Rabbit IgG antibodies against phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 μg) with Freund adjuvant. Groups of six mice (20 ± 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 μg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms

Arteriopathy diagnosis in childhood arterial ischemic stroke: results of the vascular effects of infection in pediatric stroke study.

Background and purposeAlthough arteriopathies are the most common cause of childhood arterial ischemic stroke, and the strongest predictor of recurrent stroke, they are difficult to diagnose. We studied the role of clinical data and follow-up imaging in diagnosing cerebral and cervical arteriopathy in children with arterial ischemic stroke.MethodsVascular effects of infection in pediatric stroke, an international prospective study, enrolled 355 cases of arterial ischemic stroke (age, 29 days to 18 years) at 39 centers. A neuroradiologist and stroke neurologist independently reviewed vascular imaging of the brain (mandatory for inclusion) and neck to establish a diagnosis of arteriopathy (definite, possible, or absent) in 3 steps: (1) baseline imaging alone; (2) plus clinical data; (3) plus follow-up imaging. A 4-person committee, including a second neuroradiologist and stroke neurologist, adjudicated disagreements. Using the final diagnosis as the gold standard, we calculated the sensitivity and specificity of each step.ResultsCases were aged median 7.6 years (interquartile range, 2.8-14 years); 56% boys. The majority (52%) was previously healthy; 41% had follow-up vascular imaging. Only 56 (16%) required adjudication. The gold standard diagnosis was definite arteriopathy in 127 (36%), possible in 34 (9.6%), and absent in 194 (55%). Sensitivity was 79% at step 1, 90% at step 2, and 94% at step 3; specificity was high throughout (99%, 100%, and 100%), as was agreement between reviewers (κ=0.77, 0.81, and 0.78).ConclusionsClinical data and follow-up imaging help, yet uncertainty in the diagnosis of childhood arteriopathy remains. This presents a challenge to better understanding the mechanisms underlying these arteriopathies and designing strategies for prevention of childhood arterial ischemic stroke

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Global Retinoblastoma Presentation and Analysis by National Income Level

Importance: Early diagnosis of retinoblastoma, the most common intraocular cancer, can save both a child's life and vision. However, anecdotal evidence suggests that many children across the world are diagnosed late. To our knowledge, the clinical presentation of retinoblastoma has never been assessed on a global scale. Objectives: To report the retinoblastoma stage at diagnosis in patients across the world during a single year, to investigate associations between clinical variables and national income level, and to investigate risk factors for advanced disease at diagnosis. Design, Setting, and Participants: A total of 278 retinoblastoma treatment centers were recruited from June 2017 through December 2018 to participate in a cross-sectional analysis of treatment-naive patients with retinoblastoma who were diagnosed in 2017. Main Outcomes and Measures: Age at presentation, proportion of familial history of retinoblastoma, and tumor stage and metastasis. Results: The cohort included 4351 new patients from 153 countries; the median age at diagnosis was 30.5 (interquartile range, 18.3-45.9) months, and 1976 patients (45.4) were female. Most patients (n = 3685 84.7%) were from low-and middle-income countries (LMICs). Globally, the most common indication for referral was leukocoria (n = 2638 62.8%), followed by strabismus (n = 429 10.2%) and proptosis (n = 309 7.4%). Patients from high-income countries (HICs) were diagnosed at a median age of 14.1 months, with 656 of 666 (98.5%) patients having intraocular retinoblastoma and 2 (0.3%) having metastasis. Patients from low-income countries were diagnosed at a median age of 30.5 months, with 256 of 521 (49.1%) having extraocular retinoblastoma and 94 of 498 (18.9%) having metastasis. Lower national income level was associated with older presentation age, higher proportion of locally advanced disease and distant metastasis, and smaller proportion of familial history of retinoblastoma. Advanced disease at diagnosis was more common in LMICs even after adjusting for age (odds ratio for low-income countries vs upper-middle-income countries and HICs, 17.92 95% CI, 12.94-24.80, and for lower-middle-income countries vs upper-middle-income countries and HICs, 5.74 95% CI, 4.30-7.68). Conclusions and Relevance: This study is estimated to have included more than half of all new retinoblastoma cases worldwide in 2017. Children from LMICs, where the main global retinoblastoma burden lies, presented at an older age with more advanced disease and demonstrated a smaller proportion of familial history of retinoblastoma, likely because many do not reach a childbearing age. Given that retinoblastoma is curable, these data are concerning and mandate intervention at national and international levels. Further studies are needed to investigate factors, other than age at presentation, that may be associated with advanced disease in LMICs. © 2020 American Medical Association. All rights reserved

Rabbit IgG antibodies against phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 μg) with Freund adjuvant. Groups of six mice (20 ± 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 μg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms

Pharmacological Activities Of Ba Spii Rp4 Pla2 From Bothrops Alternatus Snake Venom [actividades Farmacológicas De La Fosfolípasa A2 Ba Spii Rp4 Del Veneno De Bothrops Alternatus]

The aim of this work was to evaluate the pharmacological activities (citotoxicity, bactericidal, platelet aggregation) and the changes in the activity/stability of the isolated phospholipase A2 (PLA2) from Bothrops alternatus venom. In order to elucidate myotoxic and bactericidal activities, murine mioblast /miotubules (C2C12) and strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were used, respectively. Also, normal mammary epithelial cell culture (NMuMG) and tumoral type (LM3) were tested for a possible role in oncological therapy. With the purpose of studying the inhibitory effect of the PLA2 on platelet aggregation, washed human platelet and thrombin as physiological inductor, were used. Finally, changes in the activity and stability of the isolated PLAj at different temperatures (4-90°C) by kinetic assays were recorded; while the enzyme was subjected to radial indirect hemolysis to evaluate the effect of different pH (2.5-10). The PLAj presented dose-dependent inhibitory effect on thrombin-induced platelet aggregation. Also, it showed high structural stability when exposed to extreme conditions of temperature and pH during several hours, and catalytic activity remained almost unchanged. Nevertheless, this enzyme tested on different cells cultures did not induce cytotoxic (even at high doses) or bactericidal activities. These findings have a scientific value for toxicology since they expand the knowledge of the characterization (biological, biochemical and structural aspects) of one of the most abundant enzyme (PLA 2 Ba SpII RP4) from the protein package of B. alternatus snake venom from the northeast of Argentina. Likewise, the confirmed safety and stability of this PLA2 strongly suggest that it could be proposed as an alternative immunogen for the antiserum production against Bothrops snake venom.2312531Acosta, O.C., Teibler, P., Koscinczuk, P., Sánchez, N.M., Trulls, H., Maruñak, S., Edema and myonecrosis induced by Bothrops jararaca venom of Argentina in mice (1996) Acta Physiol Pharmacol Ther Latinoam, 46, pp. 233-238Acosta De Perez, O.C., Koscinczuk, P., Gutierrez, J.M., Actividades hemorrágica y coagulante del veneno de Bothrops alternatus de Argentina (1996) Acta Bioquimica Clinica Latinoamericana, 30 (4), pp. 401-405Acosta De Perez, O.C., Koscinczuk, P., Teibler, P., Sanchez Negrette, M., Ruiz, R., Marunak, S., Bogarin, G., Hemorrhagic, oedema-forming activities and histopathological alterations in the mouse footpad induced by Bothrops and Crotalus snake venoms from Argentina (1998) Toxicon, 36 (8), pp. 1165-1172. , DOI 10.1016/S0041-0101(98)00007-5, PII S0041010198000075Andriao-Escarso, S.H., Soares, A.M., Fontes, M.R., Fuly, A.L., Correa, F.M., Rosa, J.C., Greene, L.J., Giglio, J.R., Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from bothrops jararacussu snake venom (2002) Biochem Pharmacol, 64, pp. 723-732Born, G.V., Aggregation of blood platelets by adenosine diphosphate and its reversal (1962) Nature, 194, pp. 927-929Branson, D., Métodos en bacteriología clínica. Manual de tests y procedimientos (1974) Panamericana, Buenos Aires, p. 256Bustillo, S., Gay, C., Garcia, D.M.E., Ponce, L.A., Kier-Joffé, E.B., Acosta, O.C., Leiva, L.C., Synergism between baltergin metalloproteinase and Ba SPII RP4 PLA2 from Bothrops alternatus venom on skeletal muscle (C2C12) cells (2012) Toxicon, 59, pp. 338-343Bustillo, S., Lucero, H., Leiva, L.C., Acosta, O.C., Kier-Joffé, E.B., Gorodner, J.O., Cytotoxicity and morphological analysis of cell death induced by bothrops venoms from the northeast of Argentina (2009) J