Diversity of Micrurus Snake Species Related to Their Venom Toxic Effects and the Prospective of Antivenom Neutralization

The Elapidae family is represented in America by three genera of coral snakes: Micruroides, Leptomicrurus and Micrurus, the latter being the most abundant and diversified group. Micrurus bites can cause death by muscle paralysis and respiratory arrest few hours after envenomation. The specific treatment for Micrurus envenomation is the application of heterologous antivenom. The aim of this study was to compare the toxicity of venoms from nine species of coral snakes and analyze the neutralization potential of the Brazilian coral snake antivenom. In vitro assays showed that the majority of the Micrurus venoms are endowed with phospholipase and hyaluronidase and low proteolytic activities. These enzymes are not equally neutralized in all venoms by the therapeutic antivenom. Moreover, in vivo assays showed that some of the Micrurus venoms are extremely lethal, such as the ones from M. altirostris, M. corallinus, M. frontalis, M. lemniscatus and M. spixii. Neutralization tests, performed in vivo, showed that the therapeutic antivenom was able to neutralize better the venoms from M. frontalis, M. corallinus, and M. spixii but not from M. altirostris and M. lemniscatus. Taken together, these results suggest that modifications in the immunization antigenic mixture should occur in order to generate more comprehensive therapeutic antivenom

Sphingomyelinase D from loxosceles laeta venom induces the expression of MMP7 in human keratinocytes: contribution to dermonecrosis

Envenomation by Loxosceles spider is characterized by the development of dermonecrosis. In previous studies, we have demonstrated that increased expression/secretion of matrix metalloproteinases 2 and 9, induced by Loxosceles intermedia venom Class 2 SMases D (the main toxin in the spider venom), contribute to the development of cutaneous loxoscelism. In the present study we show that the more potent venom containing the Class 1 SMase D from Loxosceles laeta, in addition to increasing the expression/secretion of MMP2 and MMP9, also stimulates the expression of MMP7 (Matrilysin-1), which was associated with keratinocyte cell death. Tetracycline, a matrix metalloproteinase inhibitor, prevented cell death and reduced MMPs expression. Considering that L. laeta venom is more potent at inducing dermonecrosis than L. intermedia venom, our results suggest that MMP7 may play an important role in the severity of dermonecrosis induced by L. laeta spider venom SMase D. In addition, the inhibition of MMPs by e.g. tetracyclines may be considered for the treatment of the cutaneous loxoscelism

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-1β 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-1β, 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

Transcriptome analysis of Loxosceles laeta (Araneae, Sicariidae) spider venomous gland using expressed sequence tags

<p>Abstract</p> <p>Background</p> <p>The bite of spiders belonging to the genus <it>Loxosceles </it>can induce a variety of clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis, and persistent inflammation. In order to examine the transcripts expressed in venom gland of <it>Loxosceles laeta </it>spider and to unveil the potential of its products on cellular structure and functional aspects, we generated 3,008 expressed sequence tags (ESTs) from a cDNA library.</p> <p>Results</p> <p>All ESTs were clustered into 1,357 clusters, of which 16.4% of the total ESTs belong to recognized toxin-coding sequences, being the Sphingomyelinases D the most abundant transcript; 14.5% include "possible toxins", whose transcripts correspond to metalloproteinases, serinoproteinases, hyaluronidases, lipases, C-lectins, cystein peptidases and inhibitors. Thirty three percent of the ESTs are similar to cellular transcripts, being the major part represented by molecules involved in gene and protein expression, reflecting the specialization of this tissue for protein synthesis. In addition, a considerable number of sequences, 25%, has no significant similarity to any known sequence.</p> <p>Conclusion</p> <p>This study provides a first global view of the gene expression scenario of the venom gland of <it>L. laeta </it>described so far, indicating the molecular bases of its venom composition.</p

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

Administration of M. leprae Hsp65 Interferes with the Murine Lupus Progression

The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K409A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F1 mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animal's life span, and the mean survival time [MST] was determined. The treatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F1 serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K409A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule intervening in the progression of the SLE, and that the point mutated K409A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process

Premolis semirufa (Walker, 1856) Envenomation, Disease Affecting Rubber Tappers of the Amazon: Searching for Caterpillar-Bristles Toxic Components

Pararama, the popular name of the larval form of the moth Premolis semirufa inhabits rubber plantations in the Amazon region and the accidental contact of the skin with the caterpillar's bristles or cocoons results in immediate and intense heat, pain, edema, and itching. In many cases a chronic inflammatory reaction with immobilization of the joints occurs. The current study has evaluated the biological and immunochemical characteristics of the Pararama caterpillar bristles extract. Electrophoretic analysis showed the presence of several components, including a very intense 82 kDa band. This latter component was endowed with intense gelatinolytic activity, as observed in zymography assays. Further analysis revealed that the extract also contained hyaluronidase activity but is devoid of phospholipase A2 activity. In vivo assays, using mice, showed that the extract was not lethal, but caused significant edema and induced intense infiltration of inflammatory cells to the envenomation site. The extract also induced high specific antibody titers, but no autoantibodies were detected. The data obtained, so far, demonstrate the existence of a mixture of different enzymes in the bristles of Premolis semirufa caterpillar, which can act together in the generation and development of the clinical manifestations of the Pararama envenomation

A Mycobacterium leprae Hsp65 Mutant as a Candidate for Mitigating Lupus Aggravation in Mice

Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F1 mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K409A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K409A pep synthetic peptides, which cover residues 352–371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K409A+Leader pep or K409A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352–371 region. The number of interactions observed for WT is much higher than for Hsp65 K409A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F1 mice were inoculated with Hsp60 or K409A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K409A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases