The effect of phospholipase A2 from Crotalus durissus collilineatus on Leishmania (Leishmania) amazonensis infection

In this study, the effect of phospholipase A2 (PLA2) derived from Crotalus durissus collilineatus was evaluated in vitro and in vivo on experimental cutaneous leishmaniasis. The promastigote and amastigote forms treated with PLA2 presented increased growth rate. In vivo studies showed that PLA2-treated Leishmania (Leishmania) amazonensis promastigotes increased the size of lesions in BALB/c mice, and histopathological analysis showed numerous necrotic regions presenting a higher density of polymorphonuclear, mononuclear, and amastigote cells. Additionally, infected macrophages treated with PLA2 were able to generate prostaglandin E2 (PGE2). Cytokine quantification showed that the supernatant from infected macrophages presented moderate and high amounts of IL-2 and IL-10, respectively. However, in PLA2-treated infected macrophages, suppression of IL-2 levels occurred, but not of IL-10 levels. Observation also revealed that both the supernatant and lysate of L. (L.) amazonensis promastigotes exhibited PLA2 activity, which, in the presence of dexamethasone, showed no reduction in their activities; while glucocorticoid maintained the ability of promastigote forms to infect macrophages, which presented values similar to controls. In conclusion, the results indicate that PLA2 may be a progression factor for cutaneous leishmaniasis, since the PLA2 effect suppressed IL-2 levels and generated PGE2, an inflammatory lipid mediator.

Maternal and Developmental Toxicity of Ayahuasca in Wistar Rats

INTRODUCTION: Ayahuasca is a psychotropic plant beverage initially used by shamans throughout the Amazon region during traditional religious cult. In recent years, ayahuasca has also been used in ceremonies of a number of modern syncretic religious groups, including pregnant women. However, no documented study has been performed to evaluate the risk of developmental toxicity of ayahuasca. METHODS: In the present work, maternal and developmental toxicity was evaluated in Wistar rats. Ayahuasca was administered to pregnant rats in three different doses [the equivalent typical dose (TD) administered to humans, five-fold TD and 10-fold TD] during the gestational period (6-20 days). RESULTS: Dams treated with the highest ayahuasca dose showed maternal toxicity with decrease of weight gain and food intake. Visceral fetal findings were observed in all treatment groups. Skeletal findings were observed in the intermediate- and high-dose groups. The fetuses deriving from the highest dose group also presented a decrease in body weight. CONCLUSIONS: From these results, it is possible to conclude that there is a risk of maternal and developmental toxicity following ayahuasca exposure and that the level of toxicity appears to be dose-dependent. Birth Defects Res (Part B) 89:207-212, 2010. (C) 2010 Wiley-Liss, Inc.

Habitat use, diet and body size of Heard Island weevils

Habitat use, diet and body-size variation are examined in weevils from Heard Island. with specific attention being given to the Ectemnorhinus viridis species complex. E. viridis shows marked altitudinal variation in body size and vestiture, but there are no consistent associations between body size and diet. nor are there consistent among-individual differences in conventional taxonomic characters. Thus, the status of E. viridis as a single, variable species is maintained. This species occurs from sea level to 600 rn and it feeds on vascular plants and bryophytes. Canonopsis sericeus also feeds on bryophytes and vascular plants and occurs over a narrower altitudinal range. Palirhoeus eatoni is restricted to the surpralittoral zone where it feeds on marine algae and lichens. Bothrometopus brei,is and B. gracilipes both feed on cryptogams, with the former species occurring from sea level to 450 m. and the latter from 50 to 550 m above sea level. In all species, males are smaller than females and there is a size cline such that populations from higher elevations are smaller than those at lower altitudes. This cline is the reverse of that found on the Prince Edward Islands which, unlike Heard Island, lie to the north of the Antarctic Polar Frontal Zone. This difference in body-size clines between weevils on the two island groups is ascribed to the shorter growing season on the colder Heard Island. The information presented here supports previous ideas regarding the evolution of the Ectemnorhinus-group of weevils on the South Indian Ocean Province Islands, although it suggests that subsequent tests of these hypotheses would profit from the inclusion of molecular systematic work.

Fracionamento do veneno Crotalus durissus terrificus por cromatografia de exclusão molecular

Através da técnica de cromatografia de exclusão molecular utilizando Sephadex G-75 foram estudadas as diferentes frações do veneno de Crotalus durissus terrificus, uma das serpentes peçonhentas mais comuns no Brasil. Foram obtidos 4 picos (correspondentes às frações 32, 60, 86 e 103) com peso molecular variando de 4.000 a 150.000. As frações de todo o diagrama de gel filtração foram triadas através de reação de imunoeletroforese a fim de se verificar suas cargas e velocidade de migração. As linhas de precipitação encontradas foram comparadas às 11 linhas apresentadas pela reação de imunoeletroforese do veneno total contra o soro anti-crotálico. Constatou-se que as frações de um mesmo pico apresentavam características próprias com exceção da fração 54 (subida do pico II) que mostrou diferenças significativas em relação à fração 60. Após a triagem foram escolhidas as frações de cada pico onde as linhas de precipitação foram mais nítidas e intensas, para estudo de identidade através da reação de difusão radial dupla e letalidade comparada a concentração de 0,0625 mg/ml do veneno total que corresponde a DL50 em camundongo pelo método de SPEARM & KÄRBER. As frações 32, 86 e 103 correspondentes respectivamente aos picos I, III e IV apresentaram letalidade nula ou negligenciada e a fração II foi a mais tóxica.

Rabdomióilise secundária a acidente ofídico crotálico (Crotalus durissus terrificus)

Relatam-se dois casos de rabdomiólise secundária a envenenamento produzido por Crotalus durissus terrificus. O diagnóstico da rabdomiólise baseou-se na mialgia intensa e generalizada apresentada pelos pacientes e na constatação de níveis séricos elevados de CPK, TGO e DHL. A confirmação do diagnóstico foi obtida no caso n.° 2 pela detecção de mioglobina no soro através de imunoeletroforese contra soro antimioglobina humana e por biópsia muscular. Esse paciente desenvolveu também, como complicação do envenenamento ofídico, quadro clínico e laboratorial de insuficiência renal aguda. Essa complicação foi atribuída à ação nefrotóxica e hemolítica do veneno crotálico e à hipotensão arterial apresentada pelo paciente, não se afastando a possibilidade de que a rabdomiólise tenha sido um fator contribuinte para a sua instalação. Foram constatadas hipocalcemla, hiperuricemia e hiperfosfatemia grave na fase oligúria da insuficiência renal aguda, alterações peculiares quando essa condição está associada à rabdomiólise.

Afibrinogenemia following snake bite (Crotalus durissus terrificus)

This paper reports two cases of afibrinogenemia with normal platelet count following Crotalus durissus terrificus, snake bite Both patients presented high output acute renal failure and case two also had increased blood levels of CPK and LDH compatible with the diagnosis of rhabdomyolysis. Case one was given an unknown amount of antivenom and was treated with epsilonaminocaproic acid and a fresh whole blood transfusion and showed recovery of the coagulation disturbance 40 hours following these measures. Case two was given an adequate amount of crotalide antivenom and the coagulation tests performed 12 hours later showed a normal partial thromboplastin time and fibrinogen 86 mg/100ml. Case one presented no haemorrhagic disturbances. Case two presented persistent bleeding following venopuncture and after removal of impetigo crust in the legs. Acute renal failure was treated conservatively and both patients were discharged from the hospital with recovery of the renal function.

Comprometimento respiratório secundário a acidente ofídico crotálico (Crotalus durissus)

São analisados três pacientes que apresentaram comprometimento da função respiratória após acidente por Crotalus durissus. As manifestações respiratórias surgiram nas primeiras 48 horas após a picada do ofídio e consistiram de dispnéia, taquipnéia, uso da musculatura acessória da respiração (casos 1 e 2) e batimento das aletas nasais (caso 2). Dois pacientes (casos 1 e 2) apresentaram insuficiência respiratória aguda. O diagnóstico desta complicação no caso 1 foi clínico pois o paciente apresentou apnéia. O paciente do caso 2, 24 horas após o acidente ofídico apresentou dificuldade respiratória intensa e períodos de apnéia sendo intubado, permanecendo em respiração espontânea. Houve agravamento dos sinais clínicos de insuficiência respiratória e a determinação de pH e gases do sangue arterial mostrou em relação ao exame inicial elevação da pressão parcial de gás carbônico (40 mmHg para 50,3 mm Hg) caracterizando insuficiência ventilatória aguda. Ambos foram tratados com emprego de ventilação artificial mecânica, tendo o paciente do caso 1 permanecido no ventilador durante 33 dias e o do caso 2 durante 15 dias. Ambos desenvolveram insuficiência renal aguda, necessitaram de diálise peritoneal e recuperaram a função renal. A paciente do caso 3, apesar dos sintomas e sinais de comprometimento respiratório não apresentou alterações do pH e gases arteriais. Espirometria realizada 58 horas após o acidente mostrou capacidade vital forçada (CVF) e volume espirado no primeiro segundo (VEF1) abaixo do previsto (60 e 67% respectivamente). As espirometrias realizadas nos dias subseqüentes evidenciaram melhora progressiva destes parâmetros. No 10º dia após o acidente constatou-se aumento de 20% da CVF e de 17% do VEF1 comparativamente ao exame inicial. A relação entre VEF1 e a CVF manteve-se praticamente inalterada e em valores próximos ao previsto, caracterizando distúrbio ventilatório do tipo restritivo. O comprometimento respiratório nestes pacientes foi atribuído à ação da crotoxina na musculatura respiratória desde que não se encontravam presentes outras condições etiológicas que pudessem ocasioná-lo como uremia avançada, atelectasias, infecção pulmonar, hipopotassemia, congestão e edema pulmonar agudo.

