Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity.

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. The mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids.

The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase-D toxin.

Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying recombinant phospholipase-D, a potent hemolytic and phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase-D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium-sensitive fluorescent probe (Fluo-4). This calcium influx was shown to be channel-mediated rather than leak-promoted because the influx was inhibited by L-type calcium channel inhibitors but not by a T-type calcium channel blocker, sodium channel inhibitor or a specific inhibitor of calcium activated potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase-D treatment occurred in a concentration-dependent manner in the presence of L-type calcium channel blockers such as nifedipine and verapamil. The data provided herein, suggest that the brown spider venom phospholipase-D-induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as SM and LPC, generating bioactive products that stimulate a calcium influx into red blood cells mediated by the L-type channel.

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom.

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase-D (dermonecrotic toxin) from brown spider venom.

Brown spiders have world-wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase-D, causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose-dependent and time-dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP-phospholipase-D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin-treated erythrocytes reacted with annexin-V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase-D, which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine-protease inhibitor) that had no effect on hemolysis. By site-directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase-D triggers direct human blood cell hemolysis in a catalytic-dependent manner.

Biotechnological applications of brown spider (Loxosceles genus) venom toxins.

Loxoscelism (the term used to define accidents by the bite of brown spiders) has been reported worldwide. Clinical manifestations following brown spider bites are frequently associated with skin degeneration, a massive inflammatory response at the injured region, intravascular hemolysis, platelet aggregation causing thrombocytopenia and renal disturbances. The mechanisms by which the venom exerts its noxious effects are currently under investigation. The whole venom is a complex mixture of toxins enriched with low molecular mass proteins in the range of 5-40 kDa. Toxins including alkaline phosphatase, hyaluronidase, metalloproteases (astacin-like proteases), low molecular mass (5.6-7.9 kDa) insecticidal peptides and phospholipases-D (dermonecrotic toxins) have been identified in the venom. The purpose of the present review is to describe biotechnological applications of whole venom or some toxins, with especial emphasis upon molecular biology findings obtained in the last years.

Vascular permeability and vasodilation induced by the Loxosceles intermedia venom in rats: involvement of mast cell degranulation, histamine and 5-HT receptors.

The mechanisms involved in both local and systemic effects of Loxosceles intermedia (brown spider) venom (LIV) are still poorly understood. We show using rats treated with Evans blue dye (50 mg/kg, i.v.) that small doses of the LIV (0.1, 0.3, 1 and 3 microg/site) dose-dependently increase the vascular permeability in rats, an effect unchanged by indomethacin (5mg/kg, i.p.), atropine (1mg/kg, i.p.), HOE-140 (2mg/kg, s.c.) or SR140333 (0.3mg/kg, i.p.), but fully avoided by promethazine (15 mg/kg, i.p.), methysergide (2mg/kg, i.p.) and compound 48/80 (3mg/kg/day for 3 days). Addition of cumulative concentrations of LIV (0.1-5 microg) in phenylephrine-contracted aortic rings resulted in a partial ( approximately 40%) and endothelium-dependent relaxation, inhibited by the nitric oxide synthase inhibitors L-NAME (10 microM) and L-NMMA (1mM), and the guanylate cyclase inhibitors methylene blue (100 microM) and ODQ (10 microM). LIV-induced relaxation was abolished by compound 48/80 (10 microM) and pyrilamine (a selective histamine H1 receptor antagonist; 100 microM), but not by atropine (1 microM) and indomethacin (10 microM). Our results disclose that LIV increases vascular permeability and induces vascular relaxation. These effects occur due to its ability to degranulate mast cells and release mediators such as histamine and serotonin.

Activation of mononuclear bone marrow cells treated in vitro with a complex homeopathic medication.

Canova is a Brazilian homeopathic medication with immunomodulatory properties, recommended for patients where the immune system is depressed. Previous studies demonstrated that Canova induces up-regulation in numbers of leukocytes. The bone marrow microenvironment is composed of growth factors, stromal cells, extracellular matrix and progenitor cells that differentiate into mature blood cells. We now report the effect of in vitro administration of the medication on the mononuclear differentiation of the bone marrow cell. Swiss mice femurs were dissected cleaned and the cells of the marrow were flushed. The cells were plated, treated or not, incubated for different times and processed for light, transmission and scanning electron, and confocal microscopy analysis. Bone marrow cells showed an enhanced proliferation in vitro in response to Canova medication and Canova plus M-CSF and an increase was also observed in the numbers of the cell niches and ring-shaped nuclei cells. Confocal and transmission and scanning electron microscopy showed the stages of monocyte maturation, with resting and activated cells. With Canova treatment there was a marked increase in cell size, which is mainly attributable to the augmented cytoplasm, an increase in the number of mitochondria, expansion of the RER and an enlarged Golgi. The response to Canova treatment indicates that it influences mononuclear differentiation and activation of bone marrow progenitor and stromal cells.

Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom.

Injuries caused by brown spiders (Loxosceles genus) are associated with dermonecrotic lesions with gravitational spreading and systemic manifestations. The venom has a complex composition containing many different toxins, of which metalloproteases have been described in many different species of this genus. These toxins may degrade extracellular matrix constituents acting as a spreading factor. By using a cDNA library from an Loxosceles intermedia venom gland, we cloned and expressed a 900 bp cDNA, which encoded a signal peptide and a propeptide, which corresponded to a 30 kDa metalloprotease, now named LALP (Loxosceles astacin-like protease). Recombinant LALP was refolded and used to produce a polyclonal antiserum, which showed cross-reactivity with a 29 kDa native venom protein. CD analysis provided evidence that the recombinant LALP toxin was folded correctly, was still in a native conformation and had not aggregated. LALP addition to endothelial cell cultures resulted in de-adhesion of the cells, and also in the degradation of fibronectin and fibrinogen (this could be inhibited by the presence of the bivalent chelator 1,10-phenanthroline) and of gelatin in vitro. Sequence comparison (nucleotide and deduced amino acid), phylogenetic analysis and analysis of the functional recombinant toxin revealed that LALP is related in both structure and function to the astacin family of metalloproteases. This suggests that an astacin-like toxin is present in a animal venom secretion and indicates that recombinant LALP will be a useful tool for future structural and functional studies on venom and the astacin family.

Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from brown spider (Loxosceles intermedia) venom: from cloning to functional characterization.

Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. The venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D. Purified toxin induces cutaneous and systemic loxoscelism, especially necrotic lesions, hematological disturbances and renal failure. Herein, we describe cloning, heterologous expression and purification of two novel dermonecrotic toxins: LiRecDT4 and LiRecDT5. The recombinant proteins stably expressed in Escherichia coli cells were purified from culture supernatants in a single step using Ni(2+)-chelating chromatography producing soluble proteins of 34 kDa (LiRecDT4) and 37 kDa (LiRecDT5). Circular dichroism analysis evidenced correctly folding for toxins but differences in secondary structures. Both proteins were recognized by whole venom serum antibodies and by a specific antibody to dermonecrotic toxin. Also, recombinant toxins with phospholipase activity induced experimental skin lesions and caused a massive inflammatory response in rabbit skin dermis. Nevertheless, toxins displayed different effects upon platelet aggregation, increase in vascular permeability and not caused death in mice. These characteristics in combination with functional studies illustrates that a family of dermonecrotic toxins exists, and includes two novel members that are useful for future structural and functional studies. They will also be useful in biotechnological ends, for example, as inflammatory and platelet aggregating studies, as antigens for serum therapy source and for lipids biochemical research.

Hyaluronidases in Loxosceles intermedia (Brown spider) venom are endo-beta-N-acetyl-d-hexosaminidases hydrolases.

In studying Loxosceles venom, we detected degradation of purified hyaluronic acid (HA) and hydrolysis of purified chondroitin sulphate (CS) while neither dermatan sulphate, heparin or heparan sulphate were affected. In addition, with HA-degrading kinetic assays, we show that a hydrolase enzyme was involved in the HA cleavage. By use of the Reissig colorimetric reaction, we found that venom hyaluronidase is an endo-beta-N-acetyl-d-hexosaminidase that generates terminal N-acetylglucosamine residues upon cleavage of HA. Zymogram analysis of L. intermedia venom showed HA lytic activities at 41 and 43kDa, and, when CS was used as a substrate, zymograph experiments resulted in 41 and 43kDa lytic zones. Thus, these results support the hypothesis that the same molecules are involved in cleaving HA and CS residues. Experiments to compare L. intermedia electrostimulated venom and venom gland extract also demonstrated very similar HA lytic activity, suggesting again that hyaluronidases are self-components of Loxosceles spider venom instead of oral egesta contamination. HA degradation as a function of pH in these hydrolase enzymes showed no apparent activities at low or high pH, with optimal activity at 6.0-8.0 pH. Finally, we confirmed the cleaving action of the venom hyaluronidases on HA in the extracellular matrix of the dermis of rabbit by fluorescence reaction to HA and confocal microscope analysis. Thus, hyaluronidases type hydrolases endo-beta-N-acetyl-d-hexosaminidase are implicated as self-components of Loxosceles spider venom and can be involved in venom effects as spreading factors.

Biological and structural comparison of recombinant phospholipase D toxins from Loxosceles intermedia (brown spider) venom.

The clinical features of brown spider bites are the appearance of necrotic skin lesions, which can also be accompanied by systemic involvement, including weakness, vomiting, fever, convulsions, disseminated intravascular coagulation, intravascular hemolysis and renal disturbances. Severe systemic loxoscelism is much less common than the cutaneous form, but it may be the cause of clinical complications and even death following envenomation. Here, by using three recombinant dermonecrotic toxins, LiRecDT1, LiRecDT2 and LiRecDT3 (the major toxins found in the venom), we report the biological, immunological and structural differences for these members of this toxin family. Purified toxins evoked similar inflammatory reactions following injections into rabbit skin. Recombinant toxin treatments of MDCK cells with LiRecDT1 and LiRecDT2 changed cell viability, as evaluated by neutral red uptake and assessment of cell morphology through inverted microscopy, whereas LiRecDT3 caused only residual activity. Differences in cell cytotoxicity triggered by recombinant toxins were confirmed through a human red blood lysis assay, during which LiRecDT1 and LiRecDT2 caused a high degree of hemolysis compared to LiRecDT3, which induced only a small hemolytic effect. Additionally, biological differences for recombinant toxins were corroborated through mice lethality experiments, which showed animal mortality after LiRecDT1 and LiRecDT2 treatments, but an absence of lethality following LiRecDT3 exposure. Moreover, in experiments for edema, both the LiRecDT1 and the LiRecDT2 toxins evoked similar results, causing edema following toxin exposure, whereas LiRecDT3 caused only residual effects. Characterization of antigenic cross-reactivity using sera against crude venom toxins by immunoWestern blotting and immunodot blotting with recombinant LiRecDT1, LiRecDT2 and LiRecDT3 compared among themselves pointed to a higher cross-reactivity for LiRecDT1 compared to LiRecDT2 and LiRecDT3, corroborating structural and antigenic differences for these three toxins. Finally, evidence for structural differences among the recombinant toxins was strengthened by circular dichroism spectra, which suggested that the toxins were folded, and not aggregated or denatured proteins.

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland.

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni(2+) chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.

The effect of brown spider venom on endothelial cell morphology and adhesive structures.

Spiders of the Loxosceles genus have been responsible for severe clinical cases of envenomation worldwide. Accidents involving brown spiders can cause dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of animals treated by venom have shown subendothelial blebs, vacuoles and endothelial cell membrane degeneration of blood vessel walls, as well as fibrin and thrombus formation. The mechanisms by which the venom causes these disorders are poorly understood. In this work, with an endothelial cell line derived from rabbit aorta, we were able to demonstrate that venom binds to the cell surface and the extracellular matrix. Moreover, we observed that the venom also induced morphological alterations, such as cell retraction, homophilic disadhesion and an increasing in filopodia projections. We also demonstrated that toxins present in the venom disorganized focal adhesion points and actin microfilaments of endothelial cells. Nevertheless, endothelial cell viability showed no alterations compared to controls. Additionally, venom treatment changed the fibronectin matrix profile synthesized by these cells as well as cell adhesion to fibronectin. These results suggest that the deleterious effects of venom on blood vessel walls could be a consequence of the direct effect on the endothelial cell surface and adhesive structures involved in blood vessel stability. These effects indirectly lead to leukocyte and platelet activation, disseminated intravascular coagulation and an increase in vessel permeability.

Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom.

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom.

Identification and partial characterisation of hyaluronidases in Lonomia obliqua venom.

By studying Lonomia obliqua (caterpillar) venom we were able to detect a lytic activity on purified hyaluronic acid. The venom hydrolyses purified chondroitin sulphate, but was unable to degrade either heparan sulphate or dermatan sulphate. Moreover, through purified hyaluronic acid-degrading kinetic assays, we observed that this lytic activity was caused by a hydrolase rather than lyase enzyme. In addition, by using the Reissig colorimetric reaction, we detected this hyaluronic acid hydrolase action as a beta-endohexosaminidase enzyme originating terminal N-acetylglucosamine residues rather than beta-endoglucuronidase, which may originate glucuronic acid residues. Zymogram analysis of the venom detected 49 and 53 kDa molecules with hyaluronic acid lytic activity. An examination of these hyaluronic acid degrading activities as a function of pH showed that these hydrolases had no apparent activities at a pH below 5.0 and higher than 8.0 and displayed their optimal activities at pH ranging from 6.0 to 7.0. Finally, through a fluorescence reaction to hyaluronic acid and confocal microscopy, we confirmed this cleaving action upon hyaluronic acid organised on the extracellular matrix of the dermis of rabbit. The data provide experimental evidence of the presence of hyaluronidases in the L. obliqua venom, probably involved in the harmful effects of the venom.

Brown spider (Loxosceles genus) venom toxins: Evaluation of biological conservation by immune cross-reactivity.

