Adaptive evolution in the toxicity of a spider's venom enzymes.

BACKGROUND: Sphingomyelinase D is the main toxin present in the venom of Loxosceles spiders. Several isoforms present in these venoms can be structurally classified in two groups. Class I Sphingomyelinase D contains a single disulphide bridge and variable loop. Class II Sphingomyelinase D presents an additional intrachain disulphide bridge that links a flexible loop with a catalytic loop. These classes exhibit differences in their toxic potential. In this paper we address the distribution of the structural classes of SMase D within and among species of spiders and also their evolutionary origin by means of phylogenetic analyses. We also conducted tests to assess the action of natural selection in their evolution combined to structural modelling of the affected sites. RESULTS: The majority of the Class I enzymes belong to the same clade, which indicates a recent evolution from a single common ancestor. Positively selected sites are located on the catalytic interface, which contributes to a distinct surface charge distribution between the classes. Sites that may prevent the formation of an additional bridge were found in Class I enzymes. CONCLUSIONS: The evolution of Sphingomyelinase D has been driven by natural selection toward an increase in noxiousness, and this might help explain the toxic variation between classes.

Micrurus snake venoms activate human complement system and generate anaphylatoxins.

BACKGROUND: The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. RESULTS: In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. CONCLUSION: Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process.

New insights into immunopathology associated to Bothrops lanceolatus snake envenomation: focus on PLA2 toxin

The systemic increase in inflammatory mediator levels can induce diverse pathological disorders, including potentially thrombus formation, which may be lethal. Among the clinical conditions in which the formation of thrombi dictates the patient’s prognosis, envenomation by Bothrops lanceolatus should be emphasized, as it can evolve to stroke, myocardial infarction and pulmonary embolism. Despite their life-threatening potential, the immunopathological events and toxins involved in these reactions remain poorly explored. Therefore, in the present study, we examined the immunopathological events triggered by a PLA2 purified from B. lanceolatus venom, using an ex vivo human blood model of inflammation. Our results showed that the purified PLA2 from the venom of B. lanceolatus damages human erythrocytes in a dose dependent way. The cell injury was associated with a decrease in the levels of CD55 and CD59 complement regulators on the cell surface. Moreover, the generation of anaphylatoxins (C3a and C5a) and the soluble terminal complement complex (sTCC) indicates that human blood exposure to the toxin activates the complement system. Increased production of TNF-α, CXCL8, CCL2 and CCL5 followed complement activation. The venom PLA2 also triggered the generation of lipid mediators, as evidenced by the detected high levels of LTB4, PGE2 and TXB2. The scenario here observed of red blood cell damage, dysfunctions of the complement regulatory proteins, accompanied by an inflammatory mediator storm, suggests that B. lanceolatus venom PLA2 contributes to the thrombotic disorders present in the envenomed individuals.

Complement system inhibition modulates the inflammation induced by the venom of premolis semirufa, an Amazon rainforest Moth Caterpillar

The caterpillar of the Premolis semirufa moth, commonly called Pararama, is found in the Brazilian Amazon region. Contact with the hairs can cause a chronic inflammatory reaction, termed “pararamosis”. To date, there is still no specific treatment for pararamosis. In this study, we used a whole human blood model to evaluate the involvement of the complement in the proinflammatory effects of P. semirufa hair extract, as well as the anti-inflammatory potential of complement inhibitors in this process. After treatment of blood samples with the P. semirufa hair extract, there was a significant increase in the generation of soluble terminal complement complex (sTCC) and anaphylatoxins (C3a, C4a, and C5a), as well as the production of the cytokines TNF-α and IL-17 and the chemokines IL-8, RANTES, MIG, MCP-1, and IP-10. The inhibition of C3 with compstatin significantly decreased IL-17, IL-8, RANTES, and MCP-1 production. However, the use of the C5aR1 antagonist PMX205 promoted a reduction in the production of IL-8 and RANTES. Moreover, compstatin decreased CD11b, C5aR1, and TLR2 expression induced by P. semirufa hair extract in granulocytes and CD11b, TLR4, and TLR2 in monocytes. When we incubated vascular endothelial cells with extract-treated human plasma, there was an increase in IL-8 and MCP-1 production, and compstatin was able to decrease the production of these chemokines. C5aR1 antagonism also decreased the production of MCP-1 in endothelial cells. Thus, these results indicate that the extract of the Pararama bristles activates the complement system and that this action contributes to the production of cytokines and chemokines, modulation of the expression of surface markers in leukocytes, and activation of endothelial cells.

