Protective effects of mesenchymal stromal cell-derived secretome on dermonecrosis induced in rabbits by Loxosceles intermedia spider venom.

Background: Loxoscelism refers to a set of clinical manifestations caused by the bite of spiders from the Loxosceles genus. The classic clinical symptoms are characterized by an intense inflammatory reaction at the bite site followed by local necrosis and can be classified as cutaneous loxoscelism. This cutaneous form presents difficult healing, and the proposed treatments are not specific or effective. This study aimed to evaluate the protective effect of mesenchymal stromal cells-derived secretome on dermonecrosis induced by Loxosceles intermedia spider venom in rabbits. Methods: Sixteen rabbits were distributed into four groups (n = 4). Except for group 1 (G1), which received only PBS, the other three groups (G2, G3, and G4) were initially challenged with 10 µg of L. intermedia venom, diluted in 100 µL of NaCl 0.9%, by intradermic injection in the interscapular region. Thirty minutes after the challenge all groups were treated with secretome, except for group 2. Group 1 (G1-control group) received intradermal injection (ID) of 60 µg of secretome in 0.15 M PBS; Group 2 (G2) received 0.9% NaCl via ID; Group 3 (G3) received 60 µg of secretome, via ID and Group 4 (G4), received 60 µg of secretome by intravenous route. Rabbits were evaluated daily and after 15 days were euthanized, necropsied and skin samples around the necrotic lesions were collected for histological analysis. Results: Rabbits of G1 did not present edema, erythema, hemorrhagic halo, or necrosis. In animals from G2, G3, and G4, edema appeared after 6h. However, minor edema was observed in the animals of G2 and G3. Hemorrhagic halo was observed in animals, six hours and three days after, on G2, G3, and G4. Macroscopically, in G4, only one animal out of four had a lesion that evolved into a dermonecrotic wound. No changes were observed in the skin of the animals of G1, by microscopic evaluation. All animals challenged with L. intermedia venom showed similar alterations, such as necrosis and heterophilic infiltration. However, animals from G4 showed fibroblast activation, early development of connective tissue, neovascularization, and tissue re-epithelialization, indicating a more prominent healing process. Conclusion: These results suggest that secretome from mesenchymal stromal cells cultured in a xeno-free and human component-free culture media can be promising to treat dermonecrosis caused after Loxosceles spiders bite envenoming.

Comparative Biochemical, Structural, and Functional Analysis of Recombinant Phospholipases D from Three Loxosceles Spider Venoms.

Spiders of Loxosceles genus are widely distributed and their venoms contain phospholipases D (PLDs), which degrade phospholipids and trigger inflammatory responses, dermonecrosis, hematological changes, and renal injuries. Biochemical, functional, and structural properties of three recombinant PLDs from L. intermedia, L. laeta, and L. gaucho, the principal species clinically relevant in South America, were analyzed. Sera against L. gaucho and L. laeta PLDs strongly cross-reacted with other PLDs, but sera against L. intermedia PLD mostly reacted with homologous molecules, suggesting underlying structural and functional differences. PLDs presented a similar secondary structure profile but distinct melting temperatures. Different methods demonstrated that all PLDs cleave sphingomyelin and lysophosphatidylcholine, but L. gaucho and L. laeta PLDs excelled. L. gaucho PLD showed greater "in vitro" hemolytic activity. L. gaucho and L. laeta PLDs were more lethal in assays with mice and crickets. Molecular dynamics simulations correlated their biochemical activities with differences in sequences and conformations of specific surface loops, which play roles in protein stability and in modulating interactions with the membrane. Despite the high similarity, PLDs from L. gaucho and L. laeta venoms are more active than L. intermedia PLD, requiring special attention from physicians when these two species prevail in endemic regions.

Brown Spider Venom Phospholipase-D Activity upon Different Lipid Substrates.

Brown spider envenomation results in dermonecrosis, characterized by an intense inflammatory reaction. The principal toxins of brown spider venoms are phospholipase-D isoforms, which interact with different cellular membrane components, degrade phospholipids, and generate bioactive mediators leading to harmful effects. The Loxosceles intermedia phospholipase D, LiRecDT1, possesses a loop that modulates the accessibility to the active site and plays a crucial role in substrate. In vitro and in silico analyses were performed to determine aspects of this enzyme's substrate preference. Sphingomyelin d18:1/6:0 was the preferred substrate of LiRecDT1 compared to other Sphingomyelins. Lysophosphatidylcholine 16:0/0:0 was preferred among other lysophosphatidylcholines, but much less than Sphingomyelin d18:1/6:0. In contrast, phosphatidylcholine d18:1/16:0 was not cleaved. Thus, the number of carbon atoms in the substrate plays a vital role in determining the optimal activity of this phospholipase-D. The presence of an amide group at C2 plays a key role in recognition and activity. In silico analyses indicated that a subsite containing the aromatic residues Y228 and W230 appears essential for choline recognition by cation-π interactions. These findings may help to explain why different cells, with different phospholipid fatty acid compositions exhibit distinct susceptibilities to brown spider venoms.

Evaluation of the effects of Loxosceles intermedia's venom in zebrafish.

The zebrafish is an animal model of increasing use in many biomedical fields of study, including toxicology, inflammation, and tissue regeneration. In this paper, we have investigated the inflammatory effects of Loxosceles intermedia's venom (LIV) on zebrafish, as well as the effects of Maresin 2 (Mar2) and Resolvin D5 (RvD5), two specialized pro-resolving mediators (SPMs), in the context of tissue regeneration after fin fold amputation. Furthermore, increasing concentrations of LIV (250-2000 ng) were assayed for their haemolytic effects in vitro, and, afterwards, the same concentrations were evaluated in vivo, when injected intraperitoneally. LIV caused haemolysis in human red blood cells (RBCs), but not in zebrafish RBCs. The survival curve was also not altered by LIV injection, regardless of venom dosage. Histological analysis of renal and hepatic tissues, as well as the whole animal, revealed no pathological differences between LIV-injected and PBS-injected groups. Fin fold regeneration was not altered between LIV-injected and control groups, nor in the presence of MaR2 and RvD5. Results of swimming behavioral analysis also did not differ between groups. Moreover, in silico data indicated differences between human and zebrafish cell membrane lipid constitutions, such as in phospholipases D preferred substrates, that could lead to the protection of zebrafish against LIV. Although our data implies that zebrafish cannot be used as a toxicological model for LIV studies, the absence of observed toxicological effects paves the way for the comprehension of the venom's mechanism of action in mammals and the fundamental evolutionary processes involved.

Production of a novel recombinant brown spider hyaluronidase in baculovirus-infected insect cells.

Hyaluronidases are low expressed toxins of brown spider venoms, but, as highly active molecules, they present an important role as spreading factors. By degrading extracellular matrix components, these enzymes favor the diffusion of toxins in the affected tissue and at systemic level. Here, a novel isoform of hyaluronidase of Loxosceles intermedia Mello-Leitão (1934) venom was cloned, expressed in a baculovirus-insect cell expression system and fully active purified. This recombinant enzyme, named LiHyal2 (Loxosceles intermedia Hyaluronidase isoform 2), shares high identity with hyaluronidases of other spiders and scorpions. The catalytic and sugar binding amino acid residues are conserved in LiHyal2, human, and honeybee venom hyaluronidases and the molecular model of LiHyal2 shares major similarities with their crystal structures, including the active site. LiHyal2 was expressed as a 45 kDa protein and degraded hyaluronic acid (HA) and chondroitin sulphate as demonstrated by HA zymography and agarose gel electrophoresis. Lectin blot analysis revealed that LiHyal2 is post-translationally modified by the addition of high mannose N-linked carbohydrates. In vivo experiments showed that LiHyal2 potentialize dermonecrosis and edema induced by a recombinant phospholipase-D (PLD) of L. intermedia venom, as well as enhance the increase in capillary permeability triggered by this PLD, indicating that these toxins act synergistically during envenomation. Altogether, these results introduce a novel approach to express spider recombinant toxins, contribute to the elucidation of brown spider venom mechanisms and add to the development of a more specific treatment of envenomation victims.

Effects of copaiba oil on dermonecrosis induced by Loxosceles intermedia venom.

BACKGROUND: Accidents caused by spiders of the genus Loxosceles constitute an important public health problem in Brazil. The venom of Loxosceles sp induces dermonecrosis at the bite site and systemic disease in severe cases. Traditional medicine based on plant-derived products has been proven to reduce the local effects of envenomation. The present study verified the healing effects of copaiba oil on lesions induced by the venom of L. intermedia. METHODS: Cutaneous lesions were induced on the backs of rabbits by intradermal injection of L. intermedia venom. Copaiba oil was applied topically 6 hours after injection; the treatment was repeated for 30 days, after which animal skins were removed and processed for histopathological analysis. Blood samples were also collected before and 24 hours after venom inoculation to measure the hematological parameters. RESULTS: Compared to the control group, the platelet count was reduced significantly in all groups inoculated with venom, accompanied by a decreased number of heterophils in the blood. The minimum necrotic dose (MND) was defined as 2.4 µg/kg. Topical treatment with copaiba oil demonstrated a differentiated healing profile: large skin lesions were observed 10 days after venom inoculation, whereas formation of a thick crust, without scarring was observed 30 days after venom inoculation. Histopathological analysis showed no significant difference after treatment. Nevertheless, the copaiba oil treatment induced a collagen distribution similar to control skin, in marked contrast to the group that received only the spider venom injection. CONCLUSIONS: We conclude that copaiba oil may interfere in the healing process and thus propose it as a possible topical treatment for cutaneous lesions induced by L. intermedia venom.

Effects of copaiba oil on dermonecrosis induced by Loxosceles intermedia venom

Background:Accidents caused by spiders of the genus Loxosceles constitute an important public health problem in Brazil. The venom of Loxosceles sp induces dermonecrosis at the bite site and systemic disease in severe cases. Traditional medicine based on plant-derived products has been proven to reduce the local effects of envenomation. The present study verified the healing effects of copaiba oil on lesions induced by the venom of L. intermedia.Methods:Cutaneous lesions were induced on the backs of rabbits by intradermal injection of L. intermedia venom. Copaiba oil was applied topically 6 hours after injection; the treatment was repeated for 30 days, after which animal skins were removed and processed for histopathological analysis. Blood samples were also collected before and 24 hours after venom inoculation to measure the hematological parameters.Results:Compared to the control group, the platelet count was reduced significantly in all groups inoculated with venom, accompanied by a decreased number of heterophils in the blood. The minimum necrotic dose (MND) was defined as 2.4 μg/kg. Topical treatment with copaiba oil demonstrated a differentiated healing profile: large skin lesions were observed 10 days after venom inoculation, whereas formation of a thick crust, without scarring was observed 30 days after venom inoculation. Histopathological analysis showed no significant difference after treatment. Nevertheless, the copaiba oil treatment induced a collagen distribution similar to control skin, in marked contrast to the group that received only the spider venom injection.Conclusions:We conclude that copaiba oil may interfere in the healing process and thus propose it as a possible topical treatment for cutaneous lesions induced by L. intermedia venom.(AU)