Venom Anim Toxins incl Trop Dis, 15, pp. 28-42Esteso, S.C., (1985) Ofidismo en la República Argentina, p. 170. , Ed, Arpón, Córdoba (Argentina)Fox, J.W., Serrano, S.M.T., Structural considerations of the snake venom metalloproteinases, key members of the M12 reprolysin family of metalloproteinases (2005) Toxicon, 45 (8), pp. 969-985. , DOI 10.1016/j.toxicon.2005.02.012, PII S0041010105000644, Snake Toxins and HemostasisGarcia, D.M.E., Acosta, O.C., Huancahuire, S., Martins, D., Marangoni, S., Maruñak, S.L., Teibler, G.P., Ponce, L.A., Isolation and functional characterization of a new acidic PLA(2) Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina (2010) Toxicon, 56, pp. 64-74Gay, C.C., Leiva, L.C., Marunak, S., Teibler, P., Acosta De Perez, O., Proteolytic, edematogenic and myotoxic activities of a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom (2005) Toxicon, 46 (5), pp. 546-554. , DOI 10.1016/j.toxicon.2005.06.019, PII S0041010105002308Gutiérrez, J.M., Avila, C., Rojas, E., Cerdas, L., An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica (1988) Toxicon, 26, pp. 411-413Kini, R.M., Anticoagulant proteins from snake venoms: Structure, function and mechanism (2006) Biochemical Journal, 397 (3), pp. 377-387. , DOI 10.1042/BJ20060302Lobo, A., Radvanyi, F., Determination of phospholipase A2 activity by a colorimetric assay using a pH indicator (1987) Toxicon, 25, pp. 1181-1188Ohier, M., Georgieva, D., Seifert, J., Von Bergen, M., Arni, R.K., Genov, N., Betzel, C., The venomics of Bothrops alternatus is a pool of acidic proteins with predominant hemorrhagic and coagulopathic activities (2010) J Proteome Res, 9, pp. 2422-2437Perumal, R., Gopalakrishnakone, P., Ho, B., Chow, V.T., Purification, characterization and bactericidal activities of basic phospholipase A2 from the venom of Agkistrodon halys (Chinese pallas) (2008) Biochimie, 90, pp. 1372-1388Ponce-Soto, L.A., Lomonte, B., Gutierrez, J.M., Rodrigues-Simioni, L., Novello, J.C., Marangoni, S., Structural and functional properties of BaTX, a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus (2007) Biochimica et Biophysica Acta - General Subjects, 1770 (4), pp. 585-593. , DOI 10.1016/j.bbagen.2006.11.015, PII S0304416506003655Rodrigues, R.S., Izidoro, L.F.M., Teixeira, S.S., Silveira, L.B., Hamaguchi, A., Homsi-Brandeburgo, M.I., Selistre-De-Araujo, H.S., Rodrigues, V.M., Isolation and functional characterization of a new myotoxic acidic phospholipase A2 from Bothrops pauloensis snake venom (2007) Toxicon, 50 (1), pp. 153-165. , DOI 10.1016/j.toxicon.2007.03.005, PII S0041010107000967Rodrigues, V.M., Marcussi, S., Cambraia, R.S., De Araujo, A.L., Malta-Neto, N.R., Hamaguchi, A., Ferro, E.A.V., Soares, A.M., Bactericidal and neurotoxic activities of two myotoxic phospholipases A2 from Bothrops neuwiedi pauloensis snake venom (2004) Toxicon, 44 (3), pp. 305-314. , DOI 10.1016/j.toxicon.2004.06.008, PII S0041010104002600Ruiz, R., Sario, H.M., Epidemiología del accidente ofidico en la provincia de corrientes (2004) Ann Com Cientif Tecnol UNNE, (Corrientes, Argentina), V-029Santos, N.A., Silveira, L.B., Oliveira, C.Z., Bernardes, C.P., Menaldo, D.L., Fuly, A.L., Arantes, E.C., Soares, A.M., A new acidic myotoxic, anti-platelet and prostaglandin 12 inductor phospholipase A2 isolated from bothrops moojeni snake venom (2008) Toxicon, 52, pp. 908-917Smolka, M.B., Marangoni, S., Oliveira, B., Novello, J.C., Purification and partial characterization of a thrombin-like enzyme, balterobin, from the venom of Bothrops alternatus (1998) Toxicon, 36 (7), pp. 1059-1063. , DOI 10.1016/S0041-0101(98)80008-1, PII S0041010197000986Son, D.J., Park, M.H., Chae, S.J., Moon, S.O., Lee, J.W., Song, H.S., Moon, D.C., Hong, J.T., Inhibitory effect of snake venom toxin from Vipera lebetina turanica on hormone-refractory human prostate cancer cell growth: Induction of apoptosis through inactivation of nuclear factor κB (2007) Molecular Cancer Therapeutics, 6 (2), pp. 675-683. , DOI 10.1158/1535-7163.MCT-06-032

Synergism Between Baltergin Metalloproteinase And Ba Spii Rp4 Pla2 From Bothrops Alternatus Venom On Skeletal Muscle (c2c12) Cells

Acute muscle damage, myonecrosis, is one of the main characteristics of envenoming by Bothrops genus. In this invitro study we investigated the role of a metalloproteinase (baltergin) and an acidic phospholipase A2 (Ba SPII RP4) in the cytotoxicity exhibited by Bothrops alternatus venom. Baltergin metalloproteinase purified from the venom exerted a toxic effect on C2C12 myoblast cells (CC50: 583.34μg/mL) which involved morphological alterations compatible with apoptosis/anoikis. On the contrary, the most abundant PLA2 isolated from this venom did not exhibit cytotoxicity at times and doses tested. However, when myoblasts were treated with both enzymes together, synergic activity was demonstrated. Neutralization of the venom with specific antibodies (IgG anti-baltergin and IgG anti-PLA2) confirmed this synergism. © 2011 Elsevier Ltd.592338343Angulo, Y., Lomonte, B., Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms (2005) Cell Biochem. Funct., 23, pp. 307-313Bonfim, V.L., de Carvalho, D.D., Ponce-Soto, L.A., Kassab, B.H., Marangoni, S., Toxicity of phospholipases A2 D49 (6-1 and 6-2) and K49 (Bj-VII) from Bothrops jararacussu venom (2009) Cell Biol. Toxicol., 25, pp. 523-532Borkow, G., Gutierrez, J.M., Ovadia, M., Invitro activity of BaH1, the main hemorrhagic toxin of Bothrops asper snake venom on bovine endothelial cells (1995) Toxicon, 33, pp. 1387-1391Brenes, O., Munoz, E., Roldan-Rodriguez, R., Diaz, C., Cell death induced by Bothrops asper snake venom metalloproteinase on endothelial and other cell lines (2010) Exp. Mol. Pathol., 88, pp. 424-432Bustillo, S., Lucero, H., Leiva, L.C., Acosta, O., Kier Joffé, E.B., Gorodner, J.O., Cytotoxicity and morphological analysis of cell death induced by Bothrops venoms from the northeast of Argentina (2009) J. Venom Anim. Toxins Incl. Trop. Dis., 15, pp. 28-42Cintra-Francischinelli, M., Pizzo, P., Angulo, Y., Gutierrez, J.M., Montecucco, C., Lomonte, B., The C-terminal region of a Lys49 myotoxin mediates Ca(2+) influx in C2C12 myotubes (2009) ToxiconChavez-Olortegui, C., Penaforte, C.L., Silva, R.R., Ferreira, A.P., Rezende, N.A., Amaral, C.F., Diniz, C.R., An enzyme-linked immunosorbent assay (ELISA) that discriminates between the venoms of Brazilian Bothrops species and Crotalus durissus (1997) Toxicon, 35, pp. 253-260Diaz, C., Valverde, L., Brenes, O., Rucavado, A., Gutierrez, J.M., Characterization of events associated with apoptosis/anoikis induced by snake venom metalloproteinase BaP1 on human endothelial cells (2005) J. 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