Correction: Boia-Ferreira et al. TCTP from Loxosceles Intermedia (Brown Spider) Venom Contributes to the Allergic and Inflammatory Response of Cutaneous Loxoscelism. Cells 2019, 8, 1489.

In the original publication [...].

Sertraline as a potential cancer therapeutic approach: Biological relevance of TCTP in breast cancer cell lines and tumors.

PURPOSE: This study aimed to evaluate the role of Translationally Controlled Tumor Protein (TCTP) in breast cancer (BC) and investigate the effects of sertraline, a serotonin selective reuptake inhibitor (SSRI), on BC cells. The objective was to assess the potential of sertraline as a therapeutic agent in BC treatment by examining its ability to inhibit TCTP expression and exert antitumor effects. MATERIAL AND METHODS: We utilized five different BC cell lines representing the molecular heterogeneity and distinct subtypes of BC, including luminal, normal-like, HER2-positive, and triple-negative BC. These subtypes play a crucial role in determining clinical treatment strategies and prognosis. RESULTS: The highest levels of TCTP were observed in triple-negative BC cell lines, known for their aggressive behavior. Sertraline treatment reduced TCTP expression in BC cell lines, significantly impacting cell viability, clonogenicity, and migration. Additionally, sertraline sensitized triple-negative BC cell lines to cytotoxic chemotherapeutic drugs (doxorubicin and cisplatin) suggesting its potential as an adjunctive therapy to enhance the chemotherapeutic response. Bioinformatic analysis of TCTP mRNA levels in TCGA BC data revealed a negative correlation between TCTP levels and patient survival, as well as between TCTP/tpt1 and Ki67. These findings contradict our data and previous studies indicating a correlation between TCTP protein levels and aggressiveness and poor prognosis in BC. CONCLUSIONS: Sertraline shows a promise as a potential therapeutic option for BC, particularly in triple-negative BC. Its ability to inhibit TCTP expression, enhance chemotherapeutic response, highlights its potential clinical utility in BC treatment, specifically in triple-negative BC subtype.

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.

Differences in the expression of glycosaminoglycans in human fibroblasts derived from gingival overgrowths is related to TGF-beta up-regulation.

Glycosaminoglycans (GAGs) play important roles in cell behavior and have the ability to bind and modulate cytokines. Using primary cultured fibroblasts from hereditary gingival fibromatosis (HGF), normal gingiva (NG), and NG treated with cyclosporin-A (NGc) we show changes in the expression and structural characteristics of GAGs as well as in the expression of enzymes involved in their biosynthesis and degradation. In addition, we show the over-expression of TGF-beta1 and TGF-beta type II receptor in HGF and NGc. There is an increase in the GAGs retained in the cellular fraction, and the fine structure of galactosaminoglycans show a decrease in alpha-l-iduronic acid content in HGF and NGc. Elevated extracellular levels of low molecular weight hyaluronan (HA) are found in HGF due to increase in the expression of HA synthase 3 and hyaluronidases 1 and 2. The results bring new insights to the accumulation of extracellular matrix related to TGF-beta over-expression.

Engineered antigen containing epitopes from Loxosceles spp. spider toxins induces a monoclonal antibody (Lox-mAb3) against astacin-like metalloproteases.

Loxoscelism pose a health issue in the South America. The treatment for these accidents is based on the administration of antivenom produced in animals immunized with Loxosceles venom. In this work, a previously produced non-toxic multiepitopic chimeric protein (rMEPlox), composed of epitopes derived from the main toxins families (sphyngomielinase-D, metalloproteases, and hyaluronidases) of Loxosceles spider venoms, was used as antigen to produce monoclonal antibodies (mAbs). A selected anti-rMEPlox mAb (Lox-mAb3) reacted with metalloprotease from L. intermedia venom and showed cross-reactivity with metalloproteses from Brazilian and Peruvian Loxosceles laeta and Loxosceles gaucho venoms in immunoassays. The sequence recognized by Lox-mAb3 (184ENNTRTIGPFDYDSIMLYGAY205) corresponds to the C-terminal region of Astacin-like metalloprotease 1 and the amino acid sequence IGPFDYDSI, conserved among the homologs metalloproteases sequences, is important for antibody recognition. Lox-mAb3 neutralizes the fibrinogenolytic activity caused by metalloprotease from L. intermedia spider venom in vitro, which may lead to a decrease in hemorrhagic disturbances caused by Loxosceles envenomation. Our results show, for the first time, the use of a non-toxic multiepitopic protein for the production of a neutralizing monoclonal antibody against a metalloprotease of medically important Loxosceles venoms. These results contribute for the production improvement of therapeutic antivenom against loxoscelism.