Anti-SARS-CoV-2 equine F (Ab′)2 immunoglobulin as a possible therapy for COVID-19

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab′)2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab′)2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Characterization of a Gene Coding for the Complement System Component FB from Loxosceles laeta Spider Venom Glands.

The human complement system is composed of more than 30 proteins and many of these have conserved domains that allow tracing the phylogenetic evolution. The complement system seems to be initiated with the appearance of C3 and factor B (FB), the only components found in some protostomes and cnidarians, suggesting that the alternative pathway is the most ancient. Here, we present the characterization of an arachnid homologue of the human complement component FB from the spider Loxosceles laeta. This homologue, named Lox-FB, was identified from a total RNA L. laeta spider venom gland library and was amplified using RACE-PCR techniques and specific primers. Analysis of the deduced amino acid sequence and the domain structure showed significant similarity to the vertebrate and invertebrate FB/C2 family proteins. Lox-FB has a classical domain organization composed of a control complement protein domain (CCP), a von Willebrand Factor domain (vWFA), and a serine protease domain (SP). The amino acids involved in Mg2+ metal ion dependent adhesion site (MIDAS) found in the vWFA domain in the vertebrate C2/FB proteins are well conserved; however, the classic catalytic triad present in the serine protease domain is not conserved in Lox-FB. Similarity and phylogenetic analyses indicated that Lox-FB shares a major identity (43%) and has a close evolutionary relationship with the third isoform of FB-like protein (FB-3) from the jumping spider Hasarius adansoni belonging to the Family Salcitidae.

Integrative multiomics analysis of Premolis semirufa caterpillar venom in the search for molecules leading to a joint disease

The joint disease called pararamosis is an occupational disease caused by accidental contact with bristles of the caterpillar Premolis semirufa. The chronic inflammatory process narrows the joint space and causes alterations in bone structure and cartilage degeneration, leading to joint stiffness. Aiming to determine the bristle components that could be responsible for this peculiar envenomation, in this work we have examined the toxin composition of the caterpillar bristles extract and compared it with the differentially expressed genes (DEGs) in synovial biopsies of patients affected with rheumatoid arthritis (RA) and osteoarthritis (OA). Among the proteins identified, 129 presented an average of 63% homology with human proteins and shared important conserved domains. Among the human homologous proteins, we identified seven DEGs upregulated in synovial biopsies from RA or OA patients using meta-analysis. This approach allowed us to suggest possible toxins from the pararama bristles that could be responsible for starting the joint disease observed in pararamosis. Moreover, the study of pararamosis, in turn, may lead to the discovery of specific pharmacological targets related to the early stages of articular diseases.

A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

BACKGROUND: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system. RESULTS: The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. CONCLUSION: These data show that a serine protease present in the Premolis semirufa's bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

P-MAPA, a fungi-derived Immunomodulatory compound, Induces a proinflammatory response in a human whole blood model

P-MAPA is a complex compound, derived from Aspergillus oryzae cultures, that has shown immunomodulatory properties in infection and cancer animal models. Despite promising results in these models, the mechanisms of cellular activation by P-MAPA, suggested to be Toll-like receptor- (TLR-) dependent, and its effect on human immune cells, remain unclear. Using an ex vivo model of human whole blood, the effects of P-MAPA on complement system activation, production of cytokines, and the expression of complement receptors (CD11b, C5aR, and C3aR), TLR2, TLR4, and the coreceptor CD14 were analyzed in neutrophils and monocytes. P-MAPA induced complement activation in human blood, detected by increased levels of C3a, C5a, and SC5b-9 in plasma. As a consequence, CD11b expression increased and C5aR decreased upon activation, while C3aR expression remained unchanged in leukocytes. TLR2 and TLR4 expressions were not modulated by P-MAPA treatment on neutrophils, but TLR4 expression was reduced in monocytes, while CD14 expression increased in both cell types. P-MAPA also induced the production of TNF-α, IL-8, and IL-12 and oxidative burst, measured by peroxynitrite levels, in human leukocytes. Complement inhibition with compstatin showed that P-MAPA-induced complement activation drives modulation of C5aR, but not of CD11b, suggesting that P-MAPA acts through both complement-dependent and complement-independent mechanisms. Compstatin also significantly reduced the peroxynitrite generation. Altogether, our results show that P-MAPA induced proinflammatory response in human leukocytes, which is partially mediated by complement activation. Our data contribute to elucidate the complement-dependent and complement-independent mechanisms of P-MAPA, which ultimately result in immune cell activation and in its immunomodulatory properties in infection and cancer animal models

Human chondrocyte activation by toxins from premolis semirufa, an Amazon rainforest moth caterpillar: identifying an osteoarthritis signature

Pararamosis is a disease that occurs due to contact with the hairs of the larval stage of the Brazilian moth Premolis semirufa. Envenomation induces osteoarticular alterations with cartilage impairment that resembles joint synovitis. Thus, the toxic venom present in the caterpillar hairs interferes with the phenotype of the cells present in the joints, resulting in inflammation and promoting tissue injury. Therefore, to address the inflammatory mechanisms triggered by envenomation, we studied the effects of P. semirufa hair extract on human chondrocytes. We have selected for the investigation, cytokines, chemokines, matrix metalloproteinases (MMPs), complement components, eicosanoids, and extracellular matrix (ECM) components related to OA and RA. In addition, for measuring protein-coding mRNAs of some molecules associated with osteoarthritis (OA) and rheumatoid arthritis (RA), reverse transcription (RT) was performed followed by quantitative real-time PCR (RT-qPCR) and we performed the RNA-sequencing (RNA-seq) analysis of the chondrocytes transcriptome. In the supernatant of cell cultures treated with the extract, we observed increased IL-6, IL-8, MCP-1, prostaglandin E2, metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-13), and complement system components (C3, C4, and C5). We noticed a significant decrease in both aggrecan and type II collagen and an increase in HMGB1 protein in chondrocytes after extract treatment. RNA-seq analysis of the chondrocyte transcriptome allowed us to identify important pathways related to the inflammatory process of the disease, such as the inflammatory response, chemotaxis of immune cells and extracellular matrix (ECM) remodeling. Thus, these results suggest that components of Premolis semirufa hair have strong inflammatory potential and are able to induce cartilage degradation and ECM remodeling, promoting a disease with an osteoarthritis signature. Modulation of the signaling pathways that were identified as being involved in this pathology may be a promising approach to develop new therapeutic strategies for the control of pararamosis and other inflammatory joint diseases.

EcTI impairs survival and proliferation pathways in triple-negative breast cancer by modulating cell-glycosaminoglycans and inflammatory cytokines

Breast cancer is the most common malignant tumor among women worldwide, and triple-negative breast cancer is the most aggressive type of breast cancer, which does not respond to hormonal therapies. The protease inhibitor, EcTI, extracted from seeds of Enterolobium contortisiliquum, acts on the main signaling pathways of the MDA-MB-231 triple-negative breast cancer cells. This inhibitor, when bound to collagen I of the extracellular matrix, triggers a series of pathways capable of decreasing the viability, adhesion, migration, and invasion of these cells. This inhibitor can interfere in the cell cycle process through the main signaling pathways such as the adhesion, Integrin/FAK/SRC, Akt, ERK, and the cell death pathway BAX and BCL-2. It also acts by reducing the main inflammatory cytokines such as TGF-α, IL-6, IL-8, and MCP-1, besides NFκB, a transcription factor, responsible for the aggressive and metastatic characteristics of this type of tumor. Thus, the inhibitor was able to reduce the main processes of carcinogenesis of this type of cancer.

Complement system inhibition modulates the pro-inflammatory effects of a snake venom metalloproteinase

