Immunological Cross-Reactivity and Preclinical Assessment of a Colombian Anticoral Antivenom against the Venoms of Three Micrurus Species.

Snakebite accident treatment requires the administration of antivenoms that provide efficacy and effectiveness against several snake venoms of the same genus or family. The low number of immunogenic components in venom mixtures that allow the production of antivenoms consequently gives them partial neutralization and a suboptimal pharmacological response. This study evaluates the immunorecognition and neutralizing efficacy of the polyvalent anticoral antivenom from the Instituto Nacional de Salud (INS) of Colombia against the heterologous endemic venoms of Micrurus medemi, and M. sangilensis, and M. helleri by assessing immunoreactivity through affinity chromatography, ELISA, Western blot, and neutralization capability. Immunorecognition towards the venoms of M. medemi and M. sangilensis showed values of 62% and 68% of the protein composition according to the immunoaffinity matrix, respectively. The analysis by Western blot depicted the highest recognition patterns for M. medemi, followed by M. sangilensis, and finally by M. helleri. These findings suggest that the venom compositions are closely related and exhibit similar recognition by the antivenom. According to enzyme immunoassays, M. helleri requires a higher amount of antivenom to achieve recognition than the others. Besides reinforcing the evaluation of INS antivenom capability, this work recommends the use of M. helleri in the production of Colombian antisera.

Development of a high-throughput in vitro screening method for the assessment of cell-damaging activities of snake venoms.

Snakebite envenoming is a globally important public health issue that has devastating consequences on human health and well-being, with annual mortality rates between 81,000 and 138,000. Snake venoms may cause different pathological effects by altering normal physiological processes such as nervous transfer and blood coagulation. In addition, snake venoms can cause severe (local) tissue damage that may result in life-long morbidities, with current estimates pointing towards an additional 450,000 individuals that suffer from permanent disabilities such as amputations, contractions and blindness. Despite such high morbidity rates, research to date has been mainly focusing on neurotoxic and haemotoxic effects of snake venoms and considerably less on venom-induced tissue damage. The molecular mechanisms underlaying this pathology include membrane disruption and extracellular matrix degradation. This research describes methods used to study the (molecular) mechanisms underlaying venom-induced cell- and tissue damage. A selection of cellular bioassays and fluorescent microscopy were used to study cell-damaging activities of snake venoms in multi-well plates, using both crude and fractionated venoms. A panel of 10 representative medically relevant snake species was used, which cover a large part of the geographical regions most heavily affected by snakebite. The study comprises both morphological data as well as quantitative data on cell metabolism and viability, which were measured over time. Based on this data, a distinction could be made in the ways by which viper and elapid venoms exert their effects on cells. We further made an effort to characterise the bioactive compounds causing these effects, using a combination of liquid chromatography methods followed by bioassaying and protein identification using proteomics. The outcomes of this study might prove valuable for better understanding venom-induced cell- and tissue-damaging pathologies and could be used in the process of developing and improving snakebite treatments.

Biochemical characterization and assessment of leishmanicidal effects of a new L-amino acid oxidase from Crotalus durissus collilineatus snake venom (CollinLA AO-I).

This study reports the isolation of CollinLAAO-I, a new L-amino acid oxidase from Crotalus durissus collilineatus snake venom, its biochemical characterization and leishmanicidal potential in Leishmania spp. CollinLAAO-I (63.1 kDa) was successfully isolated with high purity using two chromatographic steps and represents 2.5% of total venom proteins. CollinLAAO-I displayed high enzymatic activity (4262.83 U/mg/min), significantly reducing after 28 days. The enzymatic activity of CollinLAAO-I revealed higher affinity for hydrophobic amino acids such as L-leucine, high enzymatic activity in a wide pH range (6.0-10.0), at temperatures from 0 to 25 °C, and showed complete inhibition in the presence of Na+ and K+. Cytotoxicity assays revealed IC50 of 18.49 and 11.66 µg/mL for Leishmania (L.) amazonensis and Leishmania (L.) infantum, respectively, and the cytotoxicity was completely suppressed by catalase. CollinLAAO-I significantly increased the intracellular concentration of reactive oxygen species (ROS) and reduced the mitochondrial potential of both Leishmania species. Furthermore, CollinLAAO-I decreased the parasite capacity to infect macrophages by around 70%, indicating that even subtoxic concentrations of CollinLAAO-I can interfere with Leishmania vital processes. Thus, the results obtained for CollinLAAO-I provide important support for developing therapeutic strategies against leishmaniasis.

