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Toxicon 109 (2016) 51e62

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Toxicon

journal homepage: www.elsevier .com/locate/ toxicon

Review

Understanding and confronting snakebite envenoming: The harvestof cooperation

Jos�e María Guti�errezInstituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jos�e, Costa Rica

a r t i c l e i n f o

Article history:Received 26 October 2015Received in revised form13 November 2015Accepted 18 November 2015Available online 23 November 2015

Keywords:Costa RicaToxinologySnake venomsAntivenomsMyonecrosisHemorrhageNeutralizationBothrops asper

E-mail address: [email protected].

http://dx.doi.org/10.1016/j.toxicon.2015.11.0130041-0101/© 2015 The Author. Published by Elsevier L

a b s t r a c t

During 45 years, the Instituto Clodomiro Picado (ICP, University of Costa Rica) has developed an ambi-tious scientific, technological, productive, and social program aimed at providing a better understandingof snakes and their venoms, contributing to the development, production and distribution of antivenoms,improving the prevention and management of snakebite envenomings, and strengthening human re-sources in science and technology. Among other topics, its research agenda has focused on the localtissue alterations induced by viperid snake venoms, i.e. myonecrosis, hemorrhage, dermonecrosis,extracellular matrix degradation, lymphatic vessel damage, and inflammation. In addition, the preclinicalefficacy of antivenoms has been thoroughly investigated, together with the technological development ofnovel antivenoms. ICP's project has been based on a philosophical frame characterized by: (a) An inte-grated approach for confronting the problem of snakebites, involving research, production, extensionactivities, and teaching; (b) a cooperative and team work perspective in the pursuit of scientific, tech-nological, productive, and social goals; (c) a search for excellence and continuous improvement in thequality of its activities; and (d) a vision of solidarity and compassion, based on the realization thatsnakebite envenomings mostly affect impoverished vulnerable populations in the rural settings ofdeveloping countries. A key aspect in this program has been the consolidation of international part-nerships with groups of all continents, within a frame of academic and social cooperation, some of whichare described in this review.

© 2015 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. The dawn of Costa Rican toxinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522. The philosophical basis of an ambitious project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533. Scientific research at ICP: towards understanding the complex landscape of local tissue damage induced by viperid snake venoms . . . . . . . . . . . . . 53

3.1. Myonecrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533.2. Hemorrhage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543.3. Damage to skin and to lymphatic vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563.4. Exploring the causes of deficient skeletal muscle regeneration after venom-induced myonecrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563.5. Inflammation in venom-affected tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

4. Towards a better characterization of the preclincal efficacy of antivenoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565. Confronting snakebite envenoming from a broad perspective: Central America and beyond . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

5.1. Improvements and innovation in antivenom manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575.2. A better understanding of snakebite epidemiology: providing information for public health interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585.3. Promoting prevention and appropriate management of snakebite cases through extension programs . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 585.4. Regional cooperation in Latin America . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585.5. Antivenom development for other regions of the world . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585.6. Advocacy: raising global awareness of the impact of snakebite envenoming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

td. This is an open access article u

nder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Delta:1_given namehttp://creativecommons.org/licenses/by-nc-nd/4.�0/mailto:[email protected]://crossmark.crossref.org/dialog/?doi=10.1016/j.toxicon.2015.11.013&domain=pdfwww.sciencedirect.com/science/journal/00410101www.elsevier.com/locate/toxiconhttp://dx.doi.org/10.1016/j.toxicon.2015.11.013http://creativecommons.org/licenses/by-nc-nd/4.�0/http://dx.doi.org/10.1016/j.toxicon.2015.11.013http://dx.doi.org/10.1016/j.toxicon.2015.11.013

J.M. Guti�errez / Toxicon 109 (2016) 51e6252

Ethical statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Transparency document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

1. The dawn of Costa Rican toxinology

The scientific study of venomous snakes and their venoms inCosta Rica started with the pioneering work of Clodomiro PicadoTwight (1887e1944), during the first decades of the 20th century(Fig. 1). Picado, a talented person interested in natural history, wasawarded a scholarship to perform advanced studies at La Sorbonne,from 1908 to 1913. During his last year in France, he also receivedtraining inMedical Microbiology at Institut Pasteur. Upon his returnto Costa Rica, Picado was appointed Director of the Clinical Labo-ratory of Hospital San Juan de Dios, at that time the main healthcenter in the country. In addition to introducing modern methodsfor laboratory diagnosis, which represented a leap forward in themedical practice in the country, he established an ambitiousresearch program which covered a wide array of topics, includingMedical Microbiology, Immunology, Experimental Pathophysi-ology, Clinical Chemistry, Hematology, Therapeutics, Phytopa-thology, and General Biology (Guti�errez, 1986).

One of the subjects that captured the attention of Picadowas theproblem of snakebite envenoming, which was then, as it is now, aserious public health hazard in the country. Taking an integratedapproach to the subject, he developed a serpentarium at the hos-pital, collected venoms, and studied a variety of aspects includingthe biology of snakes, the toxicology of venoms, and the therapy ofenvenomings. His contributions in the field of Toxinology are

Fig. 1. Pioneers of Toxinology in Costa Rica. Left, Clodomiro Picado Twight (1887e1944). AfteLaboratory of Hospital San Juan de Dios (San Jos�e, Costa Rica). In addition to his work on diagamong other topics, the study of snakes, snake venoms, and snakebite envenomings. RiMicrobiology (University of Costa Rica). In the decade of 1960 he was involved in an inter-inof this project, the Instituto Clodomiro Picado was created in 1970, and Bola~nos was its Dirpromoted an innovative integrated approach for confronting snakebite envenomings, involvi170e171, Guti�errez, Bothrops asper: Beauty and peril in the Neotropics, copyright 2009,permission from Elsevier.

summarized in the book Serpientes Venenosas de Costa Rica. SusVenenos. Seroterapia Antiofídica (Picado, 1931). It may be said thatPicado introduced in Costa Rica what is currently known as‘translational research’, since he went from the laboratory studiesto the implementation of practical solutions to the problem. Beingaware of the developments that had taken place since 1901 inBrazil, associated with the production of the first antivenoms inLatin America, at Instituto Butantan, under the leadership of VitalBrazil Mineiro da Campanha (Brazil, 1911), Picado hypothesizedthat the similarities between the venoms of Brazilian and CostaRican viperid snakes could imply that antivenoms manufactured inBrazil would be effective in Costa Rica. Through an agreement withInstituto Butantan, Brazilian antivenoms were imported to CostaRica, and used in the treatment of patients. The success of thisapproach initiated the modern era of snakebite envenoming ther-apy in Central America (Picado, 1931).

