A Meta-Analysis of the Protein Components in Rattlesnake Venom.

The specificity and potency of venom components give them a unique advantage in developing various pharmaceutical drugs. Though venom is a cocktail of proteins, rarely are the synergy and association between various venom components studied. Understanding the relationship between various components of venom is critical in medical research. Using meta-analysis, we observed underlying patterns and associations in the appearance of the toxin families. For Crotalus, Dis has the most associations with the following toxins: PDE; BPP; CRL; CRiSP; LAAO; SVMP P-I and LAAO; SVMP P-III and LAAO. In Sistrurus venom, CTL and NGF have the most associations. These associations can predict the presence of proteins in novel venom and understand synergies between venom components for enhanced bioactivity. Using this approach, the need to revisit the classification of proteins as major components or minor components is highlighted. The revised classification of venom components is based on ubiquity, bioactivity, the number of associations, and synergies. The revised classification can be expected to trigger increased research on venom components, such as NGF, which have high biomedical significance. Using hierarchical clustering, we observed that the genera's venom compositions were similar, based on functional characteristics rather than phylogenetic relationships.

From birth to adulthood: An analysis of the Brazilian lancehead (Bothrops moojeni) venom at different life stages.

The Brazilian lancehead (Bothrops moojeni) has a wide distribution in Brazil and represents a serious public health hazard. Previous works reported that the symptoms of snakebites caused by B. moojeni juveniles' bites were mainly related to coagulation, while those caused by adults' bites had a more prominent local damage. In this work, we analyzed the venoms of B. moojeni at different life stages to better understand the ontogeny shift in this species. Snakes were grouped by age and sex, and venom pools were formed accordingly. Compositional analyses by one-dimensional electrophoresis (1-DE), chromatography, and mass spectrometry revealed that ontogenetic changes might be mostly related to phospholipase A2 (PLA2) and metalloproteases. Regarding the venoms functional aspect, proteolytic, L-amino acid oxidase, PLA2, and coagulant in vitro activities were assayed, but only the first and the last ones showed age-related changes, with the venom of snakes up to 1 year-old displaying lower proteolytic and higher coagulant activities, while those from 2 years-old onward presented the opposite relation. The venoms of 3 years-old snakes were exceptions to the compositional and functional pattern of adults as both venoms presented profiles similar to neonates. Sex-related differences were observed in specific groups and were not age-related. In vivo experiments (median lethal dose and hemorrhagic activity) were statistically similar between neonates and adults, however we verified that the adult venom killed mice faster comparing to the neonates. All venoms were mostly recognized by the antibothropic serum and displayed similar profiles to 1-DE in western blotting. In conclusion, the Brazilian lancehead venom showed ontogenetic shift in its composition and activities. Furthermore, this change occurred in snakes from 1 to 2 years-old, and interestingly the venom pools from 3 years-old snakes had particular characteristics, which highlights the importance of comprehensive studies to better understand venom variability.

Size Matters: An Evaluation of the Molecular Basis of Ontogenetic Modifications in the Composition of Bothrops jararacussu Snake Venom.

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.

The relationship between clinics and the venom of the causative Amazon pit viper (Bothrops atrox).

Snake venoms are complex mixtures of proteins with toxic activities, with many distinct isoforms, affecting different physiological targets, comprised in a few protein families. It is currently accepted that this diversity in venom composition is an adaptive advantage for venom efficacy on a wide range of prey. However, on the other side, variability on isoforms expression has implications in the clinics of human victims of snakebites and in the efficacy of antivenoms. B. atrox snakes are responsible for most of the human accidents in Brazilian Amazon and the type and abundance of protein families on their venoms present individual variability. Thus, in this study we attempted to correlate the individual venom proteome of the snake brought to the hospital by the patient seeking for medical assistance with the clinical signs observed in the same patient. Individual variability was confirmed in venoms of the 14 snakes selected for the study. The abundance of each protein family was quite similar among the venom samples, while the isoforms composition was highly variable. Considering the protein families, the SVMP group presented the best correlation with bleeding disorders and edema. Considering individual isoforms, some isoforms of venom metalloproteinase (SVMP), C-type lectin-like toxins (CTL) and snake venom serine proteinases (SVSP) presented expression levels that with statistically significant positive correlation to signs and symptoms presented by the patients as bleeding disorders, edema, ecchymosis and blister formation. However, some unexpected data were also observed as the correlation between a CTL, CRISP or LAAO isoforms with blister formation, still to be confirmed with a larger number of samples. Although this is still a small number of patient samples, we were able to indicate that venom composition modulates clinical manifestations of snakebites, to confirm at the bedside the prominent role of SVMPs and to include new possible toxin candidates for the development of toxin inhibitors or to improve antivenom selectiveness, important actions for the next generation treatments of snakebites.

Use of a phosphopeptide as a ligand to purify phospholipase A2 from the venom of Crotalus durisuss terrificus by affinity chromatography.

The venom of Crotalus durissus terrificus (Cdt) is a source of a wide variety of toxins, some of them with interesting pharmacological applications. Of these toxins, the phospholipase A2 (PLA2) subunit of crotoxin (Ctx) has been studied for its potential as an antiviral and antibacterial agent. Peptides have proven useful ligands for the purification of numerous molecules, including antibodies, toxins, enzymes and other proteins. Here, we sought to use a phosphopeptide (P-Lys) as a ligand for PLA2 purification. P-Lys was synthesized in solid phase on Rink-Amide-ChemMatrix resin, immobilized on NHS-agarose, and then evaluated as a chromatographic matrix. Under the best conditions, total protein adsorption reached 39% and only the eluate fraction presented PLA2 activity. Analysis of the eluate by SDS-PAGE showed three bands, one corresponding to the molecular weight of PLA2 (14 kDa). Said bands were analyzed by mass spectrometry and identified as PLA2 and its multimers. The final product showed a purity of over 90%. In addition, slightly changing the process conditions also allowed the isolation of crotamine.

Crotalus oreganus concolor: Envenomation Case with Venom Analysis and a Diagnostic Conundrum of Myoneurologic Symptoms.

A case of midget-faded rattlesnake (Crotalus oreganus concolor) envenomation of an adult male professional herpetologist occurred in a rural setting and resulted in an array of venom induced myoneurologic symptoms. The patient experienced blurry vision, total body paresthesia, dyspnea, chest tightness, and waves of spastic muscle movements of the hands and feet that resembled tetany. It was not apparent whether these symptoms were potentially venom induced or were related to stress-induced physiologic responses. Local envenomation effects were minimal, and coagulation parameters remained within normal limits. Antivenom was not administered per patient concerns related to a history of acute allergic reactions to antivenom. Venom was collected from the Crotalus oreganus concolor responsible for the bite, and analysis revealed the presence of high levels of myotoxins (SR calcium pump antagonists) and concolor toxin, a presynaptic neurotoxin that can have myotoxic effects and cause respiratory paralysis; several serine proteinases associated with coagulopathies were also present in the venom profile.

Delineating the venom toxin arsenal of Malabar pit viper (Trimeresurus malabaricus) from the Western Ghats of India and evaluating its immunological cross-reactivity and in vitro cytotoxicity.

The venom protein components of Malabar pit viper (Trimeresurus malabaricus) were identified by combining SDS-PAGE and ion-exchange chromatography pre-fractionation techniques with LC-MS/MS incorporating Novor and PEAKS-assisted de novo sequencing strategies. Total 97 proteins that belong to 16 protein families such as L-amino acid oxidase, metalloprotease, serine protease, phospholipase A2, 5'-nucleotidase, C-type lectins/snaclecs and disintegrin were recognized from the venom of a single exemplar species. Of the 97 proteins, eighteen were identified through de novo approaches. Immunological cross-reactivity assessed through ELISA and western blot indicate that the Indian antivenoms binds less effectively to Malabar pit viper venom components compared to that of Russell's viper venom. The in vitro cell viability assays suggest that compared to the normal cells, MPV venom induces concentration dependent cell death in various cancer cells. Moreover, crude venom resulted in chromatin condensation and apoptotic bodies implying the induction of apoptosis. Taken together, the present study enabled in dissecting the venom proteome of Trimeresurus malabaricus and revealed the immuno-cross-reactivity profiles of commercially available Indian polyvalent antivenoms that, in turn, is expected to provide valuable insights on the need in improving antivenom preparations against its bite.

Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature

Abstract Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.(AU)

Evidence for Snake Venom Plasticity in a Long-Term Study with Individual Captive Bothrops atrox.

Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations.

Global proteomic and functional analysis of Crotalus durissus collilineatus individual venom variation and its impact on envenoming.