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.

Glycosaminoglycan chains from alpha5beta1 integrin are involved in fibronectin-dependent cell migration.

Alpha5beta1 integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745) is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [35S]sulfate metabolic labeling of the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, and flow cytometry. The alpha5beta1 integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfate chains. Xyloside inhibition of sulfate incorporation into alpha5beta1 integrin also supports that integrin is a proteoglycan. Also, cells grown with xyloside adhered on fibronectin with no alteration in alpha5beta1 integrin expression. However, haptotactic motility on fibronectin declined in cells grown with xyloside or chlorate as compared with controls. Thus, alpha5beta1 integrin is a proteoglycan and the glycosaminoglycan chains of the integrin influence cell motility on fibronectin. Similar glycosylation of alpha5beta1 integrin was observed in other normal and malignant cells, suggesting that this modification is conserved and important in the function of this integrin. Therefore, these glycosaminoglycan chains of alpha5beta1 integrin are involved in cellular migration on fibronectin.

TCTP from Loxosceles Intermedia (Brown Spider) Venom Contributes to the Allergic and Inflammatory Response of Cutaneous Loxoscelism.

LiTCTP is a toxin from the Translationally Controlled Tumor Protein (TCTP) family identified in Loxosceles brown spider venoms. These proteins are known as histamine-releasing factors (HRF). TCTPs participate in allergic and anaphylactic reactions, which suggest their potential role as therapeutic targets. The histaminergic effect of TCTP is related to its pro-inflammatory functions. An initial characterization of LiTCTP in animal models showed that this toxin can increase the microvascular permeability of skin vessels and induce paw edema in a dose-dependent manner. We evaluated the role of LiTCTP in vitro and in vivo in the inflammatory and allergic aspects that undergo the biological responses observed in Loxoscelism, the clinical condition after an accident with Loxosceles spiders. Our results showed LiTCTP recombinant toxin (LiRecTCTP) as an essential synergistic factor for the dermonecrotic toxin actions (LiRecDT1, known as the main toxin in the pathophysiology of Loxoscelism), revealing its contribution to the exacerbated inflammatory response clinically observed in envenomated patients.

Fatty acid synthase inhibition with Orlistat promotes apoptosis and reduces cell growth and lymph node metastasis in a mouse melanoma model.

Fatty acid synthase (FASN) is the enzyme responsible for the endogenous synthesis of the saturated fatty acid palmitate. In contrast to most normal cells, malignant cells depend on FASN activity for growth and survival. In fact, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Here, we show that the specific inhibition of FASN activity by the antiobesity drug Orlistat or siRNA is able to significantly reduce proliferation and promote apoptosis in the mouse metastatic melanoma cell line B16-F10. These results prompted us to verify the effect of FASN inhibition on the metastatic process in a model of spontaneous melanoma metastasis, in which B16-F10 cells injected in the peritoneal cavity of C57BL/6 mice metastasize to the mediastinal lymph nodes. We observed that mice treated with Orlistat 48 hr after the inoculation of B16-F10 cells exhibited a 52% reduction in the number of mediastinal lymph node metastases, in comparison with the control animals. These results suggest that FASN activity is essential for B16-F10 melanoma cell proliferation and survival while its inactivation by Orlistat significantly reduces their metastatic spread. The chemical inhibition of FASN activity could have a potential benefit in association with the current chemotherapy for melanoma.

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.

Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits.

Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho, and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms.

Structural and functional characterization of a P-III metalloproteinase, leucurolysin-B, from Bothrops leucurus venom.

Leucurolysin-B (leuc-B) is an hemorrhagic metalloproteinase found in the venom of Bothrops leucurus (white-tailed-jararaca) snake. By means of liquid chromatography consisting of gel filtration on Sephracryl S-200, S-300 and ion-exchange on DEAE Sepharose, leuc-B was purified to homogeneity. The proteinase has an apparent molecular mass of 55kDa as revealed by the reduced SDS-PAGE, and represents approximately 1.2% of the total protein in B. leucurus venom. The partial amino acid sequence of leuc-B was determined by automated Edman sequencing of peptides derived from digests of the S-reduced and alkylated protein with trypsin. Leuc-B exhibits the characteristic motif of metalloproteinases, HEXXHXXGXXH and a methionine-containing turn of similar conformation ("Met-turn"), which forms a hydrophobic basis for the zinc ions and the three histidine residues involved as ligands. Leuc-B has been characterized as a P-III metalloproteinase and possesses a multidomain structure including a metalloproteinase, a disintegrin-like (ECD sequence instead of the typical RGD motif) and a cysteine-rich C-terminal domain. Leuc-B contains three potential sites of N-glycosylation. The enzyme only cleaves the Ala14-Leu15 peptide bond of the oxidized insulin B-chain and preferentially hydrolyzes the Aalpha-chain of fibrinogen and the alpha-chain of fibrin. Its proteolytic activity was completely inhibited by metal chelating agents but not by other typical proteinase inhibitors. In addition, its enzymatic activity was stimulated by the divalent cations Ca2+ and Mg2+ but inhibited by Zn2+ and Cu2+. The catalytic activity of leuc-B on extracellular matrix proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites (MHD=30ng in rabbit), with alterations in platelet function. In summary, here we report the isolation and the structure-function relationship of a P-III snake venom metalloproteinase.

A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom.

Venoms are a rich source for the discovery of molecules with biotechnological applications, but their analysis is challenging even for state-of-the-art proteomics. Here we report on a large-scale proteomic assessment of the venom of Loxosceles intermedia, the so-called brown spider. Venom was extracted from 200 spiders and fractioned into two aliquots relative to a 10 kDa cutoff mass. Each of these was further fractioned and digested with trypsin (4 h), trypsin (18 h), pepsin (18 h), and chymotrypsin (18 h), then analyzed by MudPIT on an LTQ-Orbitrap XL ETD mass spectrometer fragmenting precursors by CID, HCD, and ETD. Aliquots of undigested samples were also analyzed. Our experimental design allowed us to apply spectral networks, thus enabling us to obtain meta-contig assemblies, and consequently de novo sequencing of practically complete proteins, culminating in a deep proteome assessment of the venom. Data are available via ProteomeXchange, with identifier PXD005523.

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.

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.

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.

Identification of proteases in the extract of venom glands from brown spiders.

In the present investigation, in order to dispute the rational criticism against the presence of proteolytic enzymes in the electrostimulated venom obtained from spiders of the genus Loxosceles, as a consequence of contamination with abdominal secretions, venoms of L. intermedia and L. laeta were directly collected from venom glands by microdissection and gentle homogenization. Gel electrophoresis stained by silver method carried out to compare L. intermedia electrostimulated venom and venom gland extract demonstrated no significant differences in protein profile. Zymogram analysis of L. intermedia venom gland extract detected a gelatinolytic activity in the 32-35 kDa region. The inhibitory effect of 1,10-phenanthroline on this proteolytic activity further supported its metalloprotease nature. In proteolytic digestion experiments L. intermedia venom gland extract was also able to cleave purified fibronectin and fibrinogen. The inhibitory effect of 1,10-phenanthroline on these degrading activities confirmed the presence of metalloproteases in the venom. In addition, when purified fibrinogen was incubated with L. intermedia abdominal extract, the fibrinogenolysis was completely different, generating low mass fragments that ran away from the gel, a proteolytic event not blocked by 1,10-phenanthroline. Zymogram experiments using L. laeta venom gland extracts further detected a gelatinolytic band at 32-35 kDa, also inhibited by 1,10-phenanthroline, confirming the presence of metalloproteases in both species.

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.