Loxosceles spiders are responsible for serious human envenomations worldwide. The collection of symptoms found in victims after accidents is called loxoscelism and is characterized by two clinical conditions: cutaneous loxoscelism and systemic loxocelism. The only specific treatment is serum therapy, in which an antiserum produced with Loxosceles venom is administered to the victims after spider accidents. Our aim was to improve our knowledge, regarding the immunological relationship among toxins from the most epidemiologic important species in Brazil (Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta). Immunoassays using spider venoms and L. intermedia recombinant toxins were performed and their cross-reactivity assessed. The biological conservation of the main Loxosceles toxins (Phospholipases-D, Astacin-like metalloproteases, Hyaluronidase, ICK-insecticide peptide and TCTP-histamine releasing factor) were investigated. An in silico analysis of the putative epitopes was performed and is discussed on the basis of the experimental results. Our data is an immunological investigation in light of biological conservation throughout the Loxosceles genus. The results bring out new insights on brown spider venom toxins for study, diagnosis and treatment of loxoscelism and putative biotechnological applications concerning immune conserved features in the toxins.

Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue.

Brown spider (Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowman's space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.

Brown spiders and loxoscelism.

Accidents caused by brown spiders (Loxosceles genus) are classically associated with dermonecrotic lesions and systemic manifestations including intravascular haemolysis, disseminated intravascular coagulation and acute renal failure. Systemic reactions occur in a minority of cases, but may be severe in some patients and occasionally fatal. The mechanisms by which Loxosceles venom exerts these noxious effects are currently under investigation. The venom contains several toxins, some of which have been well-characterised biochemically and biologically. The purpose of the present review is to describe some insights into loxoscelism obtained over the last ten years. The biology and epidemiology of the brown spider, the histopathology of envenomation and the immunogenicity of Loxosceles venom are reviewed, as are the clinical features, diagnosis and therapy of brown spider bites. The identification and characterisation of some toxins and the mechanism of induction of local and systemic lesions caused by brown spider venom are also discussed. Finally, the biotechnological application of some venom toxins are covered.

Hematological cell findings in bone marrow and peripheral blood of rabbits after experimental acute exposure to Loxosceles intermedia (brown spider) venom.

The purpose of this work was to find out the cellular changes occurring in bone marrow and peripheral blood after acute exposure to the venom of Loxosceles intermedia. Doses of 40 microg of venom were injected intradermally into five rabbits, and five rabbits receiving only phosphate-buffered saline (PBS) were used as controls. Bone marrow and peripheral blood samples were obtained before the envenomation and 4, 8, 12, 24 and 48 h, and 5, 10, 15, 20 and 30 days after envenomation. In bone marrow samples we assessed cellularity, nucleated red cells, megakaryocytes and neutrophils, and in peripheral blood we assessed red cells (red cell concentration, hemoglobin and hematocrit), leukocytes, neutrophils and platelets. Our objective was to find out if the venom has a direct effect on bone marrow and peripheral blood or if changes in both of them are secondary to the needs of tissues, and if there is a good correlation between histopathological and hematological findings. We found that the red cell parameters were not affected by the venom, except for nucleated red cells which decreased after venom exposure. The depression of megakaryocyte numbers and thrombocytopenia showed a strong correlation with the histopathologic changes observed in skin biopsies obtained from the rabbits. The changes in cellularity and neutrophils of bone marrow were strongly correlated with those in peripheral blood and skin. The thrombocytopenia and neutropenia in peripheral blood are due to marrow depression, which may be a consequence of an extensive migration of platelets and neutrophils to the necrotic lesion or the marrow depression may be a transitory effect of evenoming by L. intermedia.

Isolation and identification of Clostridium perfringens in the venom and fangs of Loxosceles intermedia (brown spider): enhancement of the dermonecrotic lesion in loxoscelism.

Loxoscelism or the envenoming by the brown spiders (Loxosceles genus spiders), may produce extensive dermonecrosis and hemorrhage at the bite site and, eventually, systemic reactions that may be lethal. Isolation and identification of many different bacteria, among them Clostridium perfringens, of great medical importance due to its involvement in dermonecrotizing and systemic conditions, was carried out from the venomous apparatus (fangs and venom) of spiders obtained directly from nature, through microbiological cultures in aerobic and anaerobic conditions. Working with Loxosceles intermedia venom (alone) and with the venom conjugated with Clostridium perfringens using rabbits as experimental models for dermonecrosis, allowed for the observation that venom and anaerobic bacteria conjugated resulted in a striking increase of the dermonecrotic picture when compared to venom alone, suggesting a role for Clostridium perfringens in the severe dermonecrotic picture of these patients and opening the possibility for the association of antibiotic therapy in treating loxoscelism.