Recent advancements in snake antivenom production.

Snakebite envenoming (SBE), a neglected tropical disease, claims lives of about 138,000 people globally, and antivenom is the only approved treatment worldwide. However, this century-old therapy has serious limitations, including limited efficacy and some side effects. Although alternative and adjunct therapies are being developed, their commercialization will take time. Hence, improving existing antivenom therapy is crucial for immediate reduction in the global SBE burden. The neutralization potential and immunogenicity of antivenoms depend primarily on the venom pool used for animal immunization and the production host, along with antivenom purification procedure and quality control. Enhancing antivenom quality and production capacity are also critical actions of the World Health Organization (WHO) roadmap 2021 against SBE. The present review details the latest developments in antivenom production, such as immunogen preparation, production host, antibody purification methods, antivenom testing-including alternative animal models, in vitro assays, and proteomics and in silico methodologies, and storage, reported from 2018 to 2022. Based on these reports, we propose that production of broadly specific, affordable, safe, and effective (BASE) antivenoms is fundamental to realizing the WHO roadmap and reducing the global SBE burden. This concept can also be applied during the designing of alternative antivenoms.

The Amazonian Tropical Bites Research Initiative, a hope for resolving zoonotic neglected tropical diseases in the One Health era.

BACKGROUND: Neglected tropical diseases (NTDs) disproportionately affect populations living in resource-limited settings. In the Amazon basin, substantial numbers of NTDs are zoonotic, transmitted by vertebrate (dogs, bats, snakes) and invertebrate species (sand flies and triatomine insects). However, no dedicated consortia exist to find commonalities in the risk factors for or mitigations against bite-associated NTDs such as rabies, snake envenoming, Chagas disease and leishmaniasis in the region. The rapid expansion of COVID-19 has further reduced resources for NTDs, exacerbated health inequality and reiterated the need to raise awareness of NTDs related to bites. METHODS: The nine countries that make up the Amazon basin have been considered (Bolivia, Brazil, Colombia, Ecuador, French Guiana, Guyana, Peru, Surinam and Venezuela) in the formation of a new network. RESULTS: The Amazonian Tropical Bites Research Initiative (ATBRI) has been created, with the aim of creating transdisciplinary solutions to the problem of animal bites leading to disease in Amazonian communities. The ATBRI seeks to unify the currently disjointed approach to the control of bite-related neglected zoonoses across Latin America. CONCLUSIONS: The coordination of different sectors and inclusion of all stakeholders will advance this field and generate evidence for policy-making, promoting governance and linkage across a One Health arena.

Assessment of snakebite management practices at Meserani Juu in Monduli District, Northern Tanzania.

BACKGROUND: Snakebite envenoming represents a tragically neglected tropical disease mostly affecting poor people living in remote areas of developing countries, primarily in sub-Saharan Africa. Anti-snake venom (ASV) is the only approved specific treatment for systemic envenoming from snakebite, but it remains largely unavailable in many parts of developing countries. There is paucity of data on snakebite management practice in Tanzania. This study aimed at assessing the community management practices of snakebite and availability of anti-snake venom in the public health facilities in Monduli District, Northern Tanzania. METHODS: A cross sectional study was carried out between May and June, 2018 involving 67 victims, 147 other household members, and 35 public health facilities. A structured questionnaire, respondent interview, and health facility report/document review were considered during data collection. Clean data were analyzed using SPSS version 20. RESULTS: Sixty-seven snakebite victims and 147 other household members were interviewed during a household survey. All snakebite cases reported to having visited a health facility after snakebite with the majority 55/67 (82.1%) reporting the use, prior attendance to medical care, of some form of local treatment such as tourniquets 13 (19.4%), local incision 11 (16.4%), and snakestone 7 (10.4%). None of the public health facilities in Monduli District attended a snakebite case and had never stocked anti-snake venom products. In this area, 45 snakebite cases were reported to be managed at Meserani snake park clinic where anti-snake venom products were available and provided for free in the period between January 2017 and December 2017. CONCLUSION: Majority of the snakebite cases at Meserani Juu relied on local methods for the management of snake bites of which most are of unknown efficacy and safety. Furthermore, none of the primary public health facilities in Monduli District stocked antivenom despite being a habitat for different kinds of venomous snakes. The government and local non-government organizations should collaborate so as to improve the anti-snake venom availability and the provision of snakebite preventive and management awareness programs, especially to the rural communities.

Proteomics and histological assessment of an organotypic model of human skin following exposure to Naja nigricollis venom.

Snakebite envenoming was reintroduced as a Category A Neglected Tropical Disease by the World Health Organization in 2017. Since then, increased attention has been directed towards this affliction and towards the development of a deeper understanding of how snake venoms exert their toxic effects and how antivenoms can counter them. However, most of our in vivo generated knowledge stems from the use of animal models which do not always accurately reflect how the pathogenic effects of snake venoms manifest in humans. Moreover, animal experiments are associated with pain, distress, and eventually animal sacrifice due to the toxic nature of snake venoms. Related to this, the implementation of the 3Rs principle (Replacement, Reduction, and Refinement) in the use of experimental animals in snakebite envenoming research is recommended by the World Health Organization. Therefore, more humane experimental designs and new in vitro/ex vivo alternatives for experimental animals are sought after. Here, we report the use of an organotypic model of human skin to further elucidate the pathophysiology of the dermonecrotic effects caused by the venom of the black-necked spitting cobra, Naja nigricollis, in humans. The goal of this study is to expand the repertoire of available models that can be used to study the local tissue damages induced by cytotoxic venoms.

Pharmacological re-assessment of traditional medicinal plants-derived inhibitors as antidotes against snakebite envenoming: A critical review.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional healers have used medicinal plants to treat snakebite envenomation worldwide; however, mostly without scientific validation. There have been many studies on the therapeutic potential of the natural products against snake envenomation. AIM OF THE STUDY: This review has highlighted snake venom inhibitory activity of bioactive compounds and peptides from plants that have found a traditional use in treating snakebite envenomation. We have systematically reviewed the scenario of different phases of natural snake venom inhibitors characterization covering a period from 1994 until the present and critically analysed the lacuna of the studies if any, and further scope for their translation from bench to bedside. MATERIALS AND METHODS: The medicinal plant-derived compounds used against snakebite therapy were reviewed from the available literature in public databases (Scopus, MEDLINE) from 1994 till 2020. The search words used were 'natural inhibitors against snakebite,' 'natural products as therapeutics against snakebite,' 'natural products as antidote against snake envenomation,' ' snake venom toxin natural inhibitors,' 'snake venom herbal inhibitors'. However, the scope of this review does not include computational (in silico) predictions without any wet laboratory validation and snake venom inhibitory activity of the crude plant extracts. In addition, we have also predicted the ADMET properties of the identified snake venom inhibitors to highlight their valuable pharmacokinetics for future clinical studies. RESULTS: The therapeutic application of plant-derived natural inhibitors to treat snakebite envenomation as an auxiliary to antivenom therapy has been gaining significant momentum. Pharmacological reassessment of the natural compounds derived from traditional medicinal plants has demonstrated inhibition of the principal toxic enzymes of snake venoms at various extents to curb the lethal and/or deleterious effects of venomous snakebite. Nevertheless, such molecules are yet to be commercialized for clinical application in the treatment of snakebite. There are many obstacles in the marketability of the plant-derived natural products as snake envenomation antidote and strategies must be explored for the translation of these compounds from drug candidates to their clinical application. CONCLUSION: In order to minimize the adverse implications of snake envenomation, strategies must be developed for the smooth transition of these plant-derived small molecule inhibitors from bench to bedside. In this article we have presented an inclusive review and have critically analysed natural products for their therapeutic potential against snake envenomation, and have proposed a road map for use of natural products as antidote against snakebite.

