Embriotoxic effects of Crotalus durissus terrificus snake venom

A atividade neurotóxica, miotóxica e coagulante do veneno das serpentes Crotalus durissus terrificus (VCdt) são responsáveis pelas altas taxas de mortalidade observada em acidentes envolvendo estas serpentes. Estes acidentes, quando ocorrem na gravidez, podem levar ao aborto devido à interferência com a homeostasia materna e/ou à embrioletalidade, por efeito direto do veneno. Materiais e Métodos - Este trabalho estudou os efeitos tóxicos do ( VCdt), administrado nas doses de 75 mg/kg or 200 mg/kg por via subcutânea em camundongas no terceiro dia da sua gestação (período de pré-implantação). O grupo controle foi tratado da mesma forma que os experimentais, porém com solução salina. No último dia da gestação as fêmeas foram submetidos a eutanásia e observadas as possíveis malformações ósseas e viscerais de sua prole. Resultados e Conclusão - Os resultados mostraram que a administração da menor dose do VCdt não causou alterações significantes no desenvolvimento ósseo e visceral dos animais. No entanto, quando expostos a maior dose este promoveu aumento significante das anomalias e malformações, sugerindo que o envenenamento com esta dose no período inicial da gestação altera o desenvolvimento normal da prole de camundongosIntroduction - The neurotoxic, myotoxic and coagulant activities of Crotalus durissus terrificus snake venom (VCdt) are responsible for the mortality rates observed in accidents involving the rattlesnake. Accidents during women pregnancy are a challenge, since animal venoms could led to pregnancy interruption as a consequence of maternal homeostasis disorder and/or a direct embryotoxic effect of the venom. Materials and Methods - In order to evaluate the possible embryotoxic effects of VCdt, doses of 75 mg/kg or 200 mg/kg of the venom were administered by subcutaneous route at day 3 of mice pregnancy (preimplantation period). The control group received saline in the same volume and during the same period as their respective experimental groups. The animals were submitted to euthanasia at term. Results and Conclusion - The treatment of the females during the preimplantation period did not cause significant changes in fetuses development, but the higher dose of the venom increased the number of anomalies or malformations of the fetuses. These results suggest that the VCdt in the higher dose (200 mg/kg) altered the normal development of the concept after implantatio

Walker 256 tumor growth suppression by crotoxin involves formyl peptide receptors and lipoxin a(4)

We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A(4). Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A 4 and its natural analogue 15-epi-LXA(4) 4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A 4 and 15-epi-LXA 4, which might inhibit both tumor growth and formation of new vessels via FPRs.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (fellowship-CAPES)PAP (fellowship-Secretaria da Saude do Estado de Sao Paulo)FAPESP [07/52447-8]Guggenheim FoundationSpecial Laboratory of Pain and Signaling, Butantan Institute, Avenida Vital Brazil 1500, 05503-900 São Paulo, SP, BrazilCEIS/Department of Biology, Institute of Biosciences of Rio Claro, São Paulo State University (UNESP), Rio Claro, SP, BrazilLaboratory of Pathophysiology, Butantan Institute, Avenida Vital Brazil 1500, 05503-900 São Paulo, SP, BrazilDepartment of Natural Sciences, Mathematics and Education, Agricultural Sciences Center, Federal University of São Carlos, Rodovia Anhanguera Km 174, 13600-970 Araras, SP, BrazilLaboratory of Inflammation and Vascular Pharmacology, Federal University of São Paulo, Rua São Nicolau 210, 09913-030 Diadema, SP, BrazilDepartment of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Avenida Professor Lineu Prestes 1524, 05508-900 São Paulo, SP, BrazilDepartment of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, Avenida Professor Lineu Prestes 1524, 05508-900 São Paulo, SP, BrazilLaboratory of Inflammation and Vascular Pharmacology, Federal University of São Paulo, Rua São Nicolau 210, 09913-030 Diadema, SP, BrazilFAPESP: 07/52447-8Web of Scienc

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient’s lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluationCentro para el Estudio de Venenos y Animales Venenosos/[063-2010]/CEVAP/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Peripheral sensitization increases opioid receptor expression and activation by Crotalphine in rats

Inflammation enhances the peripheral analgesic efficacy of opioid drugs, but the mechanisms involved in this phenomenon have not been fully elucidated. Crotalphine (CRP), a peptide that was first isolated from South American rattlesnake C.d. terrificus venom, induces a potent and long-lasting anti-nociceptive effect that is mediated by the activation of peripheral opioid receptors. Because the high efficacy of CRP is only observed in the presence of inflammation, we aimed to elucidate the mechanisms involved in the CRP anti-nociceptive effect induced by inflammation. Using real-time RT-PCR, western blot analysis and ELISA assays, we demonstrate that the intraplantar injection of prostaglandin E2 (PGE2) increases the mRNA and protein levels of the µ- and κ-opioid receptors in the dorsal root ganglia (DRG) and paw tissue of rats within 3 h of the injection. Using conformation state-sensitive antibodies that recognize activated opioid receptors, we show that PGE2, alone does not increase the activation of these opioid receptors but that in the presence of PGE2, the activation of specific opioid receptors by CRP and selective µ- and κ-opioid receptor agonists (positive controls) increases. Furthermore, PGE2 down-regulated the expression and activation of the δ-opioid receptor. CRP increased the level of activated mitogen-activated protein kinases in cultured DRG neurons, and this increase was dependent on the activation of protein kinase Cζ. This CRP effect was much more prominent when the cells were pretreated with PGE2. These results indicate that the expression and activation of peripheral opioid receptors by opioid-like drugs can be up- or down-regulated in the presence of an acute injury and that acute tissue injury enhances the efficacy of peripheral opioids.Fundação de Amparo a Pesquisa do Estado de São Paulo, Brazil (FAPESP, grant numbers 07/03404-4, 07/00135-2)Instituto Nacional de Ciencia e Tecnologia em Toxinologia (INCTTOX PROGRAM)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number 573790/2008-6)FAPESP (grant number 2008/57898-0)CAPES/CNPq, grant numbers 301508/2008-9 and 305345/2011-

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Pharmacological modulation of hyperalgesia induced by Bothrops asper (terciopelo) snake venom

The ability of Bothrops asper snake venom to cause hyperalgesia was investigated in rats, using the paw pressure test. Intraplantar injection of the venom (5–15 μg/paw) caused a dose and time-related hyperalgesia, which peaked 2 h after venom injection. Bothrops asper venom-induced hyperalgesia was blocked by the bradykinin B2 receptor antagonist HOE 140 and attenuated by dexamethasone, an inhibitor of phospholipase A2. Inhibition of the lipoxygenase pathway by NDGA abrogated the algogenic phenomenon. The hyperalgesic response was not modified by pretreatment with indomethacin, an inhibitor of the cyclo-oxygenase pathway, by meloxicam, an inhibitor of the type 2 cyclo-oxygenase pathway, by the PAF receptor antagonist BN52021 or by anti-TNF-α or anti-interleukin 1 antibodies. Intraplantar injection of the venom also caused an oedematogenic response which was not modified by any of these pharmacological treatments. These results suggest that hyperalgesia induced by Bothrops asper venom is, at least partially, mediated by bradykinin, phospholipase A2 activity and leukotrienes. Distinct mechanisms are involved in the development of hyperalgesia and oedema induced by the venom.Fundação de Amparo à Pesquisa do Estado de São Paulo/[98/00257-0]/FAPESP/BrasilInstituto Butantan///BrasilUniversidad de Costa Rica//UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP