用于曼德勒虫咬伤。
{"title":"Biochemical and biological characterization of the venoms of Naja kaouthia and Naja mandalayensis from Myanmar and neutralization effects of BPI cobra antivenom","authors":"Mya Nila Win , Khin Than Yee , Kyae Mhon Htwe , Ei Ei Thin , Su Mon Win , Aung Myat Kyaw , Myo Myo Aye , Kyaw Kyaw Khaing , Wai Myat Thwe , Khin Khin Htwe , Aung Zaw","doi":"10.1016/j.toxcx.2024.100196","DOIUrl":"https://doi.org/10.1016/j.toxcx.2024.100196","url":null,"abstract":"<div><p>Snakebite is a neglected public health issue, with many scientific and medical issues to be solved. Cobras are among the most common venomous snakes in Myanmar and are responsible for a considerable number of severe snakebite envenoming. There are three species of cobra (<em>Naja kaouthia</em>, <em>Naja mandalayensis</em> and <em>Ophiophagus hannah</em>) in Myanmar. The study aims to characterize the <em>N. kaouthia</em> and <em>N. mandalayensis</em> venoms and to investigate the efficacy of anti-cobra antivenom (BPI) against the two venoms. Protein components and fibrinogenolytic activity were determined by SDS-PAGE. Enzymatic activities for PLA<sub>2</sub>, protease and acetylcholinesterase were determined by spectrophotometric method. Anticoagulant activity was determined by recalcification time of citrated human plasma. Myotoxicity, necrotizing activity, median lethal dose (LD<sub>50</sub>) and median effective dose (ED<sub>50</sub>) were determined by WHO recommended methods. The SDS-PAGE displayed the proteins and enzymes containing in two venoms were different. <em>N. kaouthia</em> venom exhibited more in PLA<sub>2</sub>, acetylcholinesterase, anticoagulant, fibrinogenolytic and necrotizing activities than <em>N. mandalayensis</em> venom. <em>N.</em> mandalayensis venom had more protease activity and myotoxicity than <em>N. kaouthia</em> venom. The median lethal dose (LD<sub>50</sub>) of <em>N. kaouthia</em> and <em>N. mandalayensis</em> venom was 4.33 μg/mouse and 5.04 μg/mouse respectively. Both venoms induced fibrinogen Aα chain degradation in 30 min (<em>N. kaouthia</em>) and in 6 h (<em>N. mandalayensis</em>). The same median effective dose (ED<sub>50</sub>) (19.56 μg/mouse) showed that anti-NK antivenom can neutralize against lethal effect of <em>N. mandalayensis</em> venom. It can also neutralize the protease activity, anticoagulant activity and fibrinogenolytic activity of both venoms. Immunodiffusion and immunoblotting studies showed that the antivenom recognized its homologous venom (<em>N. kaouthia</em>) and cross-reacted against the heterologous venom (<em>N. mandalayensis</em>). The anti-NK antivenom is suitable to use for <em>N. mandalayensis</em> bite if monospecific antivenom is not available.</p></div>","PeriodicalId":37124,"journal":{"name":"Toxicon: X","volume":"22 ","pages":"Article 100196"},"PeriodicalIF":0.0,"publicationDate":"2024-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590171024000134/pdfft?md5=08cd8669236fb49e2fffc8ed64512fd9&pid=1-s2.0-S2590171024000134-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140558811","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-16DOI: 10.1016/j.toxcx.2024.100185
Julien Slagboom , Abigail H. Lewis , Wietse M. Schouten , Rien van Haperen , Mieke Veltman , Mátyás A. Bittenbinder , Freek J. Vonk , Nicholas R. Casewell , Frank Grosveld , Dubravka Drabek , Jeroen Kool
Snakebite envenoming is a priority Neglected Tropical Disease that causes an estimated 81,000–135,000 fatalities each year. The development of a new generation of safer, affordable, and accessible antivenom therapies is urgently needed. With this goal in mind, rigorous characterisation of the specific toxins in snake venom is key to generating novel therapies for snakebite. Monoclonal antibodies directed against venom toxins are emerging as potentially strong candidates in the development of new snakebite diagnostics and treatment. Venoms comprise many different toxins of which several are responsible for their pathological effects. Due to the large variability of venoms within and between species, formulations of combinations of human antibodies are proposed as the next generation antivenoms. Here a high-throughput screening method employing antibody-based ligand fishing of venom toxins in 384 filter-well plate format has been developed to determine the antibody target/s The approach uses Protein G beads for antibody capture followed by exposure to a full venom or purified toxins to bind their respective ligand toxin(s). This is followed by a washing/centrifugation step to remove non-binding toxins and an in-well tryptic digest. Finally, peptides from each well are analysed by nanoLC-MS/MS and subsequent Mascot database searching to identify the bound toxin/s for each antibody under investigation. The approach was successfully validated to rapidly screen antibodies sourced from hybridomas, derived from venom-immunised mice expressing either regular human antibodies or heavy-chain-only human antibodies (HCAbs).
{"title":"High throughput identification of human monoclonal antibodies and heavy-chain-only antibodies to treat snakebite","authors":"Julien Slagboom , Abigail H. Lewis , Wietse M. Schouten , Rien van Haperen , Mieke Veltman , Mátyás A. Bittenbinder , Freek J. Vonk , Nicholas R. Casewell , Frank Grosveld , Dubravka Drabek , Jeroen Kool","doi":"10.1016/j.toxcx.2024.100185","DOIUrl":"https://doi.org/10.1016/j.toxcx.2024.100185","url":null,"abstract":"<div><p>Snakebite envenoming is a priority Neglected Tropical Disease that causes an estimated 81,000–135,000 fatalities each year. The development of a new generation of safer, affordable, and accessible antivenom therapies is urgently needed. With this goal in mind, rigorous characterisation of the specific toxins in snake venom is key to generating novel therapies for snakebite. Monoclonal antibodies directed against venom toxins are emerging as potentially strong candidates in the development of new snakebite diagnostics and treatment. Venoms comprise many different toxins of which several are responsible for their pathological effects. Due to the large variability of venoms within and between species, formulations of combinations of human antibodies are proposed as the next generation antivenoms. Here a high-throughput screening method employing antibody-based ligand fishing of venom toxins in 384 filter-well plate format has been developed to determine the antibody target/s The approach uses Protein G beads for antibody capture followed by exposure to a full venom or purified toxins to bind their respective ligand toxin(s). This is followed by a washing/centrifugation step to remove non-binding toxins and an in-well tryptic digest. Finally, peptides from each well are analysed by nanoLC-MS/MS and subsequent Mascot database searching to identify the bound toxin/s for each antibody under investigation. The approach was successfully validated to rapidly screen antibodies sourced from hybridomas, derived from venom-immunised mice expressing either regular human antibodies or heavy-chain-only human antibodies (HCAbs).</p></div>","PeriodicalId":37124,"journal":{"name":"Toxicon: X","volume":"21 ","pages":"Article 100185"},"PeriodicalIF":0.0,"publicationDate":"2024-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S259017102400002X/pdfft?md5=423c72530030c48d98623b5513704b96&pid=1-s2.0-S259017102400002X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139936818","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-15DOI: 10.1016/j.toxcx.2024.100186
Atefeh Sedighi, Abbas Mohammadi
Alternaria species produce several mycotoxins, such as alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin (ATX), tentoxin (TTX) and tenuazonic acid (TeA). This research aimed to isolate and identify mycotoxins from highly toxic Alternaria alternata (w19) and A. tennuisima isolates and their phytotoxicity effects. Fungal metabolites were extracted from 21-day cultures of Alternaria in a Czapek broth medium with the organic solvent chloroform/acetone and identified using the HPLC method. Alternaria metabolites were infiltrated in vivo into several plant leaves for phytotoxicity detection. The study investigated the impact of temperature, time, and metabolite concentration on phytotoxicity using the detached leaf infiltration technique. Five mycotoxins (TTX, TeA, ALT, AOH, and AME) were detected in A. alternata W19 isolate with 959.24, 102.03, 24.01, 9.04, and 2.44 ppm, respectively. A. tennuisima produce these toxins in a lower concentration. Infiltration of fungal metabolites induced leaf chlorosis and necrosis, which differs based on temperature, concentration and plant species. Based on our knowledge, this is the first report of Alternaria mycotoxins in Iran and a highly toxic isolate of A. alternata with rapid phytotoxicity on a wide range of susceptible hosts.
{"title":"Phytotoxicity effect of a highly toxic isolate of Alternaria alternata metabolites from Iran","authors":"Atefeh Sedighi, Abbas Mohammadi","doi":"10.1016/j.toxcx.2024.100186","DOIUrl":"10.1016/j.toxcx.2024.100186","url":null,"abstract":"<div><p><em>Alternaria</em> species produce several mycotoxins, such as alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin (ATX), tentoxin (TTX) and tenuazonic acid (TeA). This research aimed to isolate and identify mycotoxins from highly toxic <em>Alternaria alternata</em> (w19) and <em>A</em>. <em>tennuisima</em> isolates and their phytotoxicity effects. Fungal metabolites were extracted from 21-day cultures of <em>Alternaria</em> in a Czapek broth medium with the organic solvent chloroform/acetone and identified using the HPLC method. <em>Alternaria</em> metabolites were infiltrated <em>in vivo</em> into several plant leaves for phytotoxicity detection. The study investigated the impact of temperature, time, and metabolite concentration on phytotoxicity using the detached leaf infiltration technique. Five mycotoxins (TTX, TeA, ALT, AOH, and AME) were detected in <em>A. alternata</em> W19 isolate with 959.24, 102.03, 24.01, 9.04, and 2.44 ppm, respectively. <em>A. tennuisima</em> produce these toxins in a lower concentration. Infiltration of fungal metabolites induced leaf chlorosis and necrosis, which differs based on temperature, concentration and plant species. Based on our knowledge, this is the first report of <em>Alternaria</em> mycotoxins in Iran and a highly toxic isolate of <em>A. alternata</em> with rapid phytotoxicity on a wide range of susceptible hosts.</p></div>","PeriodicalId":37124,"journal":{"name":"Toxicon: X","volume":"21 ","pages":"Article 100186"},"PeriodicalIF":0.0,"publicationDate":"2024-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590171024000031/pdfft?md5=53ea310677808485ce94516569a05a41&pid=1-s2.0-S2590171024000031-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139883425","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-15DOI: 10.1016/j.toxcx.2024.100187
Gabriela Solano , Stuart Ainsworth , Adriana Sánchez , Mauren Villalta , Paola Sánchez , Gina Durán , José María Gutiérrez , Guillermo León
As injectable therapeutics, snake antivenoms must meet specifications for endotoxin content. The Limulus amebocyte lysate (LAL) test was used to evaluate the endotoxin content in several commercially available antivenoms released for clinical use. It was found that some products have endotoxin concentrations higher than the accepted limit for these contaminants. These results emphasize the need to include endotoxin determination as part of the routine evaluation of antivenoms by manufacturers and regulatory agencies.
{"title":"Analysis of commercially available snake antivenoms reveals high contents of endotoxins in some products","authors":"Gabriela Solano , Stuart Ainsworth , Adriana Sánchez , Mauren Villalta , Paola Sánchez , Gina Durán , José María Gutiérrez , Guillermo León","doi":"10.1016/j.toxcx.2024.100187","DOIUrl":"10.1016/j.toxcx.2024.100187","url":null,"abstract":"<div><p>As injectable therapeutics, snake antivenoms must meet specifications for endotoxin content. The <em>Limulus</em> amebocyte lysate (LAL) test was used to evaluate the endotoxin content in several commercially available antivenoms released for clinical use. It was found that some products have endotoxin concentrations higher than the accepted limit for these contaminants. These results emphasize the need to include endotoxin determination as part of the routine evaluation of antivenoms by manufacturers and regulatory agencies.</p></div>","PeriodicalId":37124,"journal":{"name":"Toxicon: X","volume":"21 ","pages":"Article 100187"},"PeriodicalIF":0.0,"publicationDate":"2024-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590171024000043/pdfft?md5=522794276dbbe073ace369bf7ea55737&pid=1-s2.0-S2590171024000043-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139885271","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}