Assessment of quality and pre-clinical efficacy of a newly developed polyvalent antivenom against the medically important snakes of Sri Lanka.

Snake envenomation is a severe problem in Sri Lanka (SL) and Indian polyvalent antivenom (PAV) is mostly used for treating snakebite albeit due to geographical variation in venom composition, Indian PAV shows poor efficacy in neutralizing the lethality and toxicity of venom from the same species of snakes in SL. Therefore, the quality and in vivo venom neutralization potency of a country-specific PAV produced against the venom of the five most medically important snakes of SL (Daboia russelii, Echis carinatus, Hypnale hypnale, Naja naja, Bungarus caeruleus) was assessed. LC-MS/MS analysis of two batches of PAV showed the presence of 88.7-97.2% IgG and traces of other plasma proteins. The tested PAVs contained minor amounts of undigested IgG and F(ab')2 aggregates, showed complement activation, were devoid of IgE, endotoxin, and content of preservative was below the threshold level. Immunological cross-reactivity and in vitro neutralization of enzymatic activities, pharmacological properties demonstrated superior efficacy of SL PAV compared to Indian PAV against SL snake venoms. The in vivo neutralization study showed that the tested PAVs are potent to neutralize the lethality and venom-induced toxicity of SL snake venoms. Therefore, our study suggests that introduction of SL-specific PAV will improve snakebite management in SL.

In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

Good management practices of venomous snakes in captivity to produce biological venom-based medicines: achieving replicability and contributing to pharmaceutical industry.

One of the factors responsible for lack of reproducible findings may be attributed to the raw material used. To date, there are no apparent studies examining reproducibility using venoms for the development of new toxin-based drugs with respect to regulatory agencies' policies. For this reason, protocols were implemented to produce animal toxins with quality, traceability, and strict compliance with Good Manufacturing Practices. This required validation of the production chain from the arrival of the animal to the vivarium, followed by handling, housing, as well as compliance with respect to extraction, freeze-drying, and, finally, storage protocols, aimed at generating compounds to serve as candidate molecules applicable in clinical trials. Currently, to produce quality snake venoms to support reproductive studies, the Center for the Study of Venoms and Venomous Animals (CEVAP) from São Paulo State University (UNESP), São Paulo, Brazil has 449 microchipped snakes through rigid and standardized operating procedures for safety, health, and welfare of animals. Snakes were frequently subjected to vet clinical examination, anthelmintic, and antiparasitic treatment. Venom milk used to destroy prey was collected from each animal in individual plastic microtubes to avoid contamination and for traceability. In addition, venoms were submitted to microbiological, and biochemical toxicological analyses. It is noteworthy that investigators are responsible for caring, maintaining, and manipulating snakes and ensuring their health in captivity. This review aimed to contribute to the pharmaceutical industry the experimental experience and entire snake venom production chain required to generate quality products for therapeutic human consumption.

Venomous snakebites: Rapid action saves lives-A multifaceted community education programme increases awareness about snakes and snakebites among the rural population of Tamil Nadu, India.

The lack of public awareness surrounding the dangers of snakebite envenomation (SBE) is one of the most critical factors contributing to SBE-induced complications, and subsequently exacerbating the number of deaths and disabilities resulting from SBE. In this study, we deployed a multifaceted community education programme to educate students, healthcare professionals and members of the public in rural areas of Tamil Nadu, India about the dangers of SBE, appropriate first aid measures and the 'do's and don'ts' following a snakebite. An assessment of prior knowledge within these communities identified several misconceptions concerning snakes and SBE. Using a combination of direct engagement (estimated to reach over 200,000 people), information leaflets (200,000 distributed), posters, video documentaries, media and social media (>2.8 million engagements), over the course of one year (January to December 2019) we reached over 3 million people in rural Tamil Nadu (around 8% of population). Evaluation of community-based assemblies indicated that at least 90% of attendees were able to recall the key messages at the end of the events, and at least 85% were able to recall the key messages even after 12 months. Due to high demand, a one-day symposium was organised to provide clinical knowledge and training on SBE to 250 healthcare professionals in rural Tamil Nadu. Notably, an assessment of patient data (291 victims) collected from a snakebite referral hospital over the same 12-month period (2019) indicated that arrival time at hospital following a snakebite was significantly faster and the effective first aid measures were administered to patients who were aware of our activities compared to those that were not. Overall, our approach provides a framework on how to educate rural communities about the dangers of SBE and thereby, mitigate delayed SBE treatment leading to an overall reduction in SBE-induced mortality, morbidity, treatment costs and other socio-economic ramifications.

Recent developments in diagnostic tools and bioanalytical methods for analysis of snake venom: A critical review.

Snakebite is a neglected medical emergency causing fatalities and long-term disabilities throughout the world, especially in tropical countries. The effectiveness of therapy against snakebite is reliant on the unambiguous identification of bitten species of snake followed by immediate administration of venom-specific monovalent antivenom. However, this is a challenging task and therefore, over the several years scientists are constantly trying to address this issue by developing species-specific snake venom diagnostic kits as an alternative to classical methods of snake identification in clinics. Recently quite a few modern tools and techniques have been deployed for the development of simple, inexpensive, rapid, specific, and sensitive snake venom detection kits. However, despite these efforts a lone snakebite diagnostic kit is available until now which is a severe concern for efficacious snakebite therapy. In this article, we have reviewed the key issues pertaining to the rapid diagnosis of snake envenomation, tools and techniques developed and/or invented particularly over the past 40 years for the detection of snakebite as well as quantity of venom in the body fluids and/or tissues of victims. To overcome the practical constraints against the successful commercialization of these diagnostic kits, much more intensive studies for their improvement in terms of efficacy, affordability, storage stability, and usability, in addition to standardization of techniques for use in clinics are required to fulfil the objectives of the user-friendliness and commercial viability of snakebite diagnostic kits particularly in the rural and underdeveloped areas of tropical countries showing the maximum incidence of snakebite.

Venomics of the Duvernoy's gland secretion of the false coral snake Rhinobothryum bovallii (Andersson, 1916) and assessment of venom lethality towards synapsid and diapsid animal models.

The Duvernoy's gland secretory proteome of the false coral snake Rhinobothryum bovallii (Costa Rica), unveiled applying bottom-up venomics, comprises a handful of toxins belonging to only three protein families, three-finger toxin (3FTx), cysteine-rich secretory protein (CRISP), and snake venom metalloprotease (PIII-SVMP). Except for small differences in the relative abundance of the PIII-SVMPs, which may be due to individual variability, no evidence of geographic variability or ontogenetic changes was found among the venom proteomes of the juvenile and adult R. bovallii snakes sampled. Major monomeric (86.5%mol) and minor dimeric (2.8%mol) 3FTxs dominate the toxin arsenal of the Costa Rican false coral snake. The remaining 10.7% of the venom proteome comprises CRISP (8.2%) and PIII-SVMP (2.4%) molecules. In vivo lethality assays showed that R. bovallii produces venom that is non-toxic towards mammalian prey, and which exerts a different toxic effect on domestic chicken chicks and baby green iguana. Toxicovenomic analysis of R. bovallii venom in the iguana model identified two 3FTx RP-HPLC fractions that faithfully mimicked the irreversible immobilizing effect of the whole venom. BIOLOGICAL SIGNIFICANCE: With more than 2200 species in family Colubridae (sensu lato), rear-fanged snakes comprise approximately two-thirds of the extant species of advanced snakes. Snakebites from venomous snakes that are of medical concern are predominantly from front-fanged snakes of families Viperidae and Elapidae. On the other hand, rear-fanged snakes have been conventionally considered non-venomous, and thus their venoms have remained a largely untapped area of venomics. However, increasing documentation of life-threadening, even fatal, envenomings from rear-fanged snakes has sparked interest in their venoms. Appying bottom-up venomics we have revealed that the Duvernoy's gland secretory proteome of R. bovallii comprises a handful of toxins belonging to only three protein families, with slow-acting three-finger toxins (3FTx) that are non-toxic towards mammalian prey and show preference towards diapsid taxa representing the dominant structural and functional proteins. Our work documents for the first time 3FTxs exerting different effect in an avian model than in a reptile model. Besides, the 3FTx fractions that faithfully mimicked the irreversible iguana-immobilizing effect of the whole venom were identified through toxicovenomic analysis of R. bovallii venom on Iguana iguana. Our work underscores the importance of using biologically-relevant animal toxicity models for investigating the biological roles of venoms in an evolutionary-ecological context.

Snakebite accidents in Rio Grande do Norte state, Brazil: Epidemiology, health management and influence of the environmental scenario.

OBJECTIVES: Brazil is home to large variety of snake species, of which about 17% are venomous. A large proportion of reported snakebite accidents in the country take place in northeast Brazil. We aimed to analyse the epidemiology of snakebites as a public health concern in Rio Grande do Norte state. METHODS: A retrospective epidemiologic analysis was performed using data from the Brazilian Case Registry Database pertaining to the period 2007-2016, and considering environmental climate characteristics as an influential factor. RESULTS: A number of 3909 cases were reported in total. Among those cases, 58% involved venomous species and more than 80% of these were caused by Bothrops species. An association between environmental characteristics and the incidence of reported cases was found. The Borborema Potiguar region was the most affected area. Apart from that, deficiencies in managing the victims were described. CONCLUSION: This study highlights snakebite accidents as a public health concern in Rio Grande do Norte state. Snake bites are most likely being influenced by climate change and human activities. Continuous training of involved medical personnel could help optimise patient care and avoid under reporting in the accident reporting system.

OBJECTIFS: Le Brésil abrite de très nombreuses espèces de serpents, dont environ 17% sont venimeuses. Une grande partie des accidents rapportés de morsures de serpent dans le pays ont lieu dans le nord-est du pays. Notre objectif était d'analyser l'épidémiologie des morsures de serpents en tant que problème de santé publique dans l'Etat du Rio Grande do Norte. MÉTHODES: Une analyse épidémiologique rétrospective a été réalisée à l'aide des données de la Base de Données du Registre Brésilien des cas couvrant la période 2007-2016 et prenant en compte les caractéristiques climatiques de l'environnement comme facteur d'influence. RÉSULTATS: 3.909 cas au total ont été rapportés. 58% concernaient des espèces venimeuses et plus de 80% d'entre elles étaient causées par l'espèce Bothrops. Une association entre les caractéristiques environnementales et l'incidence des cas rapportés a été mise en évidence. La région de Borborema Potiguar était la plus touchée. Outre cela, les déficiences dans la prise en charges des victimes ont été décrites. CONCLUSION: Cette étude souligne que les accidents causés par des morsures de serpent constituent un problème de santé publique dans l'Etat de Rio Grande do Norte. Les morsures de serpents sont très probablement influencées par les changements climatiques et les activités humaines. La formation continue du personnel médical impliqué pourrait aider à optimiser les soins prodigués aux patients et à éviter une sous-notification dans le système de notification des accidents.

A comparative assessment of the effects of integrin inhibitor cilengitide on primary culture of head and neck squamous cell carcinoma (HNSCC) and HNSCC cell lines.

BACKGROUND: Integrins are highly attractive targets in oncology due to their involvement in angiogenesis in a wide spectrum of cancer entities. Among several integrin inhibitors, cilengitide is suggested to be one of the most promising inhibitors. However, little is known about the cellular processes induced during cilengitide chemotherapy in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: For the current study, 3 HNSCC cell lines, SCC4, SCC15 and SCC25; and 3 primary culture cells, TU53, TU57, and TU63 were used. CD90, cytokeratin, and vimentin were stained immunohistochemically to identify the biological characteristics of these cell lines and primary culture cells and the cytostatic effect of cilengitide was evaluated. Quantitative polymerase chain reaction (qPCR) arrays were applied to evaluate target protein genes ITGAV, ITGB3, and ITGB5 of integrin αvß3 and αvß5 at respective concentrations of 50 and 100 µM cilengitide for 72 h. RESULTS: Cilengitide has significantly inhibited the proliferation of HNSCC cells in a dose-dependent way. At the same concentration, cilengitide suppressed the proliferation of primary culture cells even more strongly than it did that of cell lines, suggesting that primary culture cells retain more of their internal biological characteristics than do cell lines. qPCR assay detected downregulation of ITGAV, ITGB3, and ITGB5 gene expression after exposure to 50 µM of cilengitide. However, after exposure to 100-µM cilengitide, expression of these genes significantly increased both in cell lines and primary culture cells. CONCLUSIONS: RGD-containing small-molecule synthetic peptides might be considered in tumor chemotherapy in the near future. The different reactions of primary culture cells and cell lines demonstrated that individualized chemotherapy plans may be a feasible option. However, research on the role of cilengitide in HNSCC therapy is still in its early stages, and further investigations are required.

Functional Application of Snake Venom Proteomics in In Vivo Antivenom Assessment.

Reverse-phase high-performance liquid chromatography is commonly employed as a decomplexing strategy in snake venom proteomics. The chromatographic fractions often contain relatively pure toxins that can be assessed functionally for toxicity level through the determination of their median lethal doses (LD50). Further, antivenom efficacy can be evaluated specifically against these venom fractions to understand the limitation of the antivenom as the treatment for snake envenomation. However, methods of toxicity assessment and antivenom evaluation vary across laboratories; hence there is a need to standardize the protocols and parameters, in particular those related to the neutralizing efficacy of antivenom. This chapter outlines the important in vivo techniques and data interpretation that can be applied in the functional study of snake venom proteomes.

Toxin-resolved antivenomics-guided assessment of the immunorecognition landscape of antivenoms.

Snakebite envenoming represents a major issue in rural areas of tropical and subtropical regions across sub-Saharan Africa, South to Southeast Asia, Latin America and Oceania. Antivenoms constitute the only scientifically validated therapy for snakebite envenomings, provided they are safe, effective, affordable, accessible and administered appropriately. However, the lack of financial incentives in a technology that has remained relatively unchanged for more than a century, has contributed to some manufacturers leaving the market and others downscaling production or increasing the prices, leading to a decline in the availability and accessibility for these life-saving antidotes to millions of rural poor most at risk from snakebites in low income countries. The shortage of antivenoms can be significantly alleviated by optimizing the use of current antivenoms (through the assessment of their specific and paraspecific efficacy against the different medically relevant homologous and heterologous snake venoms) and by generating novel polyspecific antivenoms exhibiting broad clinical spectrum and wide geographic distribution range. Research on venoms has been continuously enhanced by advances in technology. Particularly, the last decade has witnessed the development of omics strategies for unravelling the toxin composition of venoms ("venomics") and to assess the immunorecognition profile of antivenoms ("antivenomics"). Here, we review recent developments and reflect on near future innovations that promise to revolutionize the mutually enlightening relationship between evolutionary and translational venomics.

Assessment of the potential toxicological hazard of the Green Parrot Snake (Leptophis ahaetulla marginatus): Characterization of its venom and venom-delivery system.

Snakes are the major group of venomous vertebrates, and the rear-fanged snakes represent the vast majority of species and occur worldwide; however, relatively few studies have characterized their venoms and evaluated their potential hazards for humans. Herein we explore the protein composition and properties of the venom of the rear-fanged Green Parrot Snake, Leptophis ahaetulla marginatus, the most common snake found in the Iguazu National Park (Argentina), as well as the main features of its venom delivery system. This species has venom reminiscent of elapid venoms, composed mainly of components such as 3FTxs, CRiSPs and AChE, but it shows low toxicity toward mammals (LD50 > 20 µg/g mouse). The histology of its Duvernoy's venom gland is similar to that of other colubrids, with serous secretory cells arranged in densely packed secretory tubules. The posterior end of its maxilla exhibits 1-3 blade-shaped and slightly recurved fangs but without grooves. This study provides an initial analysis of the biological role of venom in Leptophis, with implications for potential symptoms that might be anticipated from bites by this species.