Individual variations studies are important to understand the snakebite envenoming and to improve the antivenom production and its effectiveness. In this way, the objective of this study was a comparative analysis of intraspecific variation in the venom composition of 22 Crotalus durissus collilineatus specimens through proteomic techniques. Venoms were fractionated by RP-FPLC, and analyzed by SDS-PAGE and mass spectrometry. Although similar, chromatographic and electrophoretic profiles showed significant qualitative and quantitative differences. Some venom components were identified for the very first time in C. d. collilineatus, such as glutathione peroxidase, nerve growth factor, 5'-nucleotidase, angiotensin-converting enzyme, carboxypeptidase, phosphodiesterase, glutaminyl cyclase and phospholipase B. Regarding hyaluronidase activity, 2 venoms did not present detectable enzyme activity in the tested amounts. Additionally, in vivo crotalic envenoming in mice showed that venoms from different specimens resulted in diversified changes of biochemical and immunological parameters, such as creatine kinase and interleukin 6. This study demonstrated significant intraspecific variations in the venom of C. d. collilineatus, which may impact the production and effectiveness of the antivenom therapy. BIOLOGICAL SIGNIFICANCE: This study performed the proteomic and functional analyzes of 22 C. d. collilineatus individual venoms and verified the occurrence of quali and quantitative variations among them. The venoms evaluated caused envenomings with different changes in biochemical and immunological parameters. These results confirm the need to use a pool of venoms with the greatest possible variability in the preparation of antivenoms, in order to improve their effectiveness. In addition, this study was able to identify for the first time 8 different proteins in this subspecies venom, increasing knowledge about its composition and showing that it is a source of these proteins with possible biotechnological applications.

Snake Venom Extracellular vesicles (SVEVs) reveal wide molecular and functional proteome diversity.

Proteins constitute almost 95% of snake venom's dry weight and are produced and released by venom glands in a solubilized form during a snake bite. These proteins are responsible for inducing several pharmacological effects aiming to immobilize and initiate the pre-digestion of the prey. This study shows that proteins can be secreted and confined in snake venom extracellular vesicles (SVEVs) presenting a size distribution between 50 nm and 500 nm. SVEVs isolated from lyophilized venoms collected from four different species of snakes (Agkistrodon contortrix contortrix, Crotalus atrox, Crotalus viridis and Crotalus cerberus oreganus) were analyzed by mass spectrometry-based proteomic, which allowed the identification of proteins belonging to eight main functional protein classes such as SVMPs, serine proteinases, PLA2, LAAO, 5'nucleotidase, C-type lectin, CRISP and Disintegrin. Biochemical assays indicated that SVEVs are functionally active, showing high metalloproteinase and fibrinogenolytic activity besides being cytotoxic against HUVEC cells. Overall, this study comprehensively depicts the protein composition of SVEVs for the first time. In addition, the molecular function of some of the described proteins suggests a central role for SVEVs in the cytotoxicity of the snake venom and sheds new light in the envenomation process.

Proteomic profile, biological activities and antigenic analysis of the venom from Bothriopsis bilineata smaragdina ("loro machaco"), a pitviper snake from Peru.

In order to determine Bothriopsis bilineata smaragdina venom (BbsV) composition, proteomic approaches were performed. Venom components were analyzed by RP-HPLC, SDS- PAGE and nano LC on line with LTQ Orbitrap XL. Results showed a total of 189 identified proteins, grouped into 11 different subgroups, which include snake venom metalloproteinases (SVMPs, 54.67%), snake C-type lectins (Snaclecs, 15.78%), snake venom serine proteinases (SVSPs, 14.69%), cystein-rich secretory proteins (CRISP, 2.61%), phospholipases A2 (PLA2, 1.14%), phosphodiesterase (PDE, 1.17%), venom endothelial growth factor (VEGF, 1.06%) 5'nucleotidases (0.33%), L-amino acid oxidases (LAAOs, 0.28%) and other proteins. In vitro enzymatic activities (SVMP, SVSP, LAAO, Hyal and PLA2) of BbsV were also analyzed. BbsV showed high SVSP activity but low PLA2 activity, when compared to other Bothrops venoms. In vivo, BbsV induced hemorrhage and edema in mice and showed intraperitoneal median lethal dose (LD50) of 92.74 (± 0.15) µg/20 g of mice. Furthermore, BbsV reduced cell viability when incubated with VERO cells. Peruvian and Brazilian bothropic antivenoms recognize BbsV proteins, as detected by ELISA and Western Blotting. Both antivenoms were able to neutralize in vivo edema and hemorrhage. SIGNIFICANCE: In Peru, snakebite is a public health problem, especially in the rain forest, as a result of progressive colonization of this geographical area. This country is the second in Latin America, after Brazil, to exhibit the largest variety of venomous snakes. B. atrox and B. b. smaragdina snakes are sympatric species in Peruvian Amazon region and are responsible for approximately 95% of the envenomings reported in this region. B. b. smaragdina may cause a smaller share (3 to 38%) of those accidents, due to its arboreal habits, that make human encounters with these snakes less likely to happen. Despite B. b. smaragdina recognized medical importance, its venom composition and biological activities have been poorly studied. Furthermore, BbsV is not a component of the antigenic pool used to produce the corresponding Peruvian bothropic antivenom (P-BAV). Our results not only provide new insights on BbsV composition and biological activity, but also demonstrate that both P-BAV and B-BAV polyvalent antivenoms have a considerable recognition of proteins from BbsV and, more importantly, neutralized hemorrhage and edema, the main local effects of bothropic envenomation.

