Production of TNF-a by primary cultures of human keratinocytes challenged with Loxosceles gaucho venom

Primary cultures of human keratinocytes were challenged with increasing doses from 10 ng/mL to 2 mg/mL of Loxosceles gaucho venom, responsible for dermonecrotic lesion in humans. TNF-a was investigated by bioassay and ELISA in the supernatant of the cultures challenged with 100 ng/mL, 500 ng/mL, 1 and 2 mg/mL of venom. TNF-a was detected by bioassay in the supernatant of cultures challenged with 100 ng/mL, after 6 h. The cytokine was detected by ELISA in the supernatant of the cells challenged with doses of l mg/mL, after 6 and 12 h. The results point out the capacity of this venom to activate the keratinocytes in primary cultures to produce TNF-a. The production of cytokines could contribute to the local inflammatory process in patients bitten by Loxosceles sp.

Snakebites by Bothrops spp in children in Campinas, São Paulo, Brazil

From January, 1984 to March, 1999, 73 children under 15 y old (ages 1-14 y, median 9 y) were admitted after being bitten by snakes of the genus Bothrops. Twenty-six percent of the children were classified as mild envenoming, 50.7% as moderate envenoming and 20.6% as severe envenoming. Two patients (2.7%) showed no signs of envenoming. Most of the patients presented local manifestations, mainly edema (94.5%), pain (94.5%) ecchymosis (73.9%) and blisters (11%). Local and/or systemic bleeding was observed in 28.8% of the patients. Before antivenom (AV) administration, blood coagulation disorders were observed in 60.7% (incoagulable blood in 39.3%) of the 56 children that received AV only in our hospital. AV early reactions, most of which were considered mild, were observed in 44.6% of these cases (in 15/30 patients not pretreated and in 10/26 patients pretreated with hydrocortisone and histamine H1 and H2 antagonists). The main clinical complications observed were local infection (15.1%), compartment syndrome (4.1%), gangrene (1.4%) and acute renal failure (1.4%). No deaths were recorded. There were no significant differences with regard to severity of envenoming versus the frequency of blood coagulation disorders among the three categories of envenoming (p = 0.75) or in the frequency of patients with AV early reactions between the groups that were and were not pretreated (p = 0.55). The frequency of local infection was significantly greater in severe cases (p < 0.001). Patients admitted more than 6 h after the bite had a higher risk of developing severe envenoming (p = 0.04).

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.

Cardiovascular profile after intravenous injection of Africanized bee venom in awake rats

The manifestations caused by Africanized bee stings depend on the sensitivity of the victim and the toxicity of the venom. Previous studies in our laboratory have demonstrated cardiac changes and acute tubular necrosis (ATN) in the kidney of rats inoculated with Africanized bee venom (ABV). The aim of the present study was to evaluate the changes in mean arterial pressure (MAP) and heart rate (HR) over a period of 24 h after intravenous injection of ABV in awake rats. A significant reduction in basal HR as well as in basal MAP occurred immediately after ABV injection in the experimental animals. HR was back to basal level 2 min after ABV injection and remained normal during the time course of the experiment, while MAP returned to basal level 10 min later and remained at this level for the next 5 h. However, MAP presented again a significant reduction by the 7th and 8th h and returned to the basal level by the 24th h. The fall in MAP may contribute to the pathogenesis of ATN observed. The fall in MAP probably is due to several factors, in addition to the cardiac changes already demonstrated, it is possible that the components of the venom themselves or even substances released in the organism play some role in vascular beds.

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.

Neurotoxic activity and ultrastructural changes in muscles caused by the brown widow spider Latrodectus geometricus venom

Brown widow spider (Latrodectus geometricus) venom (BrWSV) produces few local lesions and intense systemic reactions such as cramps, harsh muscle pains, nausea, vomiting and hypertension. Approximately 16 protein bands under reducing conditions and ~ 14 bands under non-reducing conditions on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis were observed. Neurotoxic clinical manifestations were confirmed in vivo, while proteolytic activity was demonstrated on gelatine film. Severe ultrastructural damages in mice skeletal muscles were observed at 3, 6, 12 and 24 h postinjection with at total of 45 µg of venom protein. Infiltration of eosinophils and ruptures of the cellular membranes were observed in the muscles along with swelling of the nuclear cover and interruption of the collagen periodicity. Altered mitochondrias and autophage vacuoles, nuclear indentation and mitochondria without cristae, slight increment of intermyofibrillar and subsarcolemic spaces and myelinic figures formation were also observed. In the capillary, endothelial membrane unfolding into the lumen was noticed; along with myelinic figures compatible with a toxic myopathy. Swollen sarcotubular systems with lysis of membrane, intense mitochondria autophagia and areas without pinocytic vesicles were observed. Swollen mitochondria surrounded by necrotic areas, myofibrillar disorganization and big vacuolas of the sarcotubular system, degenerated mitochondrium with formation of myelinic figure was seen. Glycogenosomes with small particulate, muscle type glycogen was noticed. Autophagic vacuole (autophagolysosomes) and necrotic areas were also noticed. These damages may be due to interactive effects of the multifactorial action of venom components. However, Latrodectus geometricus venom molecules may also be utilized as neuro therapeutic tools, as they affect neuronal activities with high affinity and selectivity. To our knowledge, the present study is the first ultrastructural report in the literature of muscle injuries and neurological and proteolytic activities caused by BrWSV.

Neutralization of bitis parviocula (Ethiopian mountain adder) venom by the south african institute of medical research (SAIMR) antivenom

BACKGROUND: The Ethiopian mountain adder (Bitis parviocula) is a viperid known only from a few locations in southwestern Ethiopia. METHODS: a total of 30 µg of B. arietans and B. parviocula venoms were run on a 10-20% Tricine gel. To assay lethality dose fifty (LD50), five groups of eight mice for each venom were used. Hemorrhagic activity for crude venom was tested. Fibrinogenolytic activity of crude venom was measured using (2.5 mg/mL) of fibrinogen solution and (0.03 mg/mL) of crude venom. Gelatinase activity of the venom was tested on a Kodak X-OMAT TM film. Crude venoms of B. parviocula and B. arietans were tested for their abilities to affect clotting time, clotting rate and platelet function on whole human blood. RESULTS: The (SAIMR) antivenom was confirmed in this study to neutralize the lethal activity of venom from Bitis parviocula. The ED50s of SAIMR antivenom on B. parviocula and B. arietans neutralized half of 18.2 and 66.7 mg of venom, respectively. The hemorrhagic activities (MHDs) of B. parviocula and B. arietans were 0.88 and 1.7 µg, respectively. Bitis arietans and B. parviocula venoms degradated α and β chains at different times. The γ chains remained unaffected. Bitis parviocula venom did not exhibit gelatinase activity, while B. arietans had a MGD of 6.9 µg. At 3 mg/mL, the crude venoms of B. parviocula and B. arietans did not significantly affect clotting time or clotting rate. CONCLUSIONS: The SAIMR antivenom is very effective in neutralizing the venom of B. parviocula and should be considered in treating envenomations by these snakes.

A study of bacterial contamination of rattlesnake venom

The authors studied the bacterial contamination of rattlesnake venom isolated from snakes in captivity and wild snakes caught recently. The captive snakes showed a relatively high incidence of bacterial contamination of their venom.

South american rattlesnake venom: its hemolytic power

The hemolytic power of rattlesnake venom (Crotalus durissus terrificus) was Studied. A high percentage of sample with negative hemolytic power was detected when sheep red blood cells were used. A large number of venoms with hemolytic power, though with a low hemolysis percentage, were detected when liquid, recently extracted venom was used. When crystallized venom was used under the same experimental conditions, a higher percentage ofpositivityfor hemolysis was obtained. When the results obtained on agar plates were compared to those obtained in test tubes, a large number of animals with a higher percentage of hemolysis were detected, though this value was not proportional to the number of animals showing positive plate hemolysis. When the hemolytic power of these venoms was tested on human red blood cells, a large percentage of animals with venoms having a low hemolytic power was also detected. Hemolytic power was much greater when human red blood cells were tested with crystallized venom. The preparation of red blood cells also had an important effect and the use of red blood cells from defibrinated blood is recommended. We conclude that rattlesnake venom has hemolytic power that increases when the venom is crystallized. Red blood cells should be properly preparedfor the lysis reactions. We suggest that the lytic power of the venom is related to venom concentration and to the purity of its fractions.

Evaluation of platelet number and function and fibrinogen level in patients bitten by snakes of the Bothrops genus

Platelet function and plasma fibrinogen levels were evaluated in 14 patients, 10 males and 4females, aged 13-59years bitten by Bothrops genus snakes. There was a statistical difference (p < 0.05) among plasma fibrinogen levels evaluated 24 and 48 hours after envenomation. There was a tendency towards normalization after 48 hours of treatment. The low platelet number was clear in 24-48 hour evaluations with a tendency towards normalization after 48 hours of treatment (p<0.05). Fibrinogen levels and fibrin degradation product (FDP) levels appeared to be altered in 83.33% of patients evaluated. The authors suggest that platelet hypoaggregation is related to decreased fibrinogen and increased FDP levels.

Acidente por serpentes do gênero Bothrops: série de 3.139 casos

Em avaliação dos prontuários médicos de 3.139 pacientes picados por serpentes do gênero Bothrops atendidos no Hospital Vital Brazil (HVB), de 1981 a 1990, observou-se maior acometimento do sexo masculino (75,7%). Em 1.412 casos (45,0%) a serpente foi identificada, sendo 1.376 B. jararaca, 20 B. jararacussu, 11 B. neuwiedi, 2 B. moojeni, 2 B. alternatus e 1 B. pradoi. As regiões anatômicas mais comumente picadas foram: pé (47,5%) e mão (21,3%). O torniquete foi realizado em 38,2% dos casos e sua freqüência diminuiu durante esse período (p < 0,001). As manifestações clínicas mais freqüentes foram: dor (95,6%), edema (95,4%), equimose (56,1%), bolha (13,8%), necrose (16,5%), abscesso (11,0%), sangramento extratecidual (12,3%), insuficiência renal (1,6%) e choque (0,7%). A coagulação sanguínea foi avaliada em 2.990 casos e estava alterada em 1.730 (57,9%). Ocorreram 21 amputações (0,7%) e 9 óbitos (0,3%). A dose de soro administrada no HVB diminuiu ao longo desse período (p < 0,001).

Fatores associados à incoagulabilidade sangüínea no envenenamento por serpentes do gênero Bothrops

Com o objetivo de conhecer fatores associados à incoagulabilidade sangüínea no envenenamento botrópico, foram obtidas informações de 2.991 prontuários médicos de pacientes atendidos no Instituto Butantan de 1981 a 1990. Associaram-se positivamente à incoagulabilidade sangüínea (p<0,05): picadas no final do ano e em extremidades dos membros inferiores; dor, edema e equimose local; hemorragia e choque; dose de antiveneno; tempo do acidente à chegada ao Instituto Butantan. Associaram-se negativamente à incoagulabilidade (p<0,05): tamanho de Bothrops jararaca; uso de torniquete; tempo entre a chegada ao Instituto Butantan e o início da administração do antiveneno. Não se associaram à incoagulabilidade (p>0,05): horário do acidente; presença de presa recém-deglutida no tubo digestivo da serpente; sexo e idade do paciente; ocorrência de bolha, necrose, abscesso e incisão local, amputação, insuficiência renal e óbito. Pode-se concluir que, embora a incoagulabilidade sangüínea apresente associação com manifestações precoces do envenenamento, não tem boa associação com a evolução clínica do paciente.

Experimental ophitoxemia produced by the opisthoglyphous lora snake (Philodryas olfersii) venom

Several colubrid snakes produce venomous oral secretions. In this work, the venom collected from Venezuelan opisthoglyphous (rear-fanged) Philodryas olfersii snake was studied. Different proteins were present in its venom and they were characterized by 20% SDS-PAGE protein electrophoresis. The secretion exhibited proteolytic (gelatinase) activity, which was partially purified on a chromatography ionic exchange mono Q2 column. Additionally, the haemorrhagic activity of Philodryas olfersii venom on chicken embryos, mouse skin and peritoneum was demonstrated. Neurotoxic symptoms were demonstrated in mice inoculated with Philodryas olfersii venom. In conclusion, Philodryas olfersii venom showed proteolytic, haemorrhagic, and neurotoxic activities, thus increasing the interest in the high toxic action of Philodryas venom.

Envenenamento por serpentes do gênero Bothrops no Estado da Bahia: aspectos epidemiológicos e clínicos

Descreve-se características clínico-epidemiológicas de 655 casos de acidentes botrópicos atendidos e/ou notificados ao Centro de Informações Antiveneno da Bahia, no Estado da Bahia, em 2001. A incidência anual no Estado foi de 5,0 acidentes/100.000 habitantes e a letalidade, 1%. A incidência foi máxima na microrregião Litoral Norte (21,9/100.000 habitantes) e no município de Itanagra (92,9/100.000 habitantes). Os acidentes foram predominantemente diurnos, acometendo membros inferiores, em homens, de 11-30 anos, trabalhadores, no ambiente rural e no período chuvoso. Atendimento médico após 13 horas da ocorrência da picada ocorreu em 19% dos casos. Predominaram os quadros clínicos moderados (47,8%), seguidos dos graves (23,6%). As manifestações clínicas locais e sistêmicas seguiram o padrão nacional para as serpentes do gênero Bothrops. Sintomatologias neurológicas, não usualmente atribuídas ao acidente botrópico, foram registrados em alguns casos. A soroterapia empregada (7,7 ampolas/paciente) foi compatível com o fato da maioria de casos serem moderados. Outros tipos de soro que não o univalente foram utilizados em 15 (2,3%) pacientes.

Comparação entre a epidemiologia do acidente e a clínica do envenenamento por serpentes do gênero Bothrops, em adultos idosos e não idosos

O objetivo do presente estudo foi conhecer diferenças epidemiológicas e clínicas do envenenamento por Bothrops spp em adultos idosos (>60 anos) e não idosos (20 a 59 anos). Os dados foram obtidos de 1.930 prontuários de pacientes atendidos no Instituto Butantan de 1981 a 1992. Quanto maior a idade do paciente maior a freqüência do acometimento das mãos em relação aos pés (p<0,05). Porcentagem pouco maior dos idosos (17%) em relação aos não idosos (11%) foi atendida >12 horas após a picada (p<0,05). A necrose foi mais comum entre idosos (p<0,05) e a insuficiência renal entre pacientes com 50 anos ou mais, em relação aos mais jovens (p<0,05). Concluiu-se que indivíduos com idade mais avançada são mais comumente picados nas mãos e menos nos pés e evoluem mais freqüentemente para necrose na região da picada e para insuficiência renal do que os mais jovens.