In addition to scientific research and the introduction of anti-venoms, Picado also worked in the political front, promoting, inassociation with the Minister of Health of Costa Rica, Sol�on Nú~nezFrutos, a unique and highly progressive legislation that, amongother issues, implemented the use of antivenoms in the country,the access of this therapy in rural settings, and the obligation ofland owners to keep a stock of antivenom in case their workerssuffered a snakebite (Picado, 1931; Guti�errez, 2010).

The legacy of Clodomiro Picado germinated in the 1960s as an

r completing advanced studies in France, Picado was appointed Director of the Clinicalnostic laboratory aspects, he developed an ambitious research programwhich included,ght, R�oger Bola~nos Herrera (1931e2007), professor of Immunology at the School ofstitutional program aimed at producing antivenoms in Costa Rica. Owing to the successector from 1970 to 1980. He performed studies on snake venoms and antivenoms, andng research, antivenom production, teaching and extension. Reprinted from Toxicon 50:and Toxicon 54: 901e903, Guti�errez, Obituary: R�oger Bola~nos, copyright 2007, with

J.M. Guti�errez / Toxicon 109 (2016) 51e62 53

interinstitutional ambitious project known as Programa de SuerosAntiofídicos (Program for Antivenoms). This effort, jointly promotedby the Ministry of Health, the University of Costa Rica and theEmbassy of the United States of America in Costa Rica, was aimed atinitiating the local production of antivenoms (Guti�errez, 2010). Itwas developed in the context of a profound transformation of thepublic health institutions in Costa Rica, as part of the social-democratic project that dominated the political landscape of thecountry in those years (Jaramillo, 1993). Under the leadership ofR�oger Bola~nos (1931e2007) (Fig. 1) and Herschel H. Flowers, theprogram succeeded in producing the first batches of polyvalent andanticoral antivenoms in 1967 (see Guti�errez (2010) for a detailedaccount of the origins and goals of the project).

The success of this initiative led to the creation of InstitutoClodomiro Picado (ICP), in 1970, as the institution responsible forthe manufacture of antivenoms for the country, with R�oger Bola~nosappointed as its first Director. Bola~nos, a professor of Immunologyat the School of Microbiology of the University of Costa Rica, pro-moted an integrated approach to the issue of snakebite envenom-ing, one that inherited and expanded the philosophy implementedby Clodomiro Picado Twight (Guti�errez, 2007, 2010). In 1972,through an agreement between the Ministry of Health and theUniversity of Costa Rica, the administration of ICP was transferredto this university; this event has had significant implications for thestrengthening of Toxinology and of antivenom production in thecountry.

2. The philosophical basis of an ambitious project

R�oger Bola~nos and his colleagues at ICP had a vision on how tostudy and confront snakebite envenoming which, since then, hasbeen the philosophical basis for the development of ICP. This phi-losophy, which fits within the main goals of the University of CostaRica, is based on four main pillars:

(a) The view that, for an effective approach to this complexpublic health problem, an integrated perspective had to beimplemented, including (i) scientific and technologicalresearch on snakes, venoms and antivenoms; (ii) develop-ment, production and distribution of antivenoms; (iii)strengthening of human resources through graduate andundergraduate teaching programs at the university; and (iv)extension programs to convey the basic knowledge of pre-vention and management of snakebite envenomings tocommunities, vulnerable social groups, and healthprofessionals.

(b) A permanent search for excellence in all aspects of ICP's ac-tivities, getting away from comfort zones, and searching fornew scientific, technological, productive and socialchallenges.

(c) The promotion of a cooperative and team work approach inthe fulfillment of objectives. The confrontation of the difficulttasks that ICP had to undertake was possible only through acollective agenda, characterized by a continued coordinationbetween the various sections and workers. This cooperativeperspectivewas implemented not only within ICP, but also inthe collaborations that this institute has established withother groups in many countries along the years.

(d) A philosophy of solidarity and compassion, based on the factthat snakebite envenomings cause much suffering amongvulnerable human groups living in impoverished rural set-tings. Thus, in the long term, the efforts carried out onvarious fronts by ICP have had the aim of reducing suchsuffering, through science, technology, and socialentrepreneurship.

3. Scientific research at ICP: towards understanding thecomplex landscape of local tissue damage induced by viperidsnake venoms

One of the most drastic consequences of viperid snakebiteenvenomings is the development of local pathological effects,characterized by edema, myonecrosis, dermonecrosis, blistering,and hemorrhage (Warrell, 2004; Cardoso et al., 2009). If not treatedpromptly and effectively, this may result in permanent tissuedamage and sequelae, with long lasting physical and psychologicaleffects in affected people. This subject has been of interest to re-searchers at ICP since the early years of the decade of 1980. After ageneral characterization of myotoxic and hemorrhagic activities ofCosta Rican viperid venoms (Guti�errez and Chaves, 1980), variousresearch projects have contributed to understanding thecomplexity of these phenomena.

3.1. Myonecrosis

When describing the myotoxic activity induced by the venom ofBothrops asper, the most important venomous snake of CentralAmerica, it was proposed that the quantification of the plasmalevels of the enzyme creatine kinase (CK) could be used as a markerto assess the extent of myonecrosis (Guti�errez et al., 1980). Theisolation of a myotoxic phospholipase A2 (PLA2), and the study of itsmechanism of action, was performed in collaboration with Char-lotte L. Ownby, the author's PhD supervisor, at Oklahoma StateUniversity (USA). It was proposed that this myotoxin induces acutemuscle damage by first affecting the integrity of skeletal muscleplasma membrane, through phospholipid hydrolysis, thus pro-moting a prominent calcium influx which sets the stage for a seriesof intracellular degenerative events that lead to irreversible celldamage (Guti�errez et al., 1984a, b).

Further studies in Costa Rica led to the isolation of several newmyotoxins from various snake venoms (see Lomonte and Rangel(2012) for a review). Surprisingly, some of these myotoxins hadPLA2 structure, but were devoid of catalytic activity (Lomonte andGuti�errez, 1989). Through a collaboration with the group of Ivan I.Kaiser, at the University ofWyoming (USA), it was found that one ofthese toxins, B. asper myotoxin II, is a Lys49 PLA2 homologue, pre-senting modifications not only at position 49 but also in someresidues of the so called ‘calcium-binding loop’ (Francis et al., 1991).This myotoxin has become a useful tool to investigate the mecha-nisms involved in skeletal muscle damage in the absence of phos-pholipid hydrolysis. Working with this toxin at the laboratory ofLars Å. Hanson, at the University of G€oteborg (Sweden), BrunoLomonte identified a stretch of hydrophobic and cationic residuesin the C-terminal region of myotoxin II which was responsible formembrane damage (Lomonte et al., 1994a). Later on, the 3Dstructure of this myotoxin was described in collaboration withRhaguvir R. Arni, of Universidade Estadual Paulista (Brazil) (Arniet al., 1995). The ability of catalytically-active and inactive myo-toxins to disrupt bilayers was then explored using liposomes asmodels; it was observed that liposomes made of negatively-charged phospholipids were more susceptible to the action ofthese toxins (Díaz et al., 1991; Rufini et al., 1992).

The use of myoblast/myotube cell cultures for assessing thecytotoxic activity of snake venommyotoxins has been instrumentalfor deciphering various aspects of their mechanism of action(Lomonte et al., 1999), such as the role of toxin dimerization in theability to induce cytotoxicity (Angulo et al., 2005), the effect ofcholesterol and membrane fluidity in the susceptibility of myo-blasts (Rangel et al., 2011), and the synergistic action of Asp49-andLys49 PLA2s (Mora-Obando et al., 2014). On the other hand, whenexploring myotoxicity induced by elapid venom PLA2s, and in


collaboration with the groups of Monica Thelestam and HansJ€ornvall, at Karolinska Institutet (Sweden), Alberto Alape-Gir�onproposed a hypothesis describing the evolutionary steps in theacquisition of myotoxicity in these PLA2s, and identified key resi-dues in these PLA2s necessary to exert acute muscle damage(Alape-Gir�on et al., 1999).

More recently, through a collaboration with the group of CesareMontecucco, at the University of Padova (Italy), new insights havebeen gained on the mechanism of action of viperid myotoxic PLA2s.Using a mass spectrometric approach to quantify phospholipidsand lysophospholipids in muscle tissue and myotubes in culture, itwas found that a catalytically-active myotoxic PLA2 induced pre-dominantly hydrolysis of phosphatidylcholine, whereas acatalytically-inactive PLA2 homologue did not, thus demonstratingthat intracellular PLA2s do not seem to play a key role in membranedamage in this model (Fern�andez et al., 2013). Moreover, usingsingle cell fluorescent measurement with Fura-2, it was found thatboth types of myotoxins induced a rapid increment in cytosoliccalcium, which was largely abrogated by eliminating extracellularcalcium (Cintra-Francischinelli et al., 2009). A possible adaptive rolefor having catalytically-active and -inactive myotoxic PLA2s in asingle venom was inferred from studies showing a synergism be-tween these two types of myotoxins in promoting damage tomuscle cell plasma membrane (Mora-Obando et al., 2014).

On the basis of these experimental observations, a unified viewof the mechanism of action of these myotoxins has been proposed(Guti�errez and Ownby, 2003; Montecucco et al., 2008; Lomonteand Rangel, 2012) (Fig. 2). Myotoxic PLA2s and PLA2 homologues

Myotoxic PLA2s and PL

Binding to ‘acceptors’ in plasma medisruption by catalytically-dependen

Loss of control of

Depolarization Ca2+ influx Loss of ionic gradien

Hypercontraction

Mechanical damageto plasma membrane

Activation of calpainsMito

MitoFlo

HydroDis

Hydrolysis of key structuralproteins (desmin, titin)

Sarcomeric disorganizationand Z line loss Widespread hydro

myofibrillar proteins (aand removal of ce

Fig. 2. Proposed sequence of cellular events occurring as a consequence of the action ofMyotoxins bind to as yet unidentified ‘acceptor’ sites at the plasma membrane, inducing a rthe case of Asp49 PLA2s) and catalyticallyeindependent (in the case of Lys49 PLA2 homologuand macromolecules. The main consequence of this alteration is a prominent Ca2þ influhypercontraction, mitochondrial alterations, and activation of cytosolic Ca2þ-dependent pirreversible damaged. Reprinted, with slight modifications, from Toxicon 42: 915e931, GutiInsights into the mechanisms of local and systemic myotoxicity, copyright 2003, with perm

interact with as yet unidentified ‘acceptors’ in skeletal muscle cellplasma membrane, provoking rapid membrane disruption bycatalytically-dependent and independent mechanisms. The mainconsequence of this membrane perturbation is a rapid influx ofextracellular calcium which, in turn, results in: (a) Hyper-contraction of myofilaments, (b) calcium uptake by mitochondria,eventually resulting in calcium overload and mitochondrial dam-age, and (c) activation of calcium-dependent intracellular pro-teinases (calpains) and probably PLA2s.

3.2. Hemorrhage

Local and systemic bleeding are two frequent and seriousmanifestations of viperid snakebite envenomings (Warrell, 2004;Cardoso et al., 2009). The ability of these venoms to induce hem-orrhage depends on the action of zinc-dependent metal-loproteinases (SVMPs) on the microvasculature (Fox and Serrano,2005; Guti�errez et al., 2005a). The characterization of hemorrhag-ic SVMPs from the venom of B. asper was undertaken in a collab-orative project between ICP and Michael Ovadia and Gadi Borkow,from the University of Tel Aviv (Israel) (Borkow et al., 1993;Guti�errez et al., 1995). A P-I SVMP isolated from this venom,named BaP1, has become a useful tool for investigating the mech-anism of microvessel damage and hemorrhage (Guti�errez et al.,1995; Watanabe et al., 2003). Its primary and tertiary structureswere described through collaborations with Raghuvir Arni (Uni-versidade Estadual Paulista, Brazil) and Jay W. Fox (University ofVirginia, USA), and later on with Torsten Lingott and Irmgard

A2 homologues

mbrane followed by membranet or –independent mechanisms

permeability

ts Efflux of cytosolicmolecules

Internalization of myotoxic PLA2

chondrial Ca2+overload Activation ofcytosolic PLA2s

Further phospholipidhydrolysis in membranes(plasma membrane, SR,

mitochondria)

chondrial swellingcculent densitiesxyapatite formationruption of cristae

Arrival of inflammatory cellslysis ofctin, myosin)ll debris

myotoxic PLA2s and PLA2 homologues from viperid venoms in skeletal muscle cells.apid and drastic membrane perturbation which is based on catalytically-dependent (ines) events. Membrane damage results in the loss of the control of permeability to ionsx which triggers a series of intracellular degenerative events, such as myofilamentroteinases and PLA2s. Muscle cells rapidly pass the ‘point of no return’ and become�errez and Ownby, Skeletal muscle degeneration induced by venom phospholipases A2:ission from Elsevier.


Merfort (University of Freiburg, Germany) (Watanabe et al., 2003;Lingott et al., 2009).

The mechanism of action of hemorrhagic SVMPs has beenexplored in depth at ICP, using a variety of experimental approachesthat include light and electron microscopy, intravital microscopy,immunohistochemistry, endothelial cell cultures, Western blotanalysis, and proteomic characterization of exudates collected inthe vicinity of damaged tissue. Light and electron microscopic ob-servations revealed a rapid disruption of capillary vessel structureafter injection of SVMPs, characterized by a drop in the number ofpinocytotic vesicles and a reduction in the width of endothelialcells, associated with a distention and eventual disruption ofcapillary wall (Moreira et al., 1994; Rucavado et al., 1995). Inter-estingly, SVMPs do not induce direct damage of endothelial cells inculture, but instead provoke the detachment of these cells, whicheventually die by apoptosis (Lomonte et al., 1994b; Rucavado et al.,1995; Díaz et al., 2005).

The action of SVMPs on the basement membrane (BM) sur-rounding endothelial cells in capillaries has been investigated byTeresa Escalante, Alexandra Rucavado, Cristina Herrera, and theauthor, in collaboration with the groups of Jay W. Fox (Universityof Virginia, USA), Sussan Nourshargh and Mathieu-Benoit Voisin(Queen Mary University of London, UK), and Eladio F. S�anchez(Fundaçao Ezequiel Dias, Brazil). By a combination of an in vitromodel, based on hydrolysis of Matrigel, a BM preparation from asarcoma cell line, and in vivo and ex vivo assessment of BM hy-drolysis, based on Western blotting and immunohistochemistryanalyses, it was found that SVMPs are able to degrade laminin,type IV collagen, nidogen, and heparan sulphate proteoglycan(Escalante et al., 2006, 2011a; Herrera et al., 2015). Whencomparing the action of P-I hemorrhagic and non-hemorrhagicSVMPs, a striking difference was noticed regarding the

Fig. 3. Proposed ‘two-step’ mechanism of action of snake venom hemorrhagic zinc-depecomponents of the basement membrane (BM) that surrounds endothelial cells in capillary bldue to the presence of exosites in the disintegrin-like and cysteine-rich domains, which enaactivity of hemorrhagic SVMPs is likely to be type IV collagen, since it plays a key role in theproteolysis of BM components, the capillary wall becomes weakened, and the action of hemshear stress, provokes the distention of endothelial cell and, eventually, the disruption in tProteomics 74: 1781e1794, Escalante et al., Key events in microvascular damage induced byElsevier.

hydrolysis of type IV collagen (Escalante et al., 2011a). It was thussuggested that enzymatic degradation of this BM component is akey step in the pathogenesis of hemorrhage, owing to the prom-inent role that this collagen plays in the mechanical stability of BMand the capillary wall. This hypothesis has been recently sup-ported by further observations with hemorrhagic P-I, P-II, and P-IIISVMPs (Herrera et al., 2015).

These studies have been complemented by a new approach toassess venom-induced tissue damage, based on the proteomiccharacterization of exudates collected in the vicinity of tissuesinjected with venoms or isolated toxins. These analyses, performedin collaborationwith JayW. Fox, provide a ‘window’ through whichmore subtle aspects of tissue damage can be explored (Escalanteet al., 2009, 2011a; Rucavado et al., 2011). In particular, fragmentsof several extracellular matrix (ECM) proteins have been detected,thus revealing hitherto unknown aspects of SVMP action in thetissues.

Taken together, these experimental findings on the mechanismof action of hemorrhagic SVMPs have been unified in a ‘two-stepmodel’ (Fig. 3). Initially, hemorrhagic SVMPs bind and hydrolyzecomponents in the BM of capillary vessels, the degradation of typeIV collagen being of particular relevance. P-II and P-III SVMPs bindmore specifically to capillary BM than P-I SVMP, owing to thepresence of exosites in the disintegrin, disintegrin-like andcysteine-rich domains of these SVMPs (Baldo et al., 2010; Herreraet al., 2015), thus explaining their generally higher hemorrhagicactivity. BM degradation results in the weakening of the mechan-ical stability of the capillary wall; as a consequence, the hemody-namic forces normally operating in the vasculature in vivo, i.e.hydrostatic pressure and shear stress, provoke the distention andthe eventual disruption of the capillary wall, leading to extravasa-tion (Guti�errez et al., 2005a; Escalante et al., 2011b).

ndent metalloproteinases (SVMPs) in capillary blood vessels. SVMPs hydrolyze keyood vessels. P-III SVMPs in general have a higher hemorrhagic potential than P-I SVMPsble them to bind to targets in the BM. The most important substrate for the proteolyticmechanical stability of the BM and hence of the capillary wall. After this first step, i.e.odynamic biophysical forces operating in the circulation, i.e. hydrostatic pressure andhe integrity of the capillary wall, resulting in extravasation. Reprinted from Journal ofsnake venom hemorrhagic metalloproteinases, copyright 2011, with permission from


3.3. Damage to skin and to lymphatic vessels

Two additional aspects of viperid venom-induced local pathol-ogy that have been investigated at ICP are the effects on skin andlymphatic vesicles. SVMPs induce blistering and dermonecrosis, asa consequence of the degradation of proteins of the der-maleepidermal junction (Jim�enez et al., 2008; Escalante et al.,2009). On the other hand, B. asper venom and a Lys49 PLA2 ho-mologue from this venom induce a reduction in the lumen of col-lecting lymphatic vessels in the mesentery, followed by the haltingof lymph flow (Mora et al., 2008). This effect is secondary to theaction of this myotoxin on smooth muscle cells of the lymphaticvessel wall.

3.4. Exploring the causes of deficient skeletal muscle regenerationafter venom-induced myonecrosis

Clinical observations indicate that regeneration of skeletalmuscle after myonecrosis in viperid snakebite envenoming is oftendeficient, thus resulting in permanent tissue loss and disability. Asuccessful muscle regenerative process depends on a number offactors, namely the removal of necrotic material, the presence ofintact blood supply and innervation, and the permanence of the BMsurrounding necrotic fibers, which serves a scaffolding role in theregenerative process (Ciciliot and Schiaffino, 2010). These re-quirements are largely hampered in muscle tissue affected byviperid snake venoms. In the case of the venom of B. asper, ourstudies in collaborationwith Alexandra Rucavado (ICP) and RosarioHern�andez and Patricia Saravia-Otten (Universidad de San Carlos,Guatemala) have shown that the density of microvessels and ofaxons within intramuscular nerves is greatly reduced, as a conse-quence of the action of tissue-damaging toxins (Guti�errez et al.,1984c; Arce et al., 1991; Hern�andez et al., 2011). The observationof TUNEL-positive regenerative cells strongly supports the viewthat the tissue microenvironment does not provide the conditionsnecessary for regeneration (Hern�andez et al., 2011). Through acollaboration with Stefano Gastaldello (Karolinska Institutet, Swe-den) and Patricia Saravia-Otten, it was found that homogenates ofmuscle tissue collected several days after injection of B. aspervenom and toxins inhibit myoblast cell proliferation and fusion intomyotubes in a cell culture model (Saravia-Otten et al., 2013). It isexpected that a more thorough understanding of the basis of thispoor regenerative process may lead the way to therapeutic in-terventions aimed at improving this outcome.

3.5. Inflammation in venom-affected tissues

The action of viperid snake venoms in the tissues is associatedwith a complex inflammatory reaction, characterized by edema,pain, and inflammatory cell infiltrate. Inflammation induced by thevenom of B. asper and by purified SVMPs and myotoxic PLA2s andPLA2 homologues has been investigated through collaborationsbetween ICP and the groups of Catarina F.P. Teixeira and Yara Cury(Instituto Butantan, Brazil) (see reviews by Teixeira et al., 2003a,2009). Edema and pain occur as a consequence of the activationof several inflammatory pathways, with the release of histamine,and the generation of prostaglandins, leukotrienes, kinins, nitricoxide, complement anaphylatoxins, and cytokines, together withthe synthesis of proteinases, mostly matrix metalloproteinases(MMPs) (Rucavado et al., 2002; Teixeira et al., 2009). These andother mediators act on venular endothelial cells and on a variety ofnociceptors in afferent neurons, provoking edema and pain. PLA2sand SVMPs also act directly on resident macrophages and otherinflammatory cells, inducing their activation (Zamun�er et al., 2001;Zuliani et al., 2005; Leiguez et al., 2011).

Whether inflammation contributes to further tissue damage inmuscle injected with the venom of B. asper is an open question thathas been explored by Fernando Chaves and colleagues at ICP, and byresearchers at Instituto Butantan (Teixeira et al., 2003b; Chaveset al., 2005, 2006). Other studies have focused on the possiblerole in venom-induced pathology of tissue components, i.e. dangersignals or DAMPs, released as a consequence of myonecrosis andECM degradation. For example, the release of ATP from damagedmuscle cells results in the expansion of myocyte damage throughthe action of this nucleotide on purinergic receptors, resulting inthe increment of cytosolic calcium and the consequent cytotoxicity(Cintra-Francischinelli et al., 2010).

The large body of experimental observations gathered over theyears in the study of venom-induced local tissue damage by re-searchers of ICP and other groups is summarized in Fig. 4. There is adirect toxic action of venom components, mostly SVMPs, PLA2s andPLA2 homologues. These early pathological events, in turn, generatean inflammatory reaction, in association with the direct effect ofvenom components on resident and inflammatory cells. Themyriad of inflammatory mediators released in the tissue set theonset of reparative and regenerative events, but might alsocontribute to further tissue damage and to impaired regeneration.A more deep understanding of this complex scenario is necessaryfor the design of knowledge-based therapeutic interventions aimedat ameliorating the magnitude of tissue damage and at improvingthe regenerative outcome. These are urgent challenges that de-mand renewed collaborative research efforts.

4. Towards a better characterization of the preclincal efficacyof antivenoms

The traditional way to evaluate the capacity of antivenoms toneutralize snake venoms is based on the lethality neutralizationtest, which is the gold standard for assessing antivenom potency(WHO, 2010; Guti�errez et al., 2013). This test involves the incuba-tion of a fixed dose of venom with various dilutions of antivenom,using a control group in which venom is incubated with salinesolution instead of antivenom. After an incubation interval, aliquotsof the mixtures, containing a ‘challenge dose’ of venom (usuallyeither 3, 4 or 5Median Lethal Doses, LD50s) are injected inmice, andlethality is observed. Antivenom efficacy is thus expressed as theMedian Effective Dose (ED50), i.e. the ratio venom/antivenom atwhich half of the injected animals survive (WHO, 2010; Guti�errezet al., 2013). Using this experimental approach, Bola~nos demon-strated that the polyvalent (viperid) antivenom manufactured atInstituto Clodomiro Picado (ICP) is effective in the neutralization oflethality of Costa Rican venoms of snakes from the family Viperidae(Bola~nos, 1971).

Following early developments, especially by David Theakstonand colleagues, at the Liverpool School of Tropical Medicine (UK)(Theakston and Reid, 1983), and by researchers at ICP, a newrationale of the assessment of the preclinical efficacy of antivenomswas developed. Since viperid, and some elapid, venoms induce avariety of pathologically- and pathophysiologically-relevant effects,in addition to lethality, it was considered that a more thoroughcharacterization of antivenoms' preclinical efficacy should be basedon the analysis of the neutralization of various toxic and enzymaticactivities (Guti�errez et al., 2013). In the early years of the 1980s, ourgroup at ICP initiated a long lasting effort to characterize the effi-cacy of ICP antivenoms (Guti�errez et al., 1981, 1985, 1996), and thenapplied these methodologies to assess other antivenoms againstvenoms from different regions of the world. Simple methods wereadapted or developed for the study of hemorrhagic, myotoxic,dermonecrotic, edema-forming, coagulant, defibrinogenating,proteolytic, hyaluronidase, and PLA2 activities of venoms (see


reviews by Guti�errez et al., 1996, 2013).Regarding the neutralization of Bothrops sp snake venoms,

which inflict the highest number of snakebites in Latin America,these studies have revealed a high extent of cross-neutralizingability of various antivenoms against several Bothrops sp venoms(see for example Bogarín et al., 2000; Rojas et al., 2005; Seguraet al., 2010a). One of the most extensive studies performed in thissubject involved the participation of ten Latin American public in-stitutions from Brazil, Argentina, Peru, Bolivia, Colombia, Panamaand Costa Rica, under the auspices of the Program CYTED (Ciencia yTecnología para el Desarrollo) (Segura et al., 2010a). In contrast to theextensive cross-neutralization of Bothrops sp venoms by anti-venoms, it was found that antivenoms produced using venoms ofthe rattlesnake Crotalus simus from Central America are ineffectivein the neutralization of venoms of subspecies of Crotalus durissusfrom South America, and viceversa (Saravia et al., 2002). Thismethodological platform has been also used to investigate thepreclinical efficacy of antivenoms used in sub-Saharan Africa(Segura et al., 2010b; S�anchez et al., 2015), and in Papua NewGuinea (Vargas et al., 2011).

A significant step forward in our understanding of antivenom'sability to bind venom components came as a corollary of the de-velopments in the field of snake venom proteomics, i.e. ‘venomics’,mostly through the work of the group of Juan J. Calvete, at theInstituto de Biomedicina de Valencia (Spain) (Calvete et al., 2007).Through an intensive collaboration with Calvete's group, scientistsat ICP have been able to characterize the proteomes of almost allthe venoms of Costa Rican snakes of the families Viperidae andElapidae (see Lomonte et al. (2014) for a review). Using the infor-mation gathered from proteomics, the methodology known as‘antivenomics’ was developed (Lomonte et al., 2008; Pla et al.,2012). The so called ‘second generation’ antivenomics protocolconsists in performing affinity chromatography, by passing snakevenoms through a Sepharose gel coupled with antivenom

Fig. 4. Scheme that summarizes the main events in the pathogenesis of local tissue damamyotoxic phospholipases A2s (PLA2s) and zinc-dependent metalloproteinases, induce directdegradation of extracellular matrix (ECM), and damage to lymphatic vessels and nerves. Sucand edema. Resident cells in the tissue, such as macrophages and mast cells, become activatedamage or may provoke further pathology. In turn, inflammation is followed by reparativprocesses in the tissue, may result in functional regeneration or in scarring and tissue loss

antibodies. In this way, venom components which are recognized,or not recognized, by antivenom antibodies can be identified andascribed to the corresponding venom protein families (Pla et al.,2012; Guti�errez et al., 2014). Using this approach, a detailed ac-count of the immunoreactivity of the polyvalent (Viperidae) anti-venom manufactured at ICP, when confronted with many snakevenoms, has been obtained (see Table 1 in Guti�errez et al., 2014).The combination of neutralization tests and antivenomics nowprovides a powerful methodological approach to analyze in detailthe preclinical efficacy of antivenoms, and to make knowledge-based decisions on how to improve antivenoms' neutralizingcoverage (Guti�errez et al., 2013).

5. Confronting snakebite envenoming from a broadperspective: Central America and beyond

5.1. Improvements and innovation in antivenom manufacture

In addition to the described research, ICP has approached theproblem of snakebite envenoming by an integrated perspectivewhich includes the development, production and distribution ofantivenoms, and the creation of extension programs and advocacyefforts at various levels. During its first decade, ICP manufacturedthe antivenoms that were needed in Costa Rica (Bola~nos andCerdas, 1980). On the basis of permanent innovation, the volumeof antivenom production steadily increased over the years, and ICPstarted to cover the antivenom needs of other Central Americancountries. These improvements have been possible due to thecommitment and dedication of the staff of the Industrial Division,the Administrative Section, and the Directors of ICP. This instituteprovides polyvalent (Viperidae) and anti-coral (Elapidae) anti-venoms to Panama, Nicaragua, Honduras, El Salvador, Guatemala,and Belize. More recently, ICP polyvalent antivenom has beendistributed to Ecuador, Peru, Colombia, Trinidad and Tobago, and

ge and inflammation induced by viperid snake venoms. Venom components, mostlypathological effects upon injection in muscle tissue, such as myonecrosis, hemorrhage,h widespread tissue pathology generates an inflammatory response which causes paind and contribute to the inflammatory scenario which might lead to resolution of tissuee and regenerative responses which, depending on the balance between the various. Modified from Guti�errez et al. (2007b).


the island of Saint Lucia.Initially, antivenoms were manufactured at ICP by a salting-out

procedure involving ammonium sulphate precipitation of plasmaproteins (Bola~nos and Cerdas, 1980). A significant innovation wasintroduced in the early 1990s by the use of caprylic acid precipi-tation to separate non-immunoglobulin from immunoglobulinproteins in equine plasma (Rojas et al., 1994), following previousdevelopments in Brazil (dos Santos et al., 1989). This protocolgenerates antivenoms of high purity and yield, and of good physico-chemical quality, thus becoming an alternative for antivenomproduction; in fact, it is being currently used in laboratories ofseveral countries in Latin America and Asia. The creation of theTechnological Development Section (SEDETEC) at ICP provided theimpetus for the improvement of the technologies for antivenommanufacture, and has generated new antivenoms for other regions.Among other achievements, Guillermo Le�on, �Alvaro Segura, Mar-i�angela Vargas, María Herrera, and Mauren Villalta, at SEDETEC,have adapted a new ‘two phase aqueous system’method for equineplasma fractionation (Vargas et al., 2015), and have introducedimprovements in aspects such as freeze-drying (Herrera et al.,2014b) and stability (Segura et al., 2009) of antivenoms, in addi-tion to investigating the mechanisms of adverse reactions to theseimmunobiologicals (Le�on et al., 2013), among other contributions.

5.2. A better understanding of snakebite epidemiology: providinginformation for public health interventions

ICP has been interested in the study of the epidemiology ofsnakebites in Costa Rica (Bola~nos, 1984; Arroyo et al., 1999; Sasaand V�azquez, 2003). In addition, the use of Geographical Informa-tion Systems (GIS) tools allowed a detailed analysis of the incidenceof snakebites in the various regions of the country, and showed thatthe highest incidence occurs in the Central and South Pacific re-gions, in the Talamanca mountain range, in the Caribbean, and insome counties of the Northern region (Hansson et al., 2013).Therefore, although the general incidence of snakebites in thecountry is around 14 per 100,000 population per year, drastic dif-ferences in incidence occur at the county and district levels, some ofwhich have incidences higher than 100 per 100,000 population peryear (Hansson et al., 2013). The study also identified locationswhere transportation to health facilities is more delayed. Anotherstudy found that the incidence of snakebites is affected by weatherfluctuations, and an association was observed between snakebitesand the cold and hot phases of El Ni~no Southern Oscillation (ENSO)(Chaves et al., 2015). Moreover, it was described that snakebitesaffect predominantly poor areas, thus reinforcing the concept thatthis is a ‘disease of poverty’ (Harrison et al., 2009).

This epidemiological information is of high value for decisionmakers at the public health system of the country, since it allowsthe identification of the most vulnerable regions that require spe-cial attention. For instance, the deployment of antivenoms to pri-mary health care centers, in addition to clinics and hospitals, can bedecided on the basis of this knowledge, in order to ensure theavailability of antivenoms where access to hospitals and clinics isdelayed. These developments underscore the relevance of estab-lishing channels of communication and coordination betweenresearch groups and public health authorities and practitioners.

5.3. Promoting prevention and appropriate management ofsnakebite cases through extension programs

Since the onset of ICP, an extension programwas established forconveying the basic concepts of prevention and early managementof snakebites in Costa Rica. It has been targeted especially at highrisk communities and groups, such as rural settings, including

those of indigenous ethnic groups, agricultural workers, in-stitutions whose staff has to travel to rural areas, and primary andsecondary education centers in areas of high incidence of snake-bites. Awide array of teaching materials has been prepared, such asmanuals in both Spanish and languages of indigenous groups, as inthe case of Cab�ecar communities (Instituto Clodomiro Picado,2009). These extension activities involve the participation of stu-dents from the University of Costa Rica. Additionally, there is acontinuous education program on the treatment of snakebiteenvenomings, directed to professionals in the medical field, mostlyphysicians and nurses. It includes teaching in university courses, aswell as seminars andworkshops in clinics and hospitals throughoutthe country. The use of information and communication technol-ogies has allowed the projection of these programs to rural hos-pitals in Costa Rica and in other Central American countries. Theseextension activities, over several decades, have improved theknowledge of the general population on how to prevent snakebitesand what to do in the event of an accident. They have alsocontributed to a better clinical management of snakebite enve-nomings in health facilities.

5.4. Regional cooperation in Latin America

Latin America has had a long tradition in antivenom production,starting with the pioneering work of Vital Brazil at InstitutoButantan (Vital-Brazil, 1987). There are antivenom manufacturinglaboratories in Mexico, Costa Rica, Colombia, Venezuela, Ecuador,Peru, Bolivia, Brazil, and Argentina (Guti�errez et al., 2007a). In thelast decade, regional integration has been promoted betweenpublic laboratories devoted to the production and quality control ofantivenoms, and ICP has been directly involved in such projects.These initiatives have been supported by the Pan American HealthOrganization (PAHO), the Japanese International CooperationAgency (JICA), the Program CYTED (Ciencia y Tecnología para elDesarrollo) and, more recently, the Organization of American States(OAS). Activities performed include workshops, technical consul-tations, training programs, and collaborative research projects (seeGuti�errez et al., 2007a, 2009). The involvement of Instituto Butan-tan, among other institutions, has been essential, especiallythrough the leadership of Fan Hui Wen. These initiatives havestrengthened the regional capacity to produce and control anti-venoms, and have consolidated a regional network of institutions(Fig. 5).

Cooperative research projects between Latin American groupshave been developed in subjects as varied as biochemistry of snakevenoms, the mechanism of action of venoms and toxins, and pre-clinical assessment and clinical evaluation of efficacy and safety ofantivenoms. For instance, through a long standing collaborationbetween ICP and Rafael Otero-Pati~no and colleagues, in Colombia, anumber of controlled, double-blind, randomized clinical trials havebeen performed in that country using the polyvalent antivenomproduced at ICP (Otero et al., 1999, 2006; Otero-Pati~no et al., 2012).

5.5. Antivenom development for other regions of the world

The experience gained by ICP in technological development, andin the manufacture and distribution of antivenoms in Latin Amer-ica, paved the way, more than a decade ago, to new projects for thedevelopment of novel antivenoms for other regions of the world.Following a WHO workshop held in Potters Bar, London, in 2001(Theakston et al., 2003), and in the light of a severe crisis in anti-venom availability in sub-Saharan Africa (Theakston and Warrell,2000), ICP joined a partnership with David G. Theakston andRobert Harrison (Liverpool School of Tropical Medicine, UK), DavidA. Warrell (University of Oxford, UK), Abdusalam Nasidi, Nandul

Fig. 6. ICP has participated in an international partnership aimed at generating anti-venoms, improving snakebite envenoming treatment, and performing toxinologicalresearch in Nigeria. This partnership, known as EchiTAb Study Group, has involved theFederal Ministry of Health of Nigeria, the Liverpool School of Tropical Medicine (UK),the University of Oxford (UK), and the company MicroPharm, together with ICP.Among the various outcomes of this cooperation, two antivenoms [EchiTAb G(Micropharm) and EchiTAb-Plus-ICP (ICP)] were developed and are regularly distrib-uted to Nigeria. In addition, several research projects have contributed to a betterunderstanding of venoms and envenomings induced by snakes in this country.