Biological and Proteolytic Variation in the Venom of Crotalus scutulatus scutulatus from Mexico.

Rattlesnake venoms may be classified according to the presence/absence and relative abundance of the neurotoxic phospholipases A 2 s (PLA 2 s), such as Mojave toxin, and snake venom metalloproteinases (SVMPs). In Mexico, studies to determine venom variation in Mojave Rattlesnakes (Crotalus scutulatus scutulatus) are limited and little is known about the biological and proteolytic activities in this species. Tissue (34) and venom (29) samples were obtained from C. s. scutulatus from different locations within their distribution in Mexico. Mojave toxin detection was carried out at the genomic (by PCR) and protein (by ELISA) levels for all tissue and venom samples. Biological activity was tested on representative venoms by measuring LD 50 and hemorrhagic activity. To determine the approximate amount of SVMPs, 15 venoms were separated by RP-HPLC and variation in protein profile and proteolytic activity was evaluated by SDS-PAGE (n = 28) and Hide Powder Azure proteolytic analysis (n = 27). Three types of venom were identified in Mexico which is comparable to the intraspecific venom diversity observed in the Sonoran Desert of Arizona, USA: Venom Type A (∼Type II), with Mojave toxin, highly toxic, lacking hemorrhagic activity, and with scarce proteolytic activity; Type B (∼Type I), without Mojave toxin, less toxic than Type A, highly hemorrhagic and proteolytic; and Type A + B, containing Mojave toxin, as toxic as venom Type A, variable in hemorrhagic activity and with intermediate proteolytic activity. We also detected a positive correlation between SVMP abundance and hemorrhagic and proteolytic activities. Although more sampling is necessary, our results suggest that venoms containing Mojave toxin and venom lacking this toxin are distributed in the northwest and southeast portions of the distribution in Mexico, respectively, while an intergradation in the middle of both zones is present.

Interlaboratory study for the establishment of Brazilian Bothrops Reference Venom and Antivenom for potency evaluation of Bothrops antivenom.

A collaborative study was performed for the establishment of the 5th lot of Brazilian Bothrops Reference Venom and the 1st lot of Brazilian Bothrops Reference Antivenom. All Brazilian manufacturers of Antibothrops Immunoglobulins and the National Control Laboratory participated of the study. The declared potency of the 5th lot of the Bothrops Reference Venom is 40.29 µg/0.5 ml, and the potency of the 1st lot of Bothrops Reference Antivenom is 6.51 mg/ml. For the potency evaluation of Bothrops Reference Venom the inter assay precision (gCV) was 3.25% in lab 01; 3.51% in INCQS; 4.71% in lab 03 and 25.11% in lab 02, and the inter laboratory precision was 13.76%. The intra assay precision of Bothrops Reference Antivenom determinations was 4.38% in INCQS; 8.47% in lab 02; 10.51% in lab 03 and 20.05% in lab 01. The inter assay precision was 3.51% in INCQS; 9.65% in lab 02; 18.03% in lab 01 and 20.23% in lab 03. The inter laboratory precision was 15.85%. Despite the high number of invalid results (55.6% for the pharmacopoeial method and 69.4% for the proposed method) the parallel line assay, have better inter laboratorial precision (gCV = 16.62%) than the pharmacopoeial potency assay (gCV = 38.28%).

Fatal stroke after Bothrops snakebite in the Amazonas state, Brazil: A case report.

Bothrops atrox is the snake responsible for the majority of snakebites in the Brazilian Amazon. Patients generally evolve to local manifestations such as edema, pain and ecchymoses. Systemic effects of B. atrox venom are usually restricted to blood incoagulability and spontaneous bleeding. However, in a few cases, bleeding in the central nervous system may occur, which can lead to sequels and deaths. Here, we report a case of a 59 year-old woman who presented edema, pain and ecchymoses on the right foot, headache, nausea, diarrhea, hypertension and blood incoagulability after the bite by Bothrops snake in the Brazilian Amazon. This case evolved with stroke resulting in death despite the antivenom and conservative therapy employed. In addition, we were able to identify the presence of venom in the patient's brain tissue after death. Direct action of toxins present in the snake's venom in the induction of systemic hemorrhage allied to blood incoagulability and hypertension presented by the patient could be involved in the mechanism of stroke in this case.

Origin and characterization of small membranous vesicles present in the venom of Crotalus durissus terrificus.

Small membranous vesicles are small closed fragments of membrane. They are released from multivesicular bodies (exosomes) or shed from the surface membrane (microvesicles). They contains various bioactive molecules and their molecular composition varies depending on their cellular origin. Small membranous vesicles have been identified in snake venoms, but the origin of these small membranous vesicles in the venom is controversial. The aim of this study was to verify the origin of the small membranous vesicles in venom of Crotalus durissus terrificus by morphological analyses using electron microscopy. In addition, the protein composition of the vesicles was analyzed by using a proteome approach. The small membranous vesicles present in the venom were microvesicles, since they originated from microvilli on the apical membrane of secretory cells. They contained cytoplasmic proteins, and proteins from the plasma membrane, endoplasmic reticulum (ER), and Golgi membrane. The release of microvesicles may be a mechanism to control the size of the cell membrane of the secretory cells after intense exocytosis. Microvesicle components that may have a role in envenoming include ecto-5'-nucleotidase, a cell membrane protein that releases adenosine, and aminopeptidase N, a cell membrane protein that may modulate the action of many peptides.

Proteomic Characterization and Comparison of Malaysian Tropidolaemus wagleri and Cryptelytrops purpureomaculatus Venom Using Shotgun-Proteomics.

Tropidolaemus wagleri and Cryptelytrops purpureomaculatus are venomous pit viper species commonly found in Malaysia. Tandem mass spectrometry analysis of the crude venoms has detected different proteins in T. wagleri and C. purpureomaculatus. They were classified into 13 venom protein families consisting of enzymatic and nonenzymatic proteins. Enzymatic families detected in T. wagleri and C. purpureomaculatus venom were snake venom metalloproteinase, phospholipase A2, ʟ-amino acid oxidase, serine proteases, 5'-nucleotidase, phosphodiesterase, and phospholipase B. In addition, glutaminyl cyclotransferase was detected in C. purpureomaculatus. C-type lectin-like proteins were common nonenzymatic components in both species. Waglerin was present and unique to T. wagleri-it was not in C. purpureomaculatus venom. In contrast, cysteine-rich secretory protein, bradykinin-potentiating peptide, and C-type natriuretic peptide were present in C. purpureomaculatus venom. Composition of the venom proteome of T. wagleri and C. purpureomaculatus provides useful information to guide production of effective antivenom and identification of proteins with potential therapeutic applications.

Tratamiento de la mordedura por víbora en España / Viper bite treatment in Spain

La mordedura de víbora es, con mucho, el accidente ofídico más frecuente en nuestro país. Causa entre 100 y 150 ingresos anuales en España, siendo difícil saber el número real de urgencias atendidas por este motivo. Una correcta clasificación del grado de envenenamiento y la utilización de antivenenos son la clave para un adecuado manejo de la situación. Actualmente las controversias sobre el uso o no de antivenenos están siendo superadas debido a su alta purificación, el descenso de las reacciones alérgicas que producen y a su eficacia terapéutica. Este artículo pretende ser una puesta al día sobre la atención en Urgencias de esta afección, exponiendo de forma clara las posibilidades de tratamiento (AU)

Viper snake bite is, by far, the most common ophidian accident in Spain. It is responsible for between 100 and 150 hospitalizations per year in this country, although it is difficult to determine the frequency of emergency admissions due to this cause. The cornerstone to their approach rests on the correct evaluation of the possible effects derived from envenomation and the use of anti-venoms. In spite of all the controversies surrounding the use of anti-venoms, they have become a powerful therapeutic weapon ever since the serum has been highly purified and the great decrease of related anaphylactic reactions. The aim of this article is to update the emergency room procedures when viper bites are suspected, and to clarify the main therapeutic recommendations (AU)

Anti-Trimeresurus albolabris venom IgY antibodies: preparation, purification and neutralization efficacy

Snakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens. Methods IgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice. Results Chickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg). Conclusions IgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabrisvenom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.

Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.

Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2ß1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms.