Venom Toxicity Research Articles

Bacterial expression of a snake venom metalloproteinase inhibitory protein from the North American opossum (D. virginiana)

A variety of opossum species are resistant to snake venoms due to the presence of antihemorrhagic and antimyotoxic acidic serum glycoproteins that inhibit several toxic venom components. Two virtually identical antihemorrhagic proteins isolated from either the North American opossum (D. virginiana) or the South American big-eared opossum (D. aurita), termed oprin or DM43 respectively, inhibit specific snake venom metalloproteinases (SVMPs). A better understanding of the structure of these proteins may provide useful insight to determine their mechanism of action and for the development of therapeutics against the global health concern of snake-bite envenomation. The aim of this work is to produce a recombinant snake venom metalloproteinase inhibitor (SVMPI) similar to the above opossum proteins in Escherichia coli and determine if this bacterially produced protein inhibits the proteolytic properties of Western Diamondback rattlesnake (C. atrox) venom. The resulting heterologous SVMPI was produced with either a 6-Histidine or maltose binding protein (MBP) affinity tag on either the C-terminus or N-terminus of the protein, respectively. The presence of the solubility enhancing MBP affinity tag resulted in significantly more soluble protein expression. The inhibitory activity was measured using two complementary assays and the MBP labeled SVMPI showed 7-fold less activity as compared to the 6-Histidine labeled SVMPI. Thus, the bacterially derived SVMPI with an unlabeled N-terminus showed high inhibitory activity (IC50 = 4.5 μM). The use of a solubility enhancing MBP fusion protein construct appears to be a productive way to express sufficient quantities of this mammalian protein in E. coli for further study.

The Curious Case of the "Neurotoxic Skink": Scientific Literature Points to the Absence of Venom in Scincidae.

In contrast to the clearly documented evolution of venom in many animal lineages, the origin of reptilian venom is highly debated. Historically, venom has been theorised to have evolved independently in snakes and lizards. However, some of the recent works have argued for the common origin of venom in “Toxicofera” reptiles, which include the order Serpentes (all snakes), and Anguimorpha and Iguania lizards. Nevertheless, in both these contrasting hypotheses, the lizards of the family Scincidae are considered to be harmless and devoid of toxic venoms. Interestingly, an unusual clinical case claiming neurotoxic envenoming by a scincid lizard was recently reported in Southern India. Considering its potentially significant medicolegal, conservation and evolutionary implications, we have summarised the scientific evidence that questions the validity of this clinical report. We argue that the symptoms documented in the patient are likely to have resulted from krait envenomation, which is far too frequent in these regions.

King Cobra and snakebite envenomation: on the natural history, human-snake relationship and medical importance of Ophiophagus hannah.

King Cobra (Ophiophagus hannah) has a significant place in many cultures, and is a medically important venomous snake in the world. Envenomation by this snake is highly lethal, manifested mainly by neurotoxicity and local tissue damage. King Cobra may be part of a larger species complex, and is widely distributed across Southeast Asia, southern China, northern and eastern regions as well as the Western Ghats of India, indicating potential geographical variation in venom composition. There is, however, only one species-specific King Cobra antivenom available worldwide that is produced in Thailand, using venom from the snake of Thai origin. Issues relating to the management of King Cobra envenomation (e.g., variation in the composition and toxicity of the venom, limited availability and efficacy of antivenom), and challenges faced in the research of venom (in particular proteomics), are rarely addressed. This article reviews the natural history and sociocultural importance of King Cobra, cases of snakebite envenomation caused by this species, current practice of management (preclinical and clinical), and major toxinological studies of the venom with a focus on venom proteomics, toxicity and neutralization. Unfortunately, epidemiological data of King Cobra bite is scarce, and venom proteomes reported in various studies revealed marked discrepancies in details. Challenges, such as inconsistency in snake venom sampling, varying methodology of proteomic analysis, lack of mechanistic and antivenomic studies, and controversy surrounding antivenom use in treating King Cobra envenomation are herein discussed. Future directions are proposed, including the effort to establish a standard, comprehensive Pan-Asian proteomic database of King Cobra venom, from which the venom variation can be determined. Research should be undertaken to characterize the toxin antigenicity, and to develop an antivenom with improved efficacy and wider geographical utility. The endeavors are aligned with the WHO´s roadmap that aims to reduce the disease burden of snakebite by 50% before 2030.

Comparative assessment of the biological toxicity of scyphozoan jellyfish species (Nemopilema nomurai, Cyanea nozakii, Aurellia aurita and Rhopilema esculentum) venoms

Jellyfish envenomation is a world-wide health problem, which often seriously affect the fishery and bathing activities. To date, few individual jellyfish venoms proteins have been thoroughly characterized yet. In this work, four species of scyphozoan jellyfish (Nemopilema nomurai, Cyanea nozakii, Aurellia aurita and Rhopilema esculentum) are compared according to their, cytotoxicity, hemolytic potency, brine shrimp toxicity and protein components. Jellyfish venoms showed higher cytotoxicity in H9C2 heart myoblast than in C2C12 skeletal myoblast, with the exception of C. nozakii venom. This result suggests that the selective cytotoxic effects may be possibly related to their in vivo effects of cardiac tissue dysfunction. On the other hand, hemolytic activity could be also observed from all tested jellyfish venoms. N. nomurai jellyfish venom displaying the greatest hemolytic activity. As an alternative method of evaluating the toxicities of jellyfish venoms, the toxicity on brine shrimp was examined with the four jellyfish venoms. From this, the venom of N. nomurai showed higher toxicity against brine shrimp than the other jellyfish venoms, which is consistent with the results of cytotoxicity assay as well as hemolysis assay of the present study. SDS-PAGE analysis of four jellyfish venoms showed the similar pattern with molecular weight of around 40 kDa, and appeared to be the major protein components. These results provided that N. nomurai jellyfish venom was potently toxic than other scyphozoan jellyfish venoms and may explain to some extent the deleterious effects associated with human envenoming.

Searching for the toxic potential of Loxosceles amazonica and Loxosceles willianilsoni spiders’ venoms

The Loxosceles genus belongs to the Sicariidae family and it comprises species whose venom can cause accidents with potentially fatal consequences. We have previously shown that SMase D is the enzyme responsible for the main pathological effects of Loxosceles venom. Despite the severity of accidents with Loxosceles, few species are considered to be of medical importance. Little is known about the venom of non-synanthropic species that live in natural environments. To contribute to a better understanding about the venom's toxicity of Loxosceles genus, the aim of this study was to (i) characterize the toxic properties of Loxosceles amazonica from two different localities and a recent described cave species Loxosceles willianilsoni and (ii) compare these venoms with that from Loxosceles laeta, which is among the most toxic ones. We show here that both L. amazonica venoms (from the two studied locations) and L. willianilsoni presented SMase D activity similar to that exhibited by L. laeta venom. Although L. amazonica and L. willianilsoni venoms were able to induce complement dependent human erythrocytes lysis, they were not able to induce cell death of human keratinocytes, as promoted by L. laeta venom, in the concentrations tested. These results indicate that other species of Loxosceles, in addition to those classified as medically important, have toxic potential to cause accidents in humans, despite interspecific variations that denote possible less toxicity.

Genetic and toxinological divergence among populations of Tityus trivittatus Kraepelin, 1898 (Scorpiones: Buthidae) inhabiting Paraguay and Argentina.

Envenoming by scorpions in genus Tityus is a public health problem in Tropical America. One of the most medically significant species is Tityus trivittatus, which is known to occur from southwest Brazil to central-northern and eastern Argentina. In this work, we studied the lethality, composition, antigenicity, and enzymatic activity of venom from a T. trivittatus population found further north in urban areas of eastern Paraguay, where it has caused serious envenomation of children. Our results indicate that the population is of medical importance as it produces a potently toxic venom with an LD50 around 1.19 mg/kg. Venom neutralization in preliminary mouse bioassays was complete when using Brazilian anti-T. serrulatus antivenom but only partial when using Argentinean anti-T. trivittatus antivenom. Venom competitive solid-phase enzyme immunoassays and immunoblotting from Argentinean and Paraguayan T. trivittatus populations indicated that antigenic differences exist across the species range. SDS-PAGE showed variations in type and relative amounts of venom proteins between T. trivitattus samples from Argentina and Paraguay. MALDI-TOF mass spectrometry indicated that while some sodium channel toxins are shared, including β-toxin Tt1g, others are population-specific. Proteolytic activity by zymography and peptide identification through nESI-MS/MS also point out that population-specific proteases may exist in T. trivitattus, which are postulated to be involved in the envenoming process. A time-calibrated molecular phylogeny of mitochondrial COI sequences revealed a significant (8.14%) genetic differentiation between the Argentinean and Paraguayan populations, which appeared to have diverged between the mid Miocene and early Pliocene. Altogether, toxinological and genetic evidence indicate that T. trivitattus populations from Paraguay and Argentina correspond to distinct, unique cryptic species, and suggest that further venom and taxonomic diversity exists in synanthropic southern South American Tityus than previously thought.

In vitro anti-venom potential of various solvent based leaf extracts of Andrographis serpyllifolia (Rottler ex Vahl) Wight against Naja naja and Daboia russelli

Ethnopharmacological relevanceSnake bite is a major occupational hazard in tropical and subtropical countries including India as per the World Health Organization. Naja naja (Indian cobra) and Daboia russelli (Russell's viper) are the two poisonous snakes commonly associated with human mortality in India. Andrographis serpyllifolia (Rottler ex Vahl) Wight has been documented in ethnobotanical records as a plant possessing potent anti-snake venom activity. Aim of the studyThe present study is aimed for systematic evaluation of in vitro anti-venom potential of various solvent based leaf extracts of A. serpyllifolia against toxic venom enzymes of Naja naja and Daboia russelli. Materials and methodsDifferent solvent based leaf extracts of A. serpyllifolia were tested against the snake venoms of Naja naja and Daboia russelli obtained from Irula Snake Catchers Industrial Co-operative Society Limited, Kancheepuram, Tamil nadu, India. Three different in vitro neutralization assays such as indirect hemolysis, procoagulent and lytic activities and seven in vitro enzyme inhibition assays such as protease, acetylcholinesterase, phosphomonoesterase, phosphodiesterase, 5′nucleotidase, phospholipase A2, hyaluronidase and post synaptic acetylcholine receptor binding activity were carried out according to standard protocols. The results were analyzed using the standard ANOVA procedures. ResultsAmong various solvent based leaf extracts of A. serpyllifolia tested, aqueous extract showed maximum neutralizing and inhibitory activities against Naja naja and Daboia russelli venoms. ConclusionsThe various in vitro enzymatic studies reveal that the aqueous leaf extract of A. serpyllifolia plant could inhibit most of the toxic enzymes of the Naja naja and Daboia russelli venoms which could be further confirmed by in vivo studies.

Biochemical characterization of Bothrops roedingeri Mertens, 1942 snake venom and its edematogenic, hemorrhagic, and myotoxic activities

ResumenIntroducción. El envenenamiento por mordedura de serpiente es considerado por la Organización Mundial de la Salud (OMS) una enfermedad tropical desatendida. Si bien los venenos de otras serpientes Bothrops se vienen estudiado ampliamente, poco se conoce del de Bothrops roedingeri.Objetivos. Caracterizar bioquímicamente el veneno total de la serpiente B. roedingeri y evaluar su actividad miotóxica, edematógena y hemorrágica.Materiales y métodos. Se hizo la caracterización enzimática del veneno de B. roedingeri determinando la actividad de la fosfolipasa A2 (PLA2) y de las enzimas proteolíticas, así como su acción fibrinogenolítica mediante electroforesis en gel de poliacrilamida con duodecilsulfato sódico (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE), y la caracterización tóxica del veneno estableciendo la dosis hemorrágica mínima, la dosis edematógena mínima y el efecto miotóxico local y sistémico. Resultados. La actividad de las PLA2 del veneno total de B. roedingeri fue de 3,45 ± 0,11 nmoles/minuto, la proteolítica, de 0,145 ± 0,009 nmoles/minuto, en tanto que el índice de coagulación del fibrinógeno fue de 6,67 ± 1,33 segundos. Por otro lado, el veneno produjo una dosis hemorrágica mínima de 24,5 µg, una dosis edematógena mínima de 15,6 µg y un pronunciado efecto miotóxico local evidenciado por la elevación de los niveles plasmáticos de creatina cinasa después de la inoculación por vía intramuscular. No se registró miotoxicidad sistémica.Conclusiones. El veneno de B. roedingeri tiene efectos hemorrágicos, edematógenos y miotóxicos locales, así como una elevada actividad de la PLA2, que sería responsable de los efectos miotóxico y edematógeno. También presentó actividad proteolítica, la cual podría afectar la coagulación, dada su capacidad para degradar el fibrinógeno y producir hemorragia por acción de las metaloproteasas.

A New Group II Phospholipase A2 from Walterinnesia aegyptia Venom with Antimicrobial, Antifungal, and Cytotoxic Potential

Many venomous species, especially snakes, contain a variety of secreted phospholipases A2 that contribute to venom toxicity and prey digestion. We characterized a novel highly toxic phospholipase A2 of group II, WaPLA2-II, from the snake venom of Saudi Walterinnesia aegyptia (W. aegyptia). The enzyme was purified using a reverse phase C18 column. It is a monomeric protein with a molecular weight of approximately 14 kDa and an NH2-terminal amino acid sequence exhibiting similarity to the PLA2 group II enzymes. WaPLA2-II, which contains 2.5% (w/w) glycosylation, reached a maximal specific activity of 1250 U/mg at pH 9.5 and 55 °C in the presence of Ca2+ and bile salts. WaPLA2-II was also highly stable over a large pH and temperature range. A strong correlation between antimicrobial and indirect hemolytic activities of WaPLA2 was observed. Additionally, WaPLA2-II was found to be significantly cytotoxic only on cancerous cells. However, chemical modification with para-Bromophenacyl bromide (p-BPB) inhibited WaPLA2-II enzymatic activity without affecting its antitumor effect, suggesting the presence of a separate ‘pharmacological site’ in snake venom phospholipase A2 via its receptor binding affinity. This enzyme is a candidate for applications including the treatment of phospholipid-rich industrial effluents and for the food production industry. Furthermore, it may represent a new therapeutic lead molecule for treating cancer and microbial infections.

Integrating Top-Down and Bottom-Up Mass Spectrometric Strategies for Proteomic Profiling of Iranian Saw-Scaled Viper, Echis carinatus sochureki, Venom.

Saw-scaled or carpet vipers (genus Echis) are considered to cause a higher global snakebite mortality than any other snake. Echis carinatus sochureki (ECS) is a widely distributed snake species, also found across the thirteen provinces of Iran, where it is assumed to be responsible for the most snakebite envenomings. Here, we collected the Iranian specimens of ECS from three different geographically distinct populations, investigated food habits, and performed toxicity assessment and venom proteome profiling to better understand saw-scaled viper life. Our results show that the prey items most commonly found in all populations were arthropods, with scorpions from the family Buthidae particularly well represented. LD50 (median lethal dose) values of the crude venom demonstrate highly comparable venom toxicities in mammals. Consistent with this finding, venom characterization via top-down and bottom-up proteomics, applied to both crude venoms and size-exclusion chromatographic fractions, revealed highly comparable venom compositions among the different populations. By combining all proteomics data, we identified 22 protein families from 102 liquid chromatography and tandem mass spectrometry (LC-MS/MS) raw files, including the most abundant snake venom metalloproteinases (SVMPs, 29-34%); phospholipase A2 (PLA2s, 26-31%); snake venom serine proteinases (SVSPs, 11-12%); l-amino acid oxidases (LAOs, 8-11%), C-type lectins/lectin-like (CTLs, 7-9%) protein families, and many newly detected ones, e.g., renin-like aspartic proteases (RLAPs), fibroblast growth factors (FGFs), peptidyl-prolyl cis-trans isomerases (PPIs), and venom vasodilator peptides (VVPs). Furthermore, we identified and characterized methylated, acetylated, and oxidized proteoforms relating to the PLA2 and disintegrin toxin families and the site of their modifications. It thus seems that post-translational modifications (PTMs) of toxins, particularly target lysine residues, may play an essential role in the structural and functional properties of venom proteins and might be able to influence the therapeutic response of antivenoms, to be investigated in future studies.

Adaptive venom evolution and toxicity in octopods is driven by extensive novel gene formation, expansion, and loss.

BackgroundCephalopods represent a rich system for investigating the genetic basis underlying organismal novelties. This diverse group of specialized predators has evolved many adaptations including proteinaceous venom. Of particular interest is the blue-ringed octopus genus (Hapalochlaena), which are the only octopods known to store large quantities of the potent neurotoxin, tetrodotoxin, within their tissues and venom gland.FindingsTo reveal genomic correlates of organismal novelties, we conducted a comparative study of 3 octopod genomes, including the Southern blue-ringed octopus (Hapalochlaena maculosa). We present the genome of this species and reveal highly dynamic evolutionary patterns at both non-coding and coding organizational levels. Gene family expansions previously reported in Octopus bimaculoides (e.g., zinc finger and cadherins, both associated with neural functions), as well as formation of novel gene families, dominate the genomic landscape in all octopods. Examination of tissue-specific genes in the posterior salivary gland revealed that expression was dominated by serine proteases in non–tetrodotoxin-bearing octopods, while this family was a minor component in H. maculosa. Moreover, voltage-gated sodium channels in H. maculosa contain a resistance mutation found in pufferfish and garter snakes, which is exclusive to the genus. Analysis of the posterior salivary gland microbiome revealed a diverse array of bacterial species, including genera that can produce tetrodotoxin, suggestive of a possible production source.ConclusionsWe present the first tetrodotoxin-bearing octopod genome H. maculosa, which displays lineage-specific adaptations to tetrodotoxin acquisition. This genome, along with other recently published cephalopod genomes, represents a valuable resource from which future work could advance our understanding of the evolution of genomic novelty in this family.

Species-specific and geographical variation in venom composition of two major cobras in Indian subcontinent: Impact on polyvalent antivenom therapy

While snakebite is a severe problem for several countries throughout the world the Indian subcontinent has witnessed the highest global incidence of snakebite which represents an occupational health hazard. Belonging to the family Elapidae, the Indian spectacled cobra (Naja naja), is one of the members of the “Big Four” venomous snakes found throughout the Indian subcontinent. Indian monocled cobra Naja kaouthia is prevalent in eastern and north-eastern India as well as in Bangladesh, Nepal, and Bhutan. Species-specific and geographical differences in venom composition of two major Indian cobras (N. naja vs. N. kaouthia) may limit the effectiveness of antivenom treatment. Therefore, exploring the venom proteomes of N. naja and N. kaouthia from different geographical locales of the Indian subcontinent, and the effect of this variation on antivenom efficacy is warranted. Proteomic analyses have demonstrated that although eastern Indian N. naja and N. kaouthia venoms have similarities, species-specific and geographical variation is evident by differences in their venom toxicity values and pathobiological effects in experimental animals. This is the major reason as to why a polyvalent antivenom against N. naja venom exhibited only partial neutralization of enzymatic activity and immunological cross-reactivity against cobra venoms from other regions of India. This suggests that addition of polyclonal antibodies against N. kaouthia venom to polyantivenom, or preparation/administration of region-specific polyantivenom will provide better treatment of cobra bites in the Indian subcontinent.

Cytotoxic effect of Montivipera bornmuelleri's venom on cancer cell lines: in vitro and in vivo studies.

Background Montivipera bornmuelleri’s venom has shown immunomodulation of cytokines release in mice and selective cytotoxicity on cancer cells in a dose-dependent manner, highlighting an anticancer potential. Here, we extend these findings by elucidating the sensitivity of murine B16 skin melanoma and 3-MCA-induced murine fibrosarcoma cell lines to M. bornmuelleri’s venom and its effect on tumor growth in vivo.MethodsThe toxicity of the venom on B16 and MCA cells was assessed using flow cytometry and xCELLigence assays. For in vivo testing, tumor growth was followed in mice after intratumoral venom injection.ResultsThe venom toxicity showed a dose-dependent cell death on both B16 and MCA cells. Interestingly, overexpression of ovalbumin increased the sensitivity of the cells to the venom. However, the venom was not able to eradicate induced-tumor growth when injected at 100 µg/kg. Our study demonstrates a cytotoxic effect of M. bornmuelleri’s venom in vitro which, however, does not translate to an anticancer action in vivo.

Toxin-like neuropeptides in the sea anemone Nematostella unravel recruitment from the nervous system to venom

The sea anemone Nematostella vectensis (Anthozoa, Cnidaria) is a powerful model for characterizing the evolution of genes functioning in venom and nervous systems. Although venom has evolved independently numerous times in animals, the evolutionary origin of many toxins remains unknown. In this work, we pinpoint an ancestral gene giving rise to a new toxin and functionally characterize both genes in the same species. Thus, we report a case of protein recruitment from the cnidarian nervous to venom system. The ShK-like1 peptide has a ShKT cysteine motif, is lethal for fish larvae and packaged into nematocysts, the cnidarian venom-producing stinging capsules. Thus, ShK-like1 is a toxic venom component. Its paralog, ShK-like2, is a neuropeptide localized to neurons and is involved in development. Both peptides exhibit similarities in their functional activities: They provoke contraction in Nematostella polyps and are toxic to fish. Because ShK-like2 but not ShK-like1 is conserved throughout sea anemone phylogeny, we conclude that the two paralogs originated due to a Nematostella-specific duplication of a ShK-like2 ancestor, a neuropeptide-encoding gene, followed by diversification and partial functional specialization. ShK-like2 is represented by two gene isoforms controlled by alternative promoters conferring regulatory flexibility throughout development. Additionally, we characterized the expression patterns of four other peptides with structural similarities to studied venom components and revealed their unexpected neuronal localization. Thus, we employed genomics, transcriptomics, and functional approaches to reveal one venom component, five neuropeptides with two different cysteine motifs, and an evolutionary pathway from nervous to venom system in Cnidaria.

Toxicity of venom from the mamushi, Gloydius blomhoffii, (Squamata, Crotalinae) to centipedes

Vipers include approximately 300 species and usually feed on vertebrates, but over 30 species of them occasionally eat centipedes. Centipedes have been also known to occur in stomach contents of a Japanese pit viper, mamushi, Gloydius blomhoffii. Toxicity of the venom of mamushi to small mammals has been well studied, but there is no information concerning its toxicity to arthropods. Here, we studied the toxicity of the raw venom to the red-headed centipede, Scolopendra subspinipes mutilans, by comparing with the toxicity to two other common prey animals, a house mouse, Mus musclus, and a pond frog, Pelophylax nigromaculatus. The lethal doses for mice weighing around 21.5 g and frogs weighing around 3.78 g were less than 5 μl (equivalent to ca. 0.23 and 1.32 μl/g, respectively), which presumably corresponds to an approximate dose of mamushi's one envenomation. On the other hand, centipedes weighing around 1.86 g needed 10–36 μl of venom to die (16.0 μl/g on average). This result suggests that the centipedes are much more resistant to the venom than other prey animals, and it is difficult for mamushi to kill or incapacitate centipedes by the venom of a single envenomation.

Widespread Evolution of Molecular Resistance to Snake Venom α-Neurotoxins in Vertebrates.

Venomous snakes are important subjects of study in evolution, ecology, and biomedicine. Many venomous snakes have alpha-neurotoxins (α-neurotoxins) in their venom. These toxins bind the alpha-1 nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction, causing paralysis and asphyxia. Several venomous snakes and their predators have evolved resistance to α-neurotoxins. The resistance is conferred by steric hindrance from N-glycosylated asparagines at amino acids 187 or 189, by an arginine at position 187 that has been hypothesized to either electrostatically repulse positively charged neurotoxins or sterically interfere with α-neurotoxin binding, or proline replacements at positions 194 or 197 of the nAChR ligand-binding domain to inhibit α-neurotoxin binding through structural changes in the receptor. Here, we analyzed this domain in 148 vertebrate species, and assessed its amino acid sequences for resistance-associated mutations. Of these sequences, 89 were sequenced de novo. We find widespread convergent evolution of the N-glycosylation form of resistance in several taxa including venomous snakes and their lizard prey, but not in the snake-eating birds studied. We also document new lineages with the arginine form of inhibition. Using an in vivo assay in four species, we provide further evidence that N-glycosylation mutations reduce the toxicity of cobra venom. The nAChR is of crucial importance for normal neuromuscular function and is highly conserved throughout the vertebrates as a result. Our research shows that the evolution of α-neurotoxins in snakes may well have prompted arms races and mutations to this ancient receptor across a wide range of sympatric vertebrates. These findings underscore the inter-connectedness of the biosphere and the ripple effects that one adaption can have across global ecosystems.

Toxicological study of bee venom (Apis mellifera mellifera) from different regions of the province of Buenos Aires, Argentina

Samples of Apis mellifera mellifera venom from different hives in two regions of the Buenos Aires province and its pool were analyzed for their lethal potency, myotoxic, defibrinogenating, hemolytic and inflammatory-edematizing activity and for the histological alterations they produce in the heart, lungs, kidneys, skeletal muscle and liver of mice. In vitro studies focused on the venom's hemolytic activity in different systems and species (horse, man, sheep and rabbit), the cytotoxicity in cellular lines, and on the proteolytic and coagulant activity in plasma and fibrinogen. Hemolytic activity, either observed in vitro or in vivo, showed similar toxicity levels for all samples. Erythrocytes of different species varied in their sensitivity to the venom pool, equines being the most sensitive and sheep the most resistant to direct hemolytic action. Local and systemic myotoxicity was evidenced by either the elevation of serum creatine kinase and/or histopathological lesions, observed in different muscles. All samples caused significant pathological alterations; pulmonary, cardiac, renal and skeletal muscle lesions were substantive and can be related to the pathophysiological mechanisms of envenomation. The venoms from different apiaries and regions of the Buenos Aires province showed very similar toxicological characteristics. These results suggest that severity of envenomation in case of a swarming could therefore be more related to the number of bees than to the differential toxicity of the venom from different regions of the province. This is the first study on the toxicity and toxicological characteristics of Apis mellifera venom in Argentina.

Immunoreactivity and neutralization capacity of Philippine cobra antivenom against Naja philippinensis and Naja samarensis venoms.

The Philippine cobra (Naja philippinensis) and Samar cobra (Naja samarensis) are two WHO Category 1 medically important venomous snakes in the Philippines. Philippine cobra antivenom (PCAV) is the only antivenom available in the country, but its neutralization capacity against the venoms of N. philippinensis and hetero-specific N. samarensis has not been reported. This knowledge gap greatly hinders the optimization of antivenom use in the region. This study examined the immunological binding and neutralization capacity of PCAV against the two cobra venoms using WHO-recommended protocols. In mice, both venoms were highly neurotoxic and lethal with a median lethal dose of 0.18 and 0.20 µg/g, respectively. PCAV exhibited strong and comparable immunoreactivity toward the venoms, indicating conserved venom antigenicity between the two allopatric species. In in vivo assay, PCAV was only moderately effective in neutralizing the toxicity of both venoms. Its potency was even lower against the hetero-specific N. samarensis venom by approximately two-fold compared with its potency against N. philippinensis venom. The results indicated that PCAV could be used to treat N. samarensis envenomation but at a higher dose, which might increase the risk of hypersensitivity and worsen the shortage of antivenom supply in the field. Antivenom manufacturing should be improved by developing a low-dose, high-efficacy product against cobra envenomation.

Human Chondrocyte Activation by Toxins From Premolis semirufa, an Amazon Rainforest Moth Caterpillar: Identifying an Osteoarthritis Signature.

Pararamosis is a disease that occurs due to contact with the hairs of the larval stage of the Brazilian moth Premolis semirufa. Envenomation induces osteoarticular alterations with cartilage impairment that resembles joint synovitis. Thus, the toxic venom present in the caterpillar hairs interferes with the phenotype of the cells present in the joints, resulting in inflammation and promoting tissue injury. Therefore, to address the inflammatory mechanisms triggered by envenomation, we studied the effects of P. semirufa hair extract on human chondrocytes. We have selected for the investigation, cytokines, chemokines, matrix metalloproteinases (MMPs), complement components, eicosanoids, and extracellular matrix (ECM) components related to OA and RA. In addition, for measuring protein-coding mRNAs of some molecules associated with osteoarthritis (OA) and rheumatoid arthritis (RA), reverse transcription (RT) was performed followed by quantitative real-time PCR (RT-qPCR) and we performed the RNA-sequencing (RNA-seq) analysis of the chondrocytes transcriptome. In the supernatant of cell cultures treated with the extract, we observed increased IL-6, IL-8, MCP-1, prostaglandin E2, metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-13), and complement system components (C3, C4, and C5). We noticed a significant decrease in both aggrecan and type II collagen and an increase in HMGB1 protein in chondrocytes after extract treatment. RNA-seq analysis of the chondrocyte transcriptome allowed us to identify important pathways related to the inflammatory process of the disease, such as the inflammatory response, chemotaxis of immune cells and extracellular matrix (ECM) remodeling. Thus, these results suggest that components of Premolis semirufa hair have strong inflammatory potential and are able to induce cartilage degradation and ECM remodeling, promoting a disease with an osteoarthritis signature. Modulation of the signaling pathways that were identified as being involved in this pathology may be a promising approach to develop new therapeutic strategies for the control of pararamosis and other inflammatory joint diseases.

IgE Effector Mechanisms, in Concert with Mast Cells, Contribute to Acquired Host Defense against Staphylococcusaureus

Allergies are considered to represent mal-directed type 2 immune responses against mostly innocuous exogenous compounds. Immunoglobulin E (IgE) antibodies are a characteristic feature of allergies and mediate hypersensitivity against allergens through activation of effector cells, particularly mast cells (MCs). Although the physiological functions of this dangerous branch of immunity have remained enigmatic, recent evidence shows that allergic immune reactions can help to protect against the toxicity of venoms. Because bacteria are a potent alternative source of toxins, we assessed the possible role of allergy-like type 2 immunity in antibacterial host defense. We discovered that the adaptive immune response against Staphylococcus aureus (SA) skin infection substantially improved systemic host defense against secondary SA infections in mice. Moreover, this acquired protection depended on IgE effector mechanisms and MCs. Importantly, our results reveal a previously unknown physiological function of allergic immune responses, IgE antibodies, and MCs in host defense against a pathogenic bacterium.