Systemic toxicity of snake venom metalloproteinases: Multi-omics analyses of kidney and blood plasma disturbances in a mouse model.

Snakebite envenomation is classified as a Neglected Tropical Disease. Bothrops jararaca venom induces kidney injury and coagulopathy. HF3, a hemorrhagic metalloproteinase of B. jararaca venom, participates in the envenomation pathogenesis. We evaluated the effects of HF3 in mouse kidney and blood plasma after injection in the thigh muscle, mimicking a snakebite. Transcriptomic analysis showed differential expression of 31 and 137 genes related to kidney pathology after 2 h and 6 h, respectively. However, only subtle changes were observed in kidney proteome, with differential abundance of 15 proteins after 6 h, including kidney injury markers. N-terminomic analysis of kidney proteins showed 420 proteinase-generated peptides compatible with meprin specificity, indicating activation of host proteinases. Plasma analysis revealed differential abundance of 90 and 219 proteins, respectively, after 2 h and 6 h, including coagulation-cascade and complement-system components, and creatine-kinase, whereas a semi-specific search of N-terminal peptides indicated activation of endogenous proteinases. HF3 promoted host reactions, altering the gene expression and the proteolytic profile of kidney tissue, and inducing plasma proteome imbalance driven by changes in abundance and proteolysis. The overall response of the mouse underscores the systemic action of a hemorrhagic toxin that transcends local tissue damage and is related to known venom-induced systemic effects.

Impact of antivenom administration on the evolution of cutaneous lesions in loxoscelism: A prospective observational study.

BACKGROUND: Spiders of the genus Loxosceles are distributed throughout tropical and temperate regions worldwide. Loxosceles spp. bites may evolve to necrosis, with or without intravascular hemolysis. There is no consensus regarding the best treatment to prevent necrosis. The objective of this study was to evaluate the factors associated with the development of necrosis and the impact that antivenom administration has on the evolution of cutaneous loxoscelism. METHODOLOGY/PRINCIPAL FINDINGS: This was a prospective observational study carried out at a referral center for envenoming. Over a 6-year period, we included 146 patients with a presumptive or definitive diagnosis of loxoscelism. Depending on the symptom severity, a polyvalent anti-arachnid antivenom was administered or not-in 74 cases (50.7%) and 72 cases (49.3%), respectively. Cutaneous and systemic manifestations were assessed at admission and weekly thereafter. Adverse reactions to the antivenom were also evaluated. Cutaneous loxoscelism was observed in 141 cases (96.6%), and the spider was identified in 29 (19.9%). The mean time from bite to antivenom administration was 41.6 ± 27.4 h. After discharge, 130 patients (90.9%) were treated with corticosteroids, antihistamines and analgesics being prescribed as needed. The probability of developing necrosis was significantly lower among the patients who were admitted earlier, as well as among those who received antivenom (p = 0.0245). Among the 74 patients receiving antivenom, early and delayed adverse reactions occurred in seven (9.5%) and four (5.4%), respectively. Local infection was observed only in three (2.3%) of the 128 patients for whom that information was available. CONCLUSIONS/SIGNIFICANCE: Necrosis after a Loxosceles sp. bite appears to more common when hospital admission is delayed or when antivenom is not administered. In addition, the administration of a polyvalent anti-arachnid antivenom appears to be safe, with a relatively low rate of adverse reactions.

The absence of thrombin-like activity in Bothrops erythromelas venom is due to the deletion of the snake venom thrombin-like enzyme gene.

Snake venom thrombin-like enzymes (SVTLEs) are serine proteinases that clot fibrinogen. SVTLEs are distributed mainly in venoms from snakes of the Viperidae family, comprising venomous pit viper snakes. Bothrops snakes are distributed throughout Central and South American and are responsible for most venomous snakebites. Most Bothrops snakes display thrombin-like activity in their venoms, but it has been shown that some species do not present it. In this work, to understand SVTLE polymorphism in Bothrops snake venoms, we studied individual samples from two species of medical importance in Brazil: Bothrops jararaca, distributed in Southeastern Brazil, which displays coagulant activity on plasma and fibrinogen, and Bothrops erythromelas, found in Northeastern Brazil, which lacks direct fibrinogen coagulant activity but shows plasma coagulant activity. We tested the coagulant activity of venoms and the presence of SVTLE genes by a PCR approach. The SVTLE gene structure in B. jararaca is similar to the Bothrops atrox snake, comprising five exons. We could not amplify SVTLE sequences from B. erythromelas DNA, except for a partial pseudogene. These genes underwent a positive selection in some sites, leading to an amino acid sequence diversification, mostly in exon 2. The phylogenetic tree constructed using SVTLE coding sequences confirms that they are related to the chymotrypsin/kallikrein family. Interestingly, we found a B. jararaca specimen whose venom lacked thrombin-like activity, and its gene sequence was a pseudogene with SVTLE structure, presenting nonsense and frameshift mutations. Our results indicate an association of the lack of thrombin-like activity in B. jararaca and B. erythromelas venoms with mutations and deletions of snake venom thrombin-like enzyme genes.

Biochemical and biological characterization of Bothriechis schlegelii snake venoms from Colombia and Costa Rica.

Snakebites inflicted by the arboreal viperid snake Bothriechis schlegelii in humans are characterized by pain, edema, and ecchymosis at the site of the bite, rarely with blisters, local necrosis, or defibrination. Herein, a comparative study of Bothriechis schlegelii snake venoms from Colombia (BsCo) and Costa Rica (BsCR) was carried out in order to compare their main activities and to verify the efficacy of Bothrops antivenom produced in Brazil to neutralize them. Biochemical (SDS-PAGE and zymography) and biological parameters (edematogenic, lethal, hemorrhagic, nociceptive, and phospholipase A2 activities) induced by BsCo and BsCR snake venoms were evaluated. The presence of antibodies in Bothrops antivenom that recognize BsCo and BsCR snake venoms by enzyme-linked immunosorbent assay and Western blotting, as well as the ability of this antivenom to neutralize the toxic activities were also verified. SDS-PAGE showed differences between venoms. Distinctive caseinolytic and hyaluronidase patterns were detected by zymography. BsCo and BsCR showed similar phospholipase A2 activity. Strong cross-reactivity between BsCo and BsCR was detected using Bothrops antivenom with many components located between 150 and 35 kDa. BsCR was more edematogenic and almost fourfold more hemorrhagic than BsCo, and both venoms induced nociception. BsCR (LD50 5.60 mg/kg) was more lethal to mice than BsCo (LD50 9.24 mg/kg). Bothrops antivenom was effective in the neutralization of lethal and hemorrhagic activities of BsCo and BsCR and was partially effective in the neutralization of edematogenic and nociceptive activities. In conclusion, geographic distribution influences the composition and activities of Bothriechis schlegelii venoms. Bothrops antivenom cross-reacted with these venoms and was partially effective in neutralizing some toxic activities of BsCo and BsCR.

Isolation and Characterization of IgM and IgY Antibodies from Plasma of Magellanic Penguins (Spheniscus magellanicus).

Infectious diseases such as aspergillosis, avian malaria, and viral infections are significant threats to the conservation of penguins, leading to morbidity and mortality of these birds both in captivity and in the wild. The immune response to such infectious diseases is dependent on different mechanisms mediated by cells and soluble components such as antibodies. Antibodies or immunoglobulins are glycoproteins that have many structural and functional features that mediate distinct effector immune functions. Three distinct classes of antibodies have been identified in birds: immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin Y (IgY). In this study we aim to establish an efficient laboratory method to obtain IgM and IgY antibodies from plasma samples of healthy adult Magellanic penguins (Spheniscus magellanicus). The protocol was developed combining plasma delipidation, sequential precipitation with caprylic acid and ammonium sulfate, and size-exclusion chromatography. The efficiency of the protocol and the identity of the purified IgM and IgY antibodies were confirmed through enzyme-linked immunosorbent assay, Western blotting, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, and lectin binding assay. Structural and physicochemical properties of IgM and IgY from Magellanic penguins were consistent with those of other avian species. This purification protocol will allow for more detailed studies on the humoral immunity of penguins and for the development of high specificity serologic assays to test Magellanic penguins for infectious pathogens.

Bothrops jararaca envenomation: Pathogenesis of hemostatic disturbances and intravascular hemolysis.

To attain fully functional biological activity, vitamin-K dependent coagulation factors (VKDCF) are γ-carboxylated prior to secretion from liver. Warfarin impairs the γ-carboxylation, and consequently their physiological function. Bothrops jararaca snake venom (BjV) contains several activators of blood coagulation, especially procoagulant enzymes (prothrombin and factor X activators) and thrombin-like enzymes. In order to clarify the relative contribution of prothrombin and factor X activators to the hemostatic disturbances occurring during experimental B. jararaca envenomation, warfarin was used to deplete VKDCF, prior to BjV administration. Male Wistar rats were pretreated with saline (Sal) or warfarin (War) and inoculated subsequently with BjV or saline, thus forming four groups: Sal + Sal (negative control), Sal + BjV (positive control), War + Sal (warfarinization control), and War + BjV. Three hours after inoculation, prothrombin and factor X levels fell 40% and 50%, respectively; levels of both factors decreased more than 97% in the War + Sal and War + BjV groups. Platelet counts dropped 93% and 76% in Sal + BjV and War + BjV, respectively, and plasma fibrinogen levels decreased 86% exclusively in Sal + BjV. After 6 and 24 h, platelet counts and fibrinogen levels increased progressively. A dramatic augmentation in plasma hemoglobin levels and the presence of schizocytes and microcytes in the Sal + BjV group indicated the development of intravascular hemolysis, which was prevented by warfarin pretreatment. Our findings show that intravascular thrombin generation has the foremost role in the pathogenesis of coagulopathy and intravascular hemolysis, but not in the development of thrombocytopenia, in B. jararaca envenomation in rats; in addition, fibrinogenases (metalloproteinases) may contribute to coagulopathy more than thrombin-like enzymes.

Mast cells and histamine play an important role in edema and leukocyte recruitment induced by Potamotrygon motoro stingray venom in mice.

This work aimed to investigate mechanisms underlying the inflammatory response caused by Potamotrygon motoro stingray venom (PmV) in mouse paws. Pre-treatment of animals with a mast cell degranulation inhibitor (cromolyn) diminished edema (62% of inhibition) and leukocyte influx into the site of PmV injection. Promethazine (histamine type 1 receptor antagonist) or thioperamide (histamine type 3 and 4 receptor antagonist) also decreased edema (up to 30%) and leukocyte numbers, mainly neutrophils (40-50 %). Cimetidine (histamine type 2 receptor antagonist) had no effect on PmV-induced inflammation. In the RBL-2H3 lineage of mast cells, PmV caused proper cell activation, in a dose-dependent manner, with release of PGD2 and PGE2. In addition, the role of COXs products on PmV inflammatory response was evaluated. Indomethacin (COX-1/COX-2 inhibitor) or etoricoxib (COX-2 inhibitor) partially diminished edema (around 20%) in PmV-injected mice. Indomethacin, but not etoricoxib, modulated neutrophil influx into the site of venom injection. In conclusion, mast cell degranulation and histamine, besides COXs products, play an important role in PmV-induced reaction. Since PmV mechanism of action remains unknown, hindering accurate treatment, clinical studies can be performed to validate the prescription of antihistaminic drugs, besides NSAIDs, to patients injured by freshwater stingrays.

Bothrops jararaca venom metalloproteinases are essential for coagulopathy and increase plasma tissue factor levels during envenomation.

BACKGROUND/AIMS: Bleeding tendency, coagulopathy and platelet disorders are recurrent manifestations in snakebites occurring worldwide. We reasoned that by damaging tissues and/or activating cells at the site of the bite and systemically, snake venom toxins might release or decrypt tissue factor (TF), resulting in activation of blood coagulation and aggravation of the bleeding tendency. Thus, we addressed (a) whether TF and protein disulfide isomerase (PDI), an oxireductase involved in TF encryption/decryption, were altered in experimental snake envenomation; (b) the involvement and significance of snake venom metalloproteinases (SVMP) and serine proteinases (SVSP) to hemostatic disturbances. METHODS/PRINCIPAL FINDINGS: Crude Bothrops jararaca venom (BjV) was preincubated with Na2-EDTA or AEBSF, which are inhibitors of SVMP and SVSP, respectively, and injected subcutaneously or intravenously into rats to analyze the contribution of local lesion to the development of hemostatic disturbances. Samples of blood, lung and skin were collected and analyzed at 3 and 6 h. Platelet counts were markedly diminished in rats, and neither Na2-EDTA nor AEBSF could effectively abrogate this fall. However, Na2-EDTA markedly reduced plasma fibrinogen consumption and hemorrhage at the site of BjV inoculation. Na2-EDTA also abolished the marked elevation in TF levels in plasma at 3 and 6 h, by both administration routes. Moreover, increased TF activity was also noticed in lung and skin tissue samples at 6 h. However, factor VII levels did not decrease over time. PDI expression in skin was normal at 3 h, and downregulated at 6 h in all groups treated with BjV. CONCLUSIONS: SVMP induce coagulopathy, hemorrhage and increased TF levels in plasma, but neither SVMP nor SVSP are directly involved in thrombocytopenia. High levels of TF in plasma and TF decryption occur during snake envenomation, like true disseminated intravascular coagulation syndrome, and might be implicated in engendering bleeding manifestations in severely-envenomed patients.

Characterization of inflammatory response induced by Potamotrygon motoro stingray venom in mice.

Freshwater stingray accidents cause intense pain followed by edema, erythema, and necrosis formation. Treatment for stingray envenomation is based on administration of analgesic, antipyretic, and anti-inflammatory drugs. This report evaluated the local inflammatory reaction-including edema formation, leukocyte recruitment, release of inflammatory mediators, and histopathological changes-after the intraplantar injection of Potamotrygon motoro stingray venom in mice. Edema was observed as soon as 15 min after venom injection, peaking at 30 min, and lasted up to 48 h. In addition, P. motoro venom increased neutrophil counts in the site of injection, at all time periods and venom doses analyzed. Increased eosinophil and lymphocyte counts were detected mainly at 24 h. Moreover, monocytes/macrophages were observed in large amounts at 24 and 48 h. Microscopically, the venom induced leukocyte migration to the injured tissue, edema, mast cell degranulation, angiogenesis, and epidermal damage. Inflammatory mediator release (IL-6, MCP-1 and KC) was detected as soon as 1 h after venom injection, and it increased significantly at 4 h. At 24 h, the venom induced only the production of MCP-1. These results show that this stingray venom evokes a complex inflammatory reaction, with rapid and persistent edema formation, leukocyte recruitment, and release of cytokines and chemokines.

Avaliação da albuminúria e da eletroforese de proteínas urinárias de cães com hiperadrenocorticismo e a relação com a pressão arterial sistêmica / Evaluation of albminuria and electrophoresis of urinary proteins from dogs with hyperadrenocorticism and relation with systemic arterial pressure