Epidemiologia e quadro clínico do acidente por cascavel sul-americana (Crotalus durissus)

Foram estudados 249 casos de acidentes por cascavel sul-americana (Crotalus durissus) atendidos no HVB-IB, São Paulo, Brasil, de 1974 a 1990. Os acidentes foram mais comuns no período da tarde, nos meses de janeiro a abril e de outubro a dezembro. Dentre cem serpentes classificadas quanto à subespécie 99 eram C. d. terrificus. Pertenciam ao sexo masculino 80,7% dos pacientes. Os membros inferiores e superiores foram picados em, respectivamente, 66,4% e 29,2% dos casos. As manifestações clínicas mais freqüentes foram dor (61,0%) e edema (55,0%) no local da picada, ptose palpebral (75,9%), escurecimento da urina atribuível à mioglobinúria (38,6%) e mialgia (36,1%). Nove pacientes foram submetidos a diálise devido a insuficiência renal aguda (3,6%), três apresentavam insuficiência respiratória que motivou intubação e/ou traqueostomia e um apresentou acidente vascular cerebral isquêmico. A alteração da coagulação sanguínea ocorreu em 48,1% dos pacientes. Oito amostras de sangue colhidas horas após a picada mostraram leucocitose sendo 6 com desvio a esquerda e, nos dias subseqüentes, tendência à normalização do número de leucócitos e aparecimento de eosinofilia. Atividade sérica da creatinoquinase apresentou-se aumentada em 20 dentre 21 pacientes, sendo maior no final das primeiras 24 horas após a picada, chegando a 2.377 vezes o valor de referência. A letalidade foi de 0,8%.

Avaliação em camundongo da eficácia do antiveneno administrado no local da inoculação intramuscular do veneno de Crotalus durissus terrificus

A eficácia do antiveneno crotálico por via intramuscular (im) no local da inoculação, também im, do veneno de Crotalus durissus terrificus foi avaliada em camundongos. Em três experimentos inocularam-se duas DLSO do veneno por via im e administrou-se o antiveneno de três formas: metade da DE50 por via intraperitoneal (ip) e metade por via im, no mesmo local, imediatamente após (1º) e 30' após (2º) a inoculação do veneno; quatro quintos de DE50 por via ip e um quinto por via im, no mesmo local e 30' após a inoculação do veneno (3º). O antiveneno ofereceu, por via ip, maior proteção aos camundongos (menor taxa de óbito em 48 horas) do que quando foi administrado, em parte, por via im, no local da inoculação do veneno (p<0,05). Infere-se, portanto, que esta via não deve ser rotineiramente utilizada em seres humanos picados por serpente.

Liver dysfunction in patients bitten by Crotalus durissus terrificus (Laurenti, 1768) snakes in Botucatu (State of São Paulo, Brazil)

Thirty-two patients bitten by venomous snakes sixteen by Bothrops spp. and sixteen by Crotalus durissus terrificus were studied. The group comprised thirty males and two females, aged eight to sixty-three years (mean 33±15). Bromsulphalein tests were increased in the majority of patients bitten by Crotalus durissus terrificus. The correlation coefficient of Spearman was positive between bromsulphalein tests and alanine aminotransferase levels, and between alanine aminotransferase and aspartate aminotransferase levels only in the Crotalus group. The only patient who died was bitten by Crotalus durissus terrificus and showed hydropic degeneration and mitochondrial injury in the liver. It was concluded that the hepatic damage might have been caused by at least two possible mechanisms: venom effect on liver mitochondria and cytokine effects on hepatocyte, specially interleukin-6.

Snakebites by Crotalus durissus ssp in children in Campinas, São Paulo, Brazil

From January, 1984 to March, 1999, 31 children under 15 y old (ages 1-14 y, median 8 y) were admitted after being bitten by rattlesnakes (Crotalus durissus ssp). One patient was classified as "dry-bite", 3 as mild envenoming, 9 as moderate envenoming and 18 as severe envenoming. Most patients had neuromuscular manifestations, such as palpebral ptosis (27/31), myalgia (23/31) and weakness (20/31). Laboratory tests suggesting rhabdomyolysis included an increase in total blood creatine kinase (CK, 28/29) and lactate dehydrogenase (LDH, 25/25) levels and myoglobinuria (14/15). The main local signs and symptoms were slight edema (20/31) and erythema (19/31). Before antivenom (AV) administration, blood coagulation disorders were observed in 20/25 children that received AV only at our hospital (incoagulable blood in 17/25). AV early reactions were observed in 20 of these 25 cases (9/9 patients not pretreated and 11/16 patients pretreated with hydrocortisone and histamine H1 and H2 antagonists). There were no significant differences in the frequency of patients with AV early reactions between the groups that were and were not pretreated (Fisher's exact test, p = 0.12). Patients admitted less than and more than 6 h after the bite showed the same risk of developing severe envenoming (Fisher's exact test, p = 1). No children of the first group (< 6 h) showed severe complications whereas 3/6 children admitted more than 6 h post-bite developed acute renal failure. Patients bitten in the legs had a higher risk of developing severe envenoming (Fisher's exact test, p = 0.04). There was a significant association between both total CK and LDH blood enzyme levels and severity (p < 0.001 for CK and p < 0.001 for LDH; Mann-Whitney U test). No deaths were recorded.

Immunohistochemical changes in kidney glomerular and tubular proteins caused by rattlesnake (Crotalus vegrandis) venom

Renal damage is an important cause of death in patients who have survived the early effects of severe crotalid envenomation. Extracellular matrix of renal tissue is altered by Crotalus toxin activities. The aim of this study was to describe how cytoskeletal proteins and basal membrane components undergo substantial alterations under the action of Crotalus vegrandis crude venom and its hemorrhagic fraction (Uracoina-1) in mice. To detect the proteins in question, the immunoperoxidase method with monoclonal and polyclonal antibodies was used. Cell types within renal lesions were characterized by phenotypic identification, by means of immunohistologic analysis of marker proteins using different primary antibodies against mesangial cells, endothelial cells, cytoskeletal proteins (intermediate filament), extracellular matrix and basal membranes. Samples for morphological study by standard procedures (biotin-streptavidin-peroxidase technique) using light microscopy were processed. Positive and negative controls for each antigen tested in the staining assay were included. After crude venom and hemorrhagic fraction inoculation of mice, the disappearance of cytoskeletal vimentin and desmin and collagen proteins in the kidney was observed. In extracellular matrix and basal membranes, collagen type IV from envenomed animals tends to disappear from 24 h to 120 h after venom injection.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.

ACUTE KIDNEY INJURY CAUSED BY Crotalus AND Bothrops SNAKE VENOM: A REVIEW OF EPIDEMIOLOGY, CLINICAL MANIFESTATIONS AND TREATMENT

SUMMARY Ophidic accidents are an important public health problem due to their incidence, morbidity and mortality. An increasing number of cases have been registered in Brazil in the last few years. Several studies point to the importance of knowing the clinical complications and adequate approach in these accidents. However, knowledge about the risk factors is not enough and there are an increasing number of deaths due to these accidents in Brazil. In this context, acute kidney injury (AKI) appears as one of the main causes of death and consequences for these victims, which are mainly young males working in rural areas. Snakes of the Bothrops and Crotalus genera are the main responsible for renal involvement in ophidic accidents in South America. The present study is a literature review of AKI caused by Bothrops and Crotalus snake venom regarding diverse characteristics, emphasizing the most appropriate therapeutic approach for these cases. Recent studies have been carried out searching for complementary therapies for the treatment of ophidic accidents, including the use of lipoic acid, simvastatin and allopurinol. Some plants, such as Apocynaceae, Lamiaceae and Rubiaceae seem to have a beneficial role in the treatment of this type of envenomation. Future studies will certainly find new therapeutic measures for ophidic accidents.