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Pilot-scale evaluation of a dynamic body-feed filtration system for primary clarification of snake antivenoms produced by the caprylic acid method 对用于初级澄清用辛酸法生产的蛇毒血清的动态主体进料过滤系统进行试验性评估
IF 3.6 Q2 TOXICOLOGY Pub Date : 2024-08-13 DOI: 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.

我们评估了动态给体过滤(DBF)在去除使用辛酸法生产蛇毒血清过程中产生的大块固体物质方面的性能。为此,在一项台式研究中对具有不同过滤特性的硅藻土进行了比较,以评估它们在去除向马高免疫血浆中添加辛酸后形成的沉淀物方面的效果。沉淀血浆浓度为 90 克/升的 C1000 硅藻土表现最佳。然后,将该工艺放大到三批 50 升高免疫马血浆。在这一试验规模中,血浆沉淀后的免疫球蛋白有 108 ± 4% 在 DBF 后被回收。使用该程序生成的抗蛇毒血清符合质量规格。与许多抗蛇毒血清生产商在工业规模上常用的开放式过滤系统相比,DBF 的产量相似,生产的滤液理化特性也相当。不过,DBF 能确保初级澄清的微生物质量,而开放式系统则无法做到这一点。这是因为1) DBF 是在深度过滤器的一次性封闭装置中进行的,可防止微生物污染,以及 2) DBF 可在几分钟内去除大块物质,而开放式过滤系统则需要 24 小时以上,因此降低了污染风险。结论是,DBF 是一种成本效益高、易于验证且符合 GMP 标准的替代方法,可用于蛇毒抗蛇毒血清生产中血浆的辛酸沉淀后的初级澄清。
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引用次数: 0
Generation of chicken-based IgY polyclonal antibodies against Dendroaspis polylepis and preclinical evaluation of envenomation-neutralizing efficacy vis-à-vis selected commercial antivenoms 产生鸡基 IgY 多克隆抗体抗多角雉(Dendroaspis polylepis),并对与选定的商业抗蛇毒血清相比的蛇毒中和效力进行临床前评估
IF 3.6 Q2 TOXICOLOGY Pub Date : 2024-06-28 DOI: 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.

黑曼巴蛇(D. polylepis)是肯尼亚发现的众多毒蛇之一,据悉也是造成一些蛇咬伤事件的原因。肯尼亚卫生部的数据显示,每年发生 15,000 起蛇咬事件。此外,肯尼亚每 15 人中就有 1 人被蛇咬伤,而不幸的是,其中每年每 147 人中就有 1 人死于蛇咬。传统上,用于治疗的抗蛇毒血清是从马或羊身上提取的,但生产过程复杂且成本高昂。替代生产方法,如使用从鸡蛋黄中提取的 IgY 抗体,可以克服传统抗蛇毒血清生产技术的缺点。在本研究中,从用多刺蛇毒免疫的鸡蛋黄中纯化出了多刺蛇特异性 IgY 多克隆抗体。随后对这些抗体与商业抗蛇毒血清 PANAF-Premium 和 VINS 的体内中和能力进行了评估。IgY 抗体是通过硫酸铵沉淀和亲和层析法纯化的,其质量和特异性由 SDS-PAGE 和 ELISA 测定。小鸡和小鼠的半数致死剂量分别为 0.54 毫克/千克和 0.34 毫克/千克。提取的 IgY 池浓度为 2.8 毫克/毫升。在非还原和还原条件下,纯化的 IgY 在 SDS-PAGE 上分别显示出约 183 kDa 的单蛋白条带和两条条带(67 kDa 和 25 kDa)。最小致畸剂量为 0.05 μg。抗多头蝙蝠 IgY 抗体和两种抗蛇毒血清证明了中和多头蝙蝠毒液毒性的能力。这项研究首次证实了针对黑曼巴毒液的IgY抗体的成功产生,并观察到了毒液的毒性作用以及抗蛇毒血清的中和能力。
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引用次数: 0
Cyanotoxin accumulation and growth patterns of biocrust communities under variable environmental conditions 不同环境条件下生物群落的蓝藻毒素积累和生长模式
Q2 TOXICOLOGY Pub Date : 2024-06-06 DOI: 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.