Fig. 5. Network of public institutions for the production and quality control of anti-venoms in Latin America. This network, supported by organizations such as CYTED(Ciencia y Tecnología para el Desarrollo), OAS (Organization of American States), andPAHO (Pan American Health Organization), as well as by the institutions themselves,has promoted a dynamic partnership based on workshops, seminars, collaborativeresearch projects, training of staff, and technical consultations. Through these coop-erative initiatives, the regional capacity to produce and control antivenoms has beengreatly improved in Latin America. ICP has played a leading role in these efforts.BIRMEX: Laboratorios de Biol�ogicos y Reactivos de M�exico; ICP: Instituto ClodomiroPicado (Costa Rica); INVIMA: Instituto Nacional de Vigilancia de Medicamentos yAlimentos (Colombia); INS: Instituto Nacional de Salud; U Panama: Universidad dePanam�a; Biotecfar (Universidad Central de Venezuela); ENFARMA (Ecuador); U de SanMarcos: Universidad Nacional Mayor de San Marcos (Perú); INLASA: Instituto Nacionalde Laboratorios de Salud (Bolivia); FUNED: Fundaç~ao Ezequiel Dias (Brazil); VitalBrazil: Instituto Vital Brazil (Brazil); INCQS: Instituto Nacional de Controle de Qual-idade em Saúde (Brazil); Butantan: Instituto Butantan (Brazil); CPPI: Centro deProduç~ao e Pesquisa de Imunobiol�ogicos (Brazil); ANLIS: Administraci�on Nacional deLaboratorios e Institutos de Salud (Argentina).


Durfa and other Nigerian colleagues (Federal Ministry of Health ofNigeria), and the company Micropharm (UK) (Fig. 6). This con-sortium, known as EchiTAb Study Group, developed several ini-tiatives aimed at improving antivenom availability for Nigeria. Onthe basis of epidemiological, clinical, and immunological knowl-edge, a new antivenomwas developed at ICP, using a mixture of thevenoms of Echis ocellatus, Bitis arietans and Naja nigricollis for im-munization, and the purification of antibodies from horse plasmaby the caprylic acid fractionation method (Guti�errez et al., 2005b).This polyspecific antivenom, initially named ‘Pan-African anti-venom’, and then known as ‘EchiTAb-Plus-ICP’, proved to beeffective at the preclinical level in the neutralization of the threevenoms used for immunization, as well as of other venoms ofmedical relevance of the genera Echis, Bitis and Naja (Guti�errezet al., 2005b; Segura et al., 2010b; Calvete et al., 2010). Then, thisantivenom was evaluated in a large clinical trial performed inNigeria, and showed satisfactory efficacy and safety profiles inenvenomings by E. ocellatus (Abubakar et al., 2010). It has beenregistered in various countries and is now regularly distributed tosub-Saharan Africa.

Following this successful experience, a new partnership wasestablished between ICP and the universities of Melbourne(Australia) and Papua New Guinea (PNG), with the participation ofDavid Williams, Kenneth Winkel, Simon Jensen, Owen Paiva andTeatulohi Matainaho, together with the group of the IndustrialDivision of ICP. The goal was to develop a potent and safe mono-specific antivenom to be used in PNG for the treatment of enve-nomings by the elapid snake Taipan (Oxyuranus scutellatus). Inparallel with the proteomic characterization of the venom (Herreraet al., 2012), the new antivenom was shown to be effective at the

preclinical level (Vargas et al., 2011; Herrera et al., 2014a). It is nowbeing tested in a clinical trial in PNG and, depending on the results,the long term purpose is to manufacture this antivenom at ICP fordistribution and use in PNG. The support of ICP's Directors, GustavoRojas, Yamileth Angulo and Alberto Alape, has played a key role inthe success of these international initiatives.

Another project has started as a collaboration between ICP, theUniversity of Peradeniya (Sri Lanka), and the non-governmentalorganization Animal Venom Research International (AVRI, USA),with the participation of Indika Gawarammana, Daniel Keyler,Kimberly McWhorter, Roy Malleappah, and Laki Wickremesinghe,and the group of the Industrial Division of ICP. As a first step, a newpolyspecific antivenom effective against the venoms of the mostimportant poisonous snakes of Sri Lanka will be developed.Following a clinical trial, scheduled to start in 2016, the project aimsat implementing the transfer of the technology used at ICP forantivenom production to Sri Lanka, with the support of the Tech-nology Transfer Unit (PROINNOVA) of the University of Costa Rica,in order to allow the local manufacture of the antivenom in thisAsian country (Keyler et al., 2013). If successful, this new model ofcooperation might be adapted, in the future, to other countries andregions.

5.6. Advocacy: raising global awareness of the impact of snakebiteenvenoming

The commitment of ICP in the search for solutions to reduce theimpact of snakebite envenoming has also involved, together withpeople of other institutions and countries, a number of advocacyefforts. These include the participation in international workshopsand congresses, and in the preparation of the WHO Guidelines forthe Production, Control and Regulation of Snake Antivenom Immu-noglobulins (WHO, 2010). Moreover, papers have been prepared topromote awareness on the problem of snakebites (Guti�errez et al.,2006, 2010, 2013), and contacts with national and internationalhealth authorities have been established. ICP has been supportiveof the Global Snakebite Initiative (GSI), which is currently imple-menting actions to confront the problem of snakebite envenoming


and its consequences (Williams et al., 2010; www.snakebiteinitative.org).

6. Concluding remarks

This overview highlights part of what ICP has achieved, in CostaRica and in partnership with groups in many countries, for a betterunderstanding of venomous snakes, venoms, and their effects atexperimental and clinical levels. These efforts at the scientific realmhave been linked to the development of novel technological plat-forms for the production and quality control of antivenoms, thuscontributing to the improvement of antivenom availability andaccessibility in Latin America, sub-Saharan Africa, and PNG.Extension programs at national and regional levels have provided abetter understanding of the basic aspects of snakebite preventionand management to communities and health professionals. Finally,as a fourth component of this multifaceted strategy, ICP hasstrengthened human resources in Costa Rica and elsewhere,through teaching activities at graduate and undergraduate levels.The involvement of students in the research projects of ICP hasbeen of paramount importance. The basic philosophy that hasguided this institutional project has been based on the search forexcellence and new challenges, a cooperative and collaborativeview of scientific work, an integrated approach to understandingand solving a complex public health problem, and a vision of soli-darity and compassion.

Ethical statement

The writing of this article followed national and internationalethical guidelines in the preparation of scientific manuscripts.

Acknowledgments

The achievements discussed in this review have been possiblethrough the commitment and dedication of the people who haveworked at ICP during 45 years. In addition, highly fruitful collabo-rative projects have been performed with colleagues of institutionsin many countries; their cooperation has been essential in thefulfillment of the goals described, and is therefore deeplyacknowledged. Thanks are due to Prof Alan Harvey for supportingthe publication of this review. Finally, the support of the authoritiesof Universidad de Costa Rica over the years, and of several inter-national agencies which have provided funding for our projects, isalso recognized.

Transparency document

Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.toxicon.2015.11.013.

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