O hiperadrenocorticismo é uma das endocrinopatias mais comuns em cães, sendo caracterizado pela exposição excessiva de glicocorticóides secretados pelas adrenais. A hipercortisolemia crônica pode promover várias complicações, incluindo hipertensão sistêmica e glomerulonefrite. A glomerulonefrite pode desencadear variáveis graus de proteinúria e uma tendência de evolução para doença renal crônica. A perda de proteínas na urina, principalmente da albumina, é uma característica das doenças glomerulares e a determinação de variáveis laboratoriais, como a razão proteína:creatinina urinária (RPC), albuminúria (teste de ELISA) e eletroforese das proteínas urinárias, são recomendadas para a elucidação do diagnóstico. Assim, o objetivo do estudo é avaliar a relação entre proteinúria e hipertensão arterial sistêmica em cães com hiperadrenocorticismo e verificar, pela avaliação da albuminúria e do peso molecular das proteínas urinárias, o segmento do néfron que foi comprometido ou lesado. Foram avaliados 30 cães com diagnóstico de hiperadrenocorticismo, subdivididos em 13 cães com hipertensão arterial sistêmica (grupo I) e 17 cães normotensos (grupo II). Foram determinados a RPC; a albuminúria pela avaliação da albumina normalizada e razão albumina:creatinina urinária (RAC) e a eletroforese de proteínas pela técnica em gel de poliacrilamida, contendo dodecil sulfato de sódio (SDS-PAGE). Os resultados foram comparados com os dados obtidos de 30 cães clinicamente saudáveis. Foi constatado que não houve influência da hipertensão arterial sistêmica nos cães com hiperadrenocorticismo em relação à quantificação da albuminúria, determinada pelo método ELISA, e nem na qualidade e quantidade das bandas de proteínas de baixo (<60 kDa) e de alto peso molecular (>60 kDa). No entanto foi determinado que cães com hiperadrenocorticismo podem desenvolver lesões glomerulares e tubulares, caracterizadas pela presença de albuminúria e de proteínas de alto e de baixo pesos moleculares, independentemente da presença de hipertensão arterial sistêmica. Conclui-se que a avaliação quantitativa (RPC e RAC) e qualitativa (SDS-PAGE) das proteínas urinárias traz informações adicionais que indicam os possíveis segmentos comprometidos dos néfrons que causaram as perdas de proteínas na urina.

Hyperadrenocorticism is one of the commonest endocrinopathies in dogs, and it is characterized by the excessive exposure of glucocorticoids excreted by adrenals. Chronic hypercortisolemia may promote several complications, including systemic hypertension and glomerulonephritis. Glomerulonephritis may initiate several variable degrees of proteinuria and leading to the development of chronic kidney disease. The loss of proteins through urine, mainly predominant albumin, is a characteristic of glomerular diseases and the determination of laboratorial variables, such as the urinary protein-to- creatinine ratio (UPC), urinary albumin-to-creatinine ratio (UAC; ELISA test) and electrophoresis of urinary proteins are recommended to elucidate the diagnosis. Therefore, the goal of this study is to evaluate the relationship between proteinuria and systemic arterial hypertension in dogs with hyperadrenocorticism and to determine through evaluation of albuminuria and molecular weight of urinary proteins, the segment of the nephron that could be damaged. Thirty dogs with hyperadrenocorticism were evaluated and subdivided into groups; 13 dogs with systemic arterial hypertension (group I) and 17 normotensive (group II). The UPC was determined, as well as UAC and the urine protein electrophoresis by polyacrylamide gel technique, containing dodecyl sodium sulphate (SDS-PAGE). The results were compared with data obtained from 30 clinically healthy dogs. No association between systemic arterial hypertension and albuminuria was detected in dogs with hyperadrenocorticism as well as no alterations of proteins patterns or molecular weights bands of low (<60 kDa) or high molecular weight (> 60 kDa) was found. However, dogs with hyperadrenocorticism may develop glomerular and tubular injuries that were characterized by the presence of albuminuria and proteins of low and high molecular weights, independently of systemic arterial hypertension. In conclusion, the quantitative (UPC and UAC) and qualitative (SDS-PAGE) evaluation of urinary proteins could add information to indicate the possible segments of the nephrons that caused the loss of those proteins.

Cloning, expression and characterization of a phospholipase D from Loxosceles gaucho venom gland.

Loxosceles venom comprises a mixture of diverse toxins that induces intense local inflammatory reaction, dermonecrotic injury, platelet aggregation, hemolytic anemia and acute renal failure. Among several toxins in the venom, phospholipases D (PLDs), also called dermonecrotic toxins, are the most important and best studied, since they account for the main effects observed in loxoscelism. Despite their importance, biological analysis of PLDs is hampered by the minute amounts normally purified from the venom, and therefore many efforts have been made to clone those toxins. However, to date, no PLD from Loxosceles gaucho has been obtained in a heterologous system. Thus, in this work we show the cloning of a PLD from L. gaucho venom gland, named LgRec1, which was successfully expressed in a bacterial system. LgRec1 evoked local reaction (edema, erythema, ecchymosis, and paleness), dermonecrosis and hemolysis. It was also able to hydrolyze sphingomyelin and promote platelet aggregation. ELISA and Western blot analysis showed that LgRec1 was recognized by an anti-L. gaucho venom serum, a commercial arachnidic antivenom as well as a monoclonal antibody raised against the dermonecrotic fraction of L. gaucho venom. In addition, LgRec1 demonstrated to be highly immunogenic and antibodies raised against this recombinant toxin inhibited local reaction (~65%) and dermonecrosis (~100%) elicited by L. gaucho whole venom. Since PLDs are considered the major components accounting for the local and systemic envenomation effects caused by spiders from genus Loxosceles, the information provided here may help to understand the mechanisms behind clinical symptomatology.

Venom proteomes of South and North American opisthoglyphous (Colubridae and Dipsadidae) snake species: a preliminary approach to understanding their biological roles.

Opisthoglyphous snake venoms remain under-explored despite being promising sources for ecological, evolutionary and biomedical/biotechnological research. Herein, we compared the protein composition and enzymatic properties of the venoms of Philodryas baroni (PbV), Philodryas olfersii olfersii (PooV) and Philodryas patagoniensis (PpV) from South America, and Hypsiglena torquata texana (HttV) and Trimorphodon biscutatus lambda (TblV) from North America. All venoms degraded azocasein, and this metalloproteinase activity was significantly inhibited by EDTA. PooV exhibited the highest level of catalytic activity towards synthetic substrates for serine proteinases. All venoms hydrolyzed acetylthiocholine at low levels, and only TblV showed phospholipase A(2) activity. 1D and 2D SDS-PAGE profile comparisons demonstrated species-specific components as well as several shared components. Size exclusion chromatograms from the three Philodryas venoms and HttV were similar, but TblV showed a notably different pattern. MALDI-TOF MS of crude venoms revealed as many as 49 distinct protein masses, assigned to six protein families. MALDI-TOF/TOF MS analysis of tryptic peptides confirmed the presence of cysteine-rich secretory proteins in all venoms, as well as a phospholipase A(2) and a three-finger toxin in TblV. Broad patterns of protein composition appear to follow phylogenetic lines, with finer scale variation likely influenced by ecological factors such as diet and habitat.

Clinical picture and laboratorial evaluation in human loxoscelism.

Loxosceles spiders are found globally, especially in South and North America. In Brazil, approximately 10,000 cases of Loxosceles spp. spider bites are reported annually. Herein we analyzed 81 patients diagnosed as either cutaneous or cutaneous-hemolytic loxoscelism, in a geographical area where most accidents are caused by Loxosceles gaucho, and we report their clinical and laboratory data obtained during week 1 and 2 after the bite. Massive hemolysis was noticed in only 2 cases, but high serum bilirubin and LDH levels, suggestive of hemolysis, were noticed in 25 cases on admission. Anemia was not frequent (14.7%), and reticulocytosis was particularly noticed during week 2 (in 56% of patients). High D-dimer levels were suggestive of endothelial cell activation and intravascular thrombin generation, but thrombocytopenia was noticed in only 17.6% of patients in week 1. Acute kidney injury (AKI) only occurred in patients with massive hemolysis. The definitive diagnosis of overt disseminated intravascular coagulation (DIC) could not be established on admission. Fever was associated with the presence of hemolysis (p = 0.03). Altogether, these findings provide evidence that mild hemolysis is frequent in loxoscelism and suggest that AKI is uncommon, exclusively occurring in patients with massive hemolysis.

Clinical picture and laboratorial evaluation in human loxoscelism

Loxosceles spiders are found globally, especially in South and North America. In Brazil, approximately 10,000 cases of Loxosceles spp. spider bites are reported annually. Herein weanalyzed 81 patients diagnosed as either cutaneous or cutaneous-hemolytic loxoscelism, in a geographical area where most accidents are caused by Loxosceles gaucho, and wereport their clinical and laboratory data obtained during week 1 and 2 after the bite. Massive hemolysis was noticed in only 2 cases, but high serum bilirubin and LDH levels, suggestive of hemolysis, were noticed in 25 cases on admission. Anemia was not frequent (14.7%), and reticulocytosis was particularly noticed during week 2 (in 56% of patients).High D-dimer levels were suggestive of endothelial cell activation and intravascular thrombin generation, but thrombocytopenia was noticed in only 17.6% of patients in week1. Acute kidney injury (AKI) only occurred in patients with massive hemolysis. The definitive diagnosis of overt disseminated intravascular coagulation (DIC) could not beestablished on admission. Fever was associated with the presence of hemolysis (p » 0.03). Altogether, these findings provide evidence that mild hemolysis is frequent in loxoscelism and suggest that AKI is uncommon, exclusively occurring in patients with massive hemolysis.

Loxosceles gaucho spider venom and its sphingomyelinase fraction trigger the main functions of human and rabbit platelets.

Loxosceles venoms can promote severe local and systemic damages. We have previously reported that Loxosceles gaucho spider venom causes a severe early thrombocytopenia in rabbits. Herein, we investigated the in vitro effects of this venom and its sphingomyelinase fraction on the main functions of platelets. Whole venom and its fraction induced aggregation of both human and rabbit platelets. Aggregation was dependent of plasma component(s) but independent of venom-induced lysophosphatidic acid generation. There was no increase in the levels of lactate dehydrogenase during platelet aggregation, ruling out the possibility of platelet lysis. The increased expression of ligand-induced binding site 1 (LIBS1) induced by L. gaucho venom and its sphingomyelinase fraction, as well as of P-selectin by the whole venom, evidenced the activation state of both human and rabbit platelets. Adhesion assays showed an irregular response when platelets were exposed to the whole venom, whereas the sphingomyelinase fraction induced a dose-dependent increase in the platelet adhesion to collagen. These findings evidence that L. gaucho venom and its sphingomyelinase fraction trigger adhesion, activation, and aggregation of both human and rabbit platelets. Thus, this work justifies the use of rabbits to investigate Loxosceles venom-induced platelet disturbances, and it also supports research on the role of platelets in the pathogenesis of loxoscelism.

Comparative analysis of newborn and adult Bothrops jararaca snake venoms.

Different clinical manifestations have been reported to occur in patients bitten by newborn and adult Bothrops jararaca snakes. Herein, we studied the chemical composition and biological activities of B. jararaca venoms and their immunoneutralization by commercial antivenin at these ontogenetic stages. Important differences in protein profiles were noticed both in SDS-PAGE and two-dimensional electrophoresis. Newborn venom showed lower proteolytic activity on collagen and fibrinogen, diminished hemorrhagic activity in mouse skin and hind paws, and lower edematogenic, ADPase and 5'-nucleotidase activities. However, newborn snake venom showed higher l-amino oxidase, hyaluronidase, platelet aggregating, procoagulant and protein C activating activities. The adult venom is more lethal to mice than the newborn venom. In vitro and in vivo immunoneutralization tests showed that commercial Bothrops sp antivenin is less effective at neutralizing newborn venoms. These findings indicate remarkable differences in biological activities of B. jararaca venom over its development. We suggest that not only venom from adult specimens, but also from specimens at other ontogenetic stages should be included in the venom pool used for raising antibodies. Thus, Bothrops antivenin can efficaciously neutralize proteins lacking in the adult venom pool, especially those that promote more intense hemostatic disturbances in victims of newborn snakes.

Comparative analysis of newborn and adult Bothrops jararacasnake venoms

Different clinical manifestations have been reported to occur in patients bitten by newborn and adult Bothrops jararaca snakes. Herein, we studied the chemical composition and biological activities of B. jararaca venoms and their immunoneutralization by commercialantivenin at these ontogenetic stages. Important differences in protein profiles were noticed both in SDS-PAGE and two-dimensional electrophoresis. Newborn venom showed lower proteolytic activity on collagen and fibrinogen, diminished hemorrhagic activity in mouse skin and hind paws, and lower edematogenic, ADPase and 50-nucleotidase activities. However, newborn snake venom showed higher L-amino oxidase, hyaluronidase,platelet aggregating, procoagulant and protein C activating activities. The adult venom is more lethal to mice than the newborn venom. In vitro and in vivo immunoneutralization tests showed that commercial Bothrops sp antivenin is less effective at neutralizing newborn venoms. These findings indicate remarkable differences in biological activities ofB. jararaca venom over its development. We suggest that not only venom from adult specimens, but also from specimens at other ontogenetic stages should be included in the venom pool used for raising antibodies. Thus, Bothrops antivenin can efficaciouslyneutralize proteins lacking in the adult venom pool, especially those that promote more intense hemostatic disturbances in victims of newborn snakes.

Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom.

Patients bitten by Loxosceles spiders generally manifest marked local inflammatory reaction and dermonecrosis. This report evaluated edema formation, leukocyte infiltration and release of inflammatory mediators at the injection site of Loxosceles gaucho venom. BALB/c mice were i.d. injected with venom and thereafter paws were disrupted and homogenized to obtain differential counts of migrated cells, as well to assay the levels of cytokines, chemokines and lipid mediators. Increased footpad thickness was detected as soon as 30 min after venom injection, and 24h later was similar to that of the control group. Loxosceles venom mildly augmented the recruitment of leukocytes to the footpad in comparison with PBS-injected mice. Moreover, it stimulated the release of IL-6, MCP-1 and KC at 2 and 24h after venom injection. In addition, higher levels of PGE(2) were detected 30 min after venom injection in comparison with control group. However, the venom failed to increase levels of IL-1 beta, TNF-alpha, TXB(2) and LTB(4). Our results demonstrate that L. gaucho venom evokes an early complex inflammatory reaction, stimulating the secretion of pro-inflammatory cytokines and lipid mediators (PGE(2)), and recruiting leukocytes to the footpad which contribute to the local reaction induced by L. gaucho venom.

Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom

Patients bitten by Loxosceles spiders generally manifest marked local inflammatory reaction and dermonecrosis. This report evaluated edema formation, leukocyte infiltration and release of inflammatory mediators at the injection site of Loxosceles gaucho venom. BALB/c mice were i.d. injected with venom and thereafter paws were disrupted and homogenizedto obtain differential counts of migrated cells, as well to assay the levels of cytokines, chemokines and lipid mediators. Increased footpad thickness was detected as soon as30 min after venom injection, and 24 h later was similar to that of the control group. Loxosceles venom mildly augmented the recruitment of leukocytes to the footpad in comparison with PBS-injected mice. Moreover, it stimulated the release of IL-6, MCP-1 and KC at 2 and 24 h after venom injection. In addition, higher levels of PGE2 were detected30 min after venom injection in comparison with control group. However, the venom failed to increase levels of IL-1b, TNF-a, TXB2 and LTB4. Our results demonstrate that L. gaucho venom evokes an early complex inflammatory reaction, stimulating the secretionof pro-inflammatory cytokines and lipid mediators (PGE2), and recruiting leukocytes to the $footpad which contribute to the local reaction induced by L. gaucho venom.