生物簇在沙漠的土壤表面占主导地位,由多种微生物群落组成,提供重要的生态系统服务。生物簇中的蓝藻会产生许多次级代谢产物,包括神经毒素 BMAA、AEG、DAB、anatoxin-a(S)(胍基毒素)和微囊藻毒素肝毒素,所有这些毒素都已知或怀疑会导致人类和其他动物患病。我们在沙漠相关的光照梯度下,以毫米尺度研究了蓝藻的生长情况以及这些毒素在生物簇中的流行情况。与之前的研究不同,我们发现水合作用对蓝藻的生长和毒素积累有全面的积极影响,氮与蓝藻的生长或毒素产生无关,而磷的富集对蓝藻脑苷脂(AEG)和蓝藻生物氨(BMAA)的浓度有负面影响。过度光照与 AEG 呈正相关,而与所有其他毒素和生长呈负相关。碱性 pH 只对 BMAA 的积累产生负面影响。Anatoxin-a(S) (鸟苷酸毒素)与任何测试变量均无相关性,而在任何样本中均未检测到微囊藻毒素。关于毒素库,AEG 和 BMAA 可以很好地预测毒素库中是否存在毒素。在一个新的概念化方案中,我们将生物簇生长和毒素池积累的各个方面与干旱相关的荒漠化驱动因素结合起来。
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引用次数: 0
Cytokines and their regulators in rat lung following scorpion envenomation 蝎子中毒后大鼠肺中的细胞因子及其调节因子
Q2 TOXICOLOGY Pub Date : 2024-04-08 DOI: 10.1016/j.toxcx.2024.100198
Valery Gunas , Oleksandr Maievskyi , Tatyana Synelnyk , Nataliia Raksha , Tetiana Vovk , Tetiana Halenova , Olexiy Savchuk , Igor Gunas

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.

目前,不发达的热带和亚热带国家有 20 多亿居民面临被蝎子蜇伤的危险。由于蝎子蜇伤可导致呼吸系统和/或心血管系统并发症,每年造成 2000-3000 人死亡。蝎子中毒造成肺损伤的发病机制通常是综合性的。呼吸衰竭可能是心源性的,与毒液的神经毒素作用有关。然而,某些毒液成分可刺激促炎症信号级联,继而合成细胞因子,招募和激活免疫细胞,参与肺损伤的炎症反应。雷龙属("死亡潜行者")蝎子是最危险的节肢动物之一。迄今为止,已描述了该属的 22 个物种,但对毒液成分和毒液对组织的损害机制进行了一定程度研究的只有 L. quinquestriatus、L. hebraeus 和 L. abdullahbayrami。L.macroctenus种类的蝎子被认为是非常危险的,但其毒液导致肺部组织炎症的可能性迄今尚未得到研究。因此,在本研究中,我们重点评估了大蝎子中毒后大鼠肺匀浆中细胞因子及其调节因子--转录因子(HIF-1α和NF-κB)和生长因子(FGF-2、VEGF和EGF)--的水平。研究结果显示,大多数促炎细胞因子(IL-6、IL-8、IL-1β 和 TNF-α)的水平都有所下降,而抗炎细胞因子(IL-4 和 IL-10)和干扰素-γ的含量则同时上升。此外,所有被研究的转录因子和生长因子的水平也有所提高。检测到的变化高峰出现在注射毒液后 24 小时,而在注射毒液后 72 小时,所有指标值都趋于正常。因此,我们的结果并没有显示出注射了大爪蟾毒液的大鼠肺部出现了典型的炎症过程。然而,所获得的数据表明,毒液对大鼠肺部的细胞因子谱及其调节因子含量都有影响,这是研究毒液成分导致先天性免疫反应发生某些改变的一个特征。但是,我们发现这些变化的机制还需要进一步研究。
{"title":"Cytokines and their regulators in rat lung following scorpion envenomation","authors":"Valery Gunas ,&nbsp;Oleksandr Maievskyi ,&nbsp;Tatyana Synelnyk ,&nbsp;Nataliia Raksha ,&nbsp;Tetiana Vovk ,&nbsp;Tetiana Halenova ,&nbsp;Olexiy Savchuk ,&nbsp;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}
引用次数: 0
Development of a membrane-disruption assay using phospholipid vesicles as a proxy for the detection of cellular membrane degradation 开发以磷脂囊泡为代表的膜破坏测定法,用于检测细胞膜降解情况
Q2 TOXICOLOGY Pub Date : 2024-04-07 DOI: 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。
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引用次数: 0
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 在确定毒液引起的毒性和抗蛇毒血清的临床前疗效时,评估用盐水蒿毒性试验替代小鼠致死试验的效果
Q2 TOXICOLOGY Pub Date : 2024-04-03 DOI: 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.

蛇毒研究通常使用小鼠,但小鼠成本高昂,而且会给研究人员带来痛苦。甲壳类动物盐蒿可以替代小鼠,但支持其用于蛇毒研究的数据有限。本研究的目的是评估在评估蛇毒的毒性和抗蛇毒血清的临床前疗效时,盐水蒿是否适合作为小鼠的替代品。研究人员在小鼠体内(腹腔注射和静脉注射)评估了撒哈拉以南非洲地区 22 种具有重要医疗价值的蛇的毒液的毒性,并在盐水蛙体内进行了评估。随后,研究了商用抗蛇毒血清中和这些毒液对小鼠和蝾螈毒性的能力。小鼠的静脉注射中位致死剂量(LD50s)与静脉注射 LD50s 之间呈正相关(r = 0.804; p < 0.0001),小鼠的静脉注射 LD50s 与静脉注射 LD50s 之间呈中度相关(r = 0.804; p < 0.0001)。小鼠的静脉注射半数致死剂量与盐藻类的中位致死浓度(LC50s)之间存在中度相关性(r = 0.606;p = 0.003),小鼠的口服半数致死剂量与盐藻类的 LC50s 之间存在中度相关性(r = 0.426;p = 0.048)。此外,小鼠口服中位有效剂量(ED50s)与静脉注射 ED50s 之间有很强的相关性(r = 0.941,p < 0.0001),小鼠口服 ED50s 与静脉注射 ED50s 之间也有很强的相关性(r = 0.941,p < 0.0001)。小鼠的静脉注射 ED50s 与盐肤木的 ED50s 之间(r = 0.818,p < 0.0001),以及小鼠的静脉注射 ED50s 与盐肤木的 ED50s 之间(r = 0.972,p < 0.0001)。这些研究结果表明,在蛇毒研究中,丹顶鹤有望减少对小鼠的依赖。未来的研究应以这些发现为基础,解决潜在的局限性,并扩大盐湖蛙在蛇毒研究和抗蛇毒血清开发中的应用范围。
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引用次数: 0
Biochemical and biological characterization of the venoms of Naja kaouthia and Naja mandalayensis from Myanmar and neutralization effects of BPI cobra antivenom 缅甸 Naja kaouthia 和 Naja mandalayensis 毒液的生化和生物学特征以及 BPI 眼镜蛇抗蛇毒血清的中和效果
Q2 TOXICOLOGY Pub Date : 2024-04-03 DOI: 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。
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引用次数: 0
High throughput identification of human monoclonal antibodies and heavy-chain-only antibodies to treat snakebite 高通量鉴定治疗蛇咬伤的人类单克隆抗体和纯重链抗体
Q2 TOXICOLOGY Pub Date : 2024-02-16 DOI: 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).

蛇咬伤是一种优先防治的被忽视热带病,估计每年造成 81,000 至 135,000 人死亡。开发新一代更安全、更经济、更易获得的抗蛇毒血清疗法迫在眉睫。为实现这一目标,对蛇毒中的特异性毒素进行严格鉴定是开发新型蛇咬伤疗法的关键。针对毒液毒素的单克隆抗体正在成为开发新型蛇咬伤诊断和治疗方法的有力候选药物。毒液由多种不同的毒素组成,其中有几种是造成病理效应的原因。由于毒液在物种内部和物种之间存在很大的变异性,因此有人建议将人类抗体的组合配方作为下一代抗蛇毒血清。在此,我们开发了一种高通量筛选方法,采用基于抗体的毒液毒素配体钓法,在 384 滤孔板格式中确定抗体靶标。该方法使用蛋白 G 珠捕获抗体,然后将其暴露于全毒液或纯化毒素中,以结合各自的配体毒素。然后进行洗涤/离心步骤以去除不结合的毒素,并进行孔内胰蛋白酶消化。最后,通过纳米液相色谱-质谱-质谱(nanoLC-MS/MS)和随后的 Mascot 数据库搜索分析各孔中的肽段,以确定所研究抗体的结合毒素。该方法已成功用于快速筛选来自杂交瘤的抗体,杂交瘤来自表达普通人类抗体或纯重链人类抗体(HCAbs)的毒液免疫小鼠。
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引用次数: 0
Phytotoxicity effect of a highly toxic isolate of Alternaria alternata metabolites from Iran 伊朗高毒分离的交替孢属代谢物的植物毒性效应
Q2 TOXICOLOGY Pub Date : 2024-02-15 DOI: 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.

交替缠绕菌会产生几种霉菌毒素,如交替二醇(AOH)、交替二醇单甲醚(AME)、赤烯(ALT)、变构毒素(ATX)、担子毒素(TTX)和天脲酸(TeA)。本研究旨在从剧毒交替Alternaria(w19)和A. tennuisima分离物中分离和鉴定霉菌毒素及其植物毒性效应。在 Czapek 肉汤培养基中,用有机溶剂氯仿/丙酮从培养 21 天的交替丝核菌中提取真菌代谢物,并采用高效液相色谱法进行鉴定。将交替孢霉代谢物渗入几种植物叶片中进行植物毒性检测。该研究利用离体叶片浸润技术研究了温度、时间和代谢物浓度对植物毒性的影响。在交替蚁 W19 分离物中检测到五种霉菌毒素(TTX、TeA、ALT、AOH 和 AME),含量分别为 959.24、102.03、24.01、9.04 和 2.44 ppm。A. tennuisima 产生这些毒素的浓度较低。真菌代谢物的渗入会导致叶片萎黄和坏死,不同的温度、浓度和植物种类会导致叶片萎黄和坏死。据我们所知,这是伊朗首次报道交替孢霉毒素,也是交替孢霉的一种高毒分离物,对多种易感寄主具有快速植物毒性。
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引用次数: 0
Analysis of commercially available snake antivenoms reveals high contents of endotoxins in some products 对市售蛇类抗蛇毒血清的分析表明,一些产品的内毒素含量很高
Q2 TOXICOLOGY Pub Date : 2024-02-15 DOI: 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.

作为注射治疗药物,蛇类抗蛇毒血清必须符合内毒素含量规范。我们使用鹅卵石红细胞裂解液(LAL)测试法来评估几种已上市并用于临床的抗蛇毒血清中的内毒素含量。结果发现,一些产品的内毒素含量高于这些污染物的公认限值。这些结果表明,生产商和监管机构有必要将内毒素测定作为抗蛇毒血清常规评估的一部分。
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引用次数: 0
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