Envenomation by Bothrops snakes causes prominent local effects, including pain, oedema, local bleeding, blistering and necrosis, and systemic manifestations, such as hemorrhage, hypotension, shock and acute renal failure. These snake venoms are able to activate the complement system and induce the generation of anaphylatoxins, whose mechanisms include the direct cleavage of complement components by snake venom metalloproteinases and serine proteinases present in the venoms. A metalloproteinase able to activate the three complement pathways and generate active anaphylatoxins, named C-SVMP, was purified from the venom of Bothrops pirajai. Considering the inflammatory nature of Bothrops venoms and the complement-activation property of C-SVMP, in the present work, we investigated the inflammatory effects of C-SVMP in a human whole blood model. The role of the complement system in the inflammatory process and its modulation by the use of compstatin were also investigated. C-SVMP was able to activate the complement system in the whole blood model, generating C3a/C3a desArg, C5a/C5a desArg and SC5b-9. This protein was able to promote an increase in the expression of CD11b, CD14, C3aR, C5aR1, TLR2, and TLR4 markers in leukocytes. Inhibition of component C3 by compstatin significantly reduced the production of anaphylatoxins and the Terminal Complement Complex (TCC) in blood plasma treated with the toxin, as well as the expression of CD11b, C3aR, and C5aR on leukocytes. C-SVMP was able to induce increased production of the cytokines IL-1ß and IL-6 and the chemokines CXCL8/IL-8, CCL2/MCP-1, and CXCL9/MIG in the human whole blood model. The addition of compstatin to the reactions caused a significant reduction in the production of IL-1ß, CXCL8/IL-8, and CCL2/MCP-1 in cells treated with C-SVMP. We therefore conclude that C-SVMP is able to activate the complement system, which leads to an increase in the inflammatory process. The data obtained with the use of compstatin indicate that complement inhibition may significantly control the inflammatory process initiated by Bothrops snake venom toxins.

Enzymatic properties of venoms from Brazilian scorpions of Tityus genus and the neutralisation potential of therapeutical antivenoms.

Tityus scorpion stings are an important public health problem in Brazil, where the incidence of such stings exceeds the incidence of the health problems caused by other venomous animals, including snakes. In this study, we have analysed specific enzymatic activities of the venom from the Brazilian scorpions of Tityus genus, i.e., Tityus serrulatus, Tityus bahiensis and Tityus stigmurus. The data presented here revealed that Tityus spp. venoms exhibited significant hyaluronidase activity but no phospholipase activity. All the venom samples exhibited the ability to hydrolyse Abz-FLRRV-EDDnp and dynorphin 1-13 substrates. These activities were inhibited by 1,10-phenanthroline but not by PMSF, indicating the presence of metalloproteinases in the Tityus spp. venoms. The venom peptidase activity on Abz-FLRRV-EDDnp and on dynorphin 1-13 was partially inhibited by therapeutic Brazilian anti-scorpion and anti-arachnidic antivenoms. Dynorphin 1-13 (YGGFLRRIRPKLK) contains two scissile bonds between the residues Leu-Arg and Arg-Arg that are susceptible to cleavage by the Tityus venom metallopeptidase(s). Their cleavage releases leu-enkephalin, an important bioactive peptide. The detection of metalloproteinase(s) with specificity for both dynorphin 1-13 degradation and leu-enkephalin releasing can be important for the mechanistic understanding of hypotension and bradycardia induction in cases of scorpion stings, whereas hyaluronidases might contribute to the diffusion of the toxins present in these venoms. Furthermore, the limited inhibition of the toxic enzymatic activities by commercial antivenoms illustrates the necessity of improvements in current antivenom preparation.

Venom from Bothrops lanceolatus, a snake species native to Martinique, potently activates the complement system

Bothrops lanceolatus snake venom causes systemic thrombotic syndrome but also local inflammation involving extensive oedema, pain, and haemorrhage. Systemic thrombotic syndrome may lead to fatal pulmonary embolism and myocardial and cerebral infarction. Here, we investigated the ability of B. lanceolatus venom to activate the Complement system (C) in order to improve the understanding of venom-induced local inflammation. Data presented show that B. lanceolatus venom is able to activate all C-pathways. In human serum, the venom strongly induced the generation of anaphylatoxins, such as C5a and C4a, and the Terminal Complement complex. The venom also induced cleavage of purified human components C3, C4, and C5, with the production of biologically active C5a. Furthermore, the venom enzymatically inactivated the soluble C-regulator and the C1-inhibitor (C1-INH), and significantly increased the expression of bound C-regulators, such as MCP and CD59, on the endothelial cell membrane. Our observations that B. lanceolatus venom activates the three Complement activation pathways, resulting in anaphylatoxins generation, may suggest that this could play an important role in local inflammatory reaction and systemic thrombosis caused by the venom. Inactivation of C1-INH, which is also an important inhibitor of several coagulation proteins, may also contribute to inflammation and thrombosis. Thus, further in vivo studies may support the idea that therapeutic management of systemic B. lanceolatus envenomation could include the use of Complement inhibitors as adjunct therapy.

P-I snake venom metalloproteinase is able to activate the complement system by direct cleavage of central components of the cascade.

BACKGROUND: Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom. RESULTS: Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity. CONCLUSION: We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.

Leptospira interrogans outer membrane protein LipL21 is a potent inhibitor of neutrophil myeloperoxidase

Leptospirosis is a widespread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. After entrance in the host, pathogenic leptospires evade the host natural defense mechanisms in order to propagate and disseminate to multiple organs. Myeloperoxidase is an enzyme stored in neutrophils azurophilic granules, and is released upon neutrophil activation to produce mainly hypochlorous acid, a strong oxidant and potent antimicrobial agent. In the present investigation, we studied the modulation of myeloperoxidase activity by L. interrogans serovar Copenhageni. We show that leptospires and their culture supernatants are able to inhibit both peroxidase and chlorination activities of myeloperoxidase, without interfering with neutrophil degranulation. By leptospiral outer membrane protein extraction and fractionation, we identified the proteins LipL21 and LipL45 as myeloperoxidase inhibitors, constituting new Leptospira virulence factors. Accordingly, we propose a function for the protein LipL21, one of the most expressed leptospiral outer membrane proteins. Our results show a novel innate immune evasion mechanism by which leptospires interfere with the host response in order to cope with the host oxidative stress and efficiently achieve dissemination and colonization.

Premolis semirufa (Walker, 1856) envenomation, disease affecting rubber tappers of the Amazon: searching for caterpillar-bristles toxic components.

BACKGROUND: The caterpillar of the moth Premolis semirufa (Lepidoptera: Arctiidae), commonly named Pararama, is endemic of the Amazon basin. Accidental contact with these caterpillar bristles causes local symptoms such as intense heat, pain, edema and itching which last for three to seven days; however, after multiples contacts, it may induce joint-space narrowing and bone alteration, as well as degeneration of the articular cartilage and immobilization of the affected joints. Specific treatment for this disease does not exist, but corticosteroids are frequently administered. Despite of the public health hazard of Premolis semirufa caterpillar poisoning, little is known about the nature of the toxic components involved in the induction of the pathology. METHODOLOGY/PRINCIPAL FINDINGS: Here we have investigated the biological and immunochemical characteristics of the caterpillar's bristles components. Analysis of the bristles extract in in vitro assays revealed the presence of proteolytic and hyaluronidase activities but no phospholipase A(2) activity. In vivo, it was observed that the bristles extract is not lethal but can induce an intense inflammatory process, characterized by the presence of neutrophils in the paw tissues of injected mice. Furthermore, the bristles components stimulated an intense and specific antibody response but autoantibodies such as anti-DNA or anti-collagen type II were not detected. CONCLUSION: The results suggest that Premolis semirufa caterpillar bristles secretion contains a mixture of different enzymes that may act together in the generation and development of the clinical manifestations of the Pararama envenomation. Moreover, the high immunogenicity of the caterpillar bristles components, as shown by the generation of high antibody titers, may also contribute to the induction and establishment of the inflammatory disease.

Human complement activation and anaphylatoxins generation induced by snake venom toxins from Bothrops genus.

Snake venoms are a complex mixture of components, which have a wide range of actions both on prey and human victims. The genus Bothrops causes the vast majority of snakebites in Central and South America, being responsible for 80% of snake envenomations in Brazil. Envenomations are characterized by prominent local effects, including oedema, haemorrhage and necrosis, which can lead to permanent disability. Systemic manifestations such as haemorrhage, coagulopathy, shock and acute renal failure may also occur. In the present study we have investigated the action of venoms from 19 species of snakes from the genus Bothrops, occurring in Brazil, on the complement system in in vitro studies. All venoms were able to activate the classical complement pathway, in the absence of sensitizing antibody. This activation was in part associated with the cleavage of C1-Inhibitor by proteases present in these venoms, which disrupts complement activation control. No modification of the membrane bound complement regulators, such as DAF, CR1 and CD59 was detected, after treatment of human erythrocytes with the snake venoms. Some of the Bothrops venoms were also able to activate alternative and lectin pathways, as measured in haemolytic and ELISA assays. C3a, C4a and C5a were generated in sera treated with the venoms, not only through C-activation, but also by the direct cleavage of complement components, as determined using purified C3 and C4. Metallo- and/or serine-protease inhibitors prevented cleavage of C3 and C4. These results suggest that Bothrops venoms can activate the complement system, generating a large amount of anaphylatoxins, which may play an important role in the inflammatory process presented in humans after snake envenomations, and they may also assist, due to their vasodilatory effects, to enhance the spreading of other venom components.