Effects of copaiba oil on dermonecrosis induced by Loxosceles intermedia venom

Accidents caused by spiders of the genus Loxosceles constitute an important public health problem in Brazil. The venom of Loxosceles sp induces dermonecrosis at the bite site and systemic disease in severe cases. Traditional medicine based on plant-derived products has been proven to reduce the local effects of envenomation. The present study verified the healing effects of copaiba oil on lesions induced by the venom of L. intermedia. Methods: Cutaneous lesions were induced on the backs of rabbits by intradermal injection of L. intermedia venom. Copaiba oil was applied topically 6 hours after injection; the treatment was repeated for 30 days, after which animal skins were removed and processed for histopathological analysis. Blood samples were also collected before and 24 hours after venom inoculation to measure the hematological parameters. Results: Compared to the control group, the platelet count was reduced significantly in all groups inoculated with venom, accompanied by a decreased number of heterophils in the blood. The minimum necrotic dose (MND) was defined as 2.4 μg/kg. Topical treatment with copaiba oil demonstrated a differentiated healing profile: large skin lesions were observed 10 days after venom inoculation, whereas formation of a thick crust, without scarring was observed 30 days after venom inoculation. Histopathological analysis showed no significant difference after treatment. Nevertheless, the copaiba oil treatment induced a collagen distribution similar to control skin, in marked contrast to the group that received only the spider venom injection. Conclusions: We conclude that copaiba oil may interfere in the healing process and thus propose it as a possible topical treatment for cutaneous lesions induced by L. intermedia venom.(AU)

Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D.

Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.

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.

Characterization of Brown spider (Loxosceles intermedia) hemolymph: cellular and biochemical analyses.

This is the first study on the hemolymph from a spider of the Loxosceles genus. These animals are responsible for a great number of envenomation cases worldwide. Several studies on Loxosceles venoms have been published, and the knowledge about the venom and its toxins is considerable, not only regarding the biological and biochemical characterization, but also regarding structural, genetic and phylogenetic approaches. However, the literature on Loxosceles hemolymph is nonexistent. The main goal of the present study was to characterize biochemically the hemolymph content, and especially, to identify its different hemocytes. Moreover, many papers have already shown molecules whose source is the hemolymph and their very interesting activities and biomedical applications, for example, antifungal and antibacterial activities. A 2D-SDS-PAGE of brown spider hemolymph showed approximately 111 spots for pH 3-10 and 150 spots for pH 4-7. A lectin-blotting assay showed that hemolymph carbohydrate residues were similar to those found in venom. Several types of TAG and DAG phospholipids were found in the hemolymph and characterized by HPTLC and mass spectrometry. Four different hemocytes were characterized in Loxosceles intermedia hemolymph: prohemocyte, plasmatocyte, granulocyte and adipohemocyte. This paper opens new possibilities on toxinology, studying an unknown biological material, and it characterizes a source of molecules with putative biotechnological applications.

A novel ICK peptide from the Loxosceles intermedia (brown spider) venom gland: cloning, heterologous expression and immunological cross-reactivity approaches.

The venom of a Loxosceles spider is composed of a complex mixture of biologically active components, consisting predominantly of low molecular mass molecules (3-45 kDa). Transcriptome analysis of the Loxosceles intermedia venom gland revealed ESTs with similarity to the previously described LiTx peptides. Sequences similar to the LiTx3 isoform were the most abundant, representing approximately 13.9% of all ESTs and 32% of the toxin-encoding messengers. These peptides are grouped in the ICK (Inhibitor Cystine Knot) family, which contains single chain molecules with low molecular mass (3-10 kDa). Due to their high number of cysteine residues, ICK peptides form intramolecular disulfide bridges. The aims of this study were to clone and express a novel ICK peptide isoform, as well as produce specific hyperimmune serum for immunoassays. The corresponding cDNA was amplified by PCR using specific primers containing restriction sites for the XhoI and BamHI enzymes; this PCR product was then ligated in the pET-14b vector and transformed into E. coli AD494 (DE3) cells. The peptide was expressed by IPTG induction for 4 h at 30 °C and purified by affinity chromatography with Ni-NTA resin. Hyperimmune serum to the recombinant peptide was produced in rabbits and was able to specifically recognize both the purified recombinant peptide and the native form present in the venom. Furthermore, the recombinant peptide was recognized by antisera raised against L. intermedia, L. gaucho and L. laeta whole venoms. The recombinant peptide obtained will enable future studies to characterize its biological activity, as well as investigations regarding possible biotechnological applications.

Evaluation of the efficacy of vacuum cleaners for the integrated control of brown spider Loxosceles intermedia

Some venomous spiders of the genus Loxosceles can reach high population densities inside and around houses. In Brazil, most spider accidents are related to Loxosceles intermedia. Control of loxoscelism should utilize integrated pest management tools, such as vacuum cleaners, to eliminate egg sacs, webs and spiders. The present study tested the efficacy of one type of vacuum cleaner (for professional and domestic use) in the control of L. intermedia populations. Cockroaches (Pycnoscelus surinamensis) were used in some tests for comparison. Vacuuming using standard accessories or a paper tube resulted in the death of all female (n=60), male (n=60), young (n=60) and just-hatched (n=60) L. intermedia, and all egg sacs (n=5) were destroyed. The removal of the plastic plate present at the bottom of the vacuuming tube inside the machine allowed some spiders to survive the vacuuming process. When kept inside a vacuum bag full of dust and debris, adult females (n=10) survived for 10 days; however, significant mortality was observed among male (n=10) and young individuals (n=10). Addition of cornstarch to the vacuum bag did not affect the spiders (n=20). Vacuum cleaners, such as the one used in the present investigation, are promising tools for integrated management of L. intermedia and other spiders in domestic environments.

Evaluation of the efficacy of vacuum cleaners for the integrated control of brown spider Loxosceles intermedia

Some venomous spiders of the genus Loxosceles can reach high population densities inside and around houses. In Brazil, most spider accidents are related to Loxosceles intermedia. Control of loxoscelism should utilize integrated pest management tools, such as vacuum cleaners, to eliminate egg sacs, webs and spiders. The present study tested the efficacy of one type of vacuum cleaner (for professional and domestic use) in the control of L. intermedia populations. Cockroaches (Pycnoscelus surinamensis) were used in some tests for comparison. Vacuuming using standard accessories or a paper tube resulted in the death of all female (n=60), male (n=60), young (n=60) and just-hatched (n=60) L. intermedia, and all egg sacs (n=5) were destroyed. The removal of the plastic plate present at the bottom of the vacuuming tube inside the machine allowed some spiders to survive the vacuuming process. When kept inside a vacuum bag full of dust and debris, adult females (n=10) survived for 10 days; however, significant mortality was observed among male (n=10) and young individuals (n=10). Addition of cornstarch to the vacuum bag did not affect the spiders (n=20). Vacuum cleaners, such as the one used in the present investigation, are promising tools for integrated management of L. intermedia and other spiders in domestic environments.(AU)

Evaluation of fixative solutions for ultrastructural analysis of brown spider Loxosceles intermedia (araneae: sicariidae) tissues

In view of the widely varying compositions of fixative solutions used for studying spiders, five different fixative formulas were tested for fixing male brown-spider (Loxosceles intermedia) gonad tissues. The brown spider represents a public health problem in Curitiba (Paraná State, Brazil). Morphological study of its gonads may aid in understanding the reproductive strategies of this species, and possibly in developing a reproduction control program. The fixatives tested contained glutaraldehyde alone or combined with paraformaldehyde, and the buffers cacodylate or phosphate, with or without the addition of sucrose or sodium chloride as osmolytes. Those containing 2.5% glutaraldehyde and 2% paraformaldehyde in 100 mM phosphate buffer with 200 mM sucrose, or in 200 mM sodium cacodylate, satisfactorily preserved mitochondria, the Golgi apparatus, and the membranes in general. These formulas were nearly isosmotic (439 mOsm/kg H2O and 455 mOsm/kg H2O respectively) to brown spider hemolymph (478 mOsm/kg H2O). With respective to the fixative agents, a glutaraldehyde-paraformaldehyde combination resulted in optimal fixation of Loxosceles intermedia cells. For other species of spiders, hemolymph osmolality should be considered, but the fixative formulas cited above would also probably yield good results.

Dada a variabilidade na composição de soluções fixadoras utilizadas em aranhas, cinco diferentes fixadores foram elaborados para a análise ultra-estrutural dos tecidos da aranha marrom Loxosceles intermedia. A aranha marrom representa um problema de saúde pública na cidade de Curitiba, e o estudo morfológico de suas gônadas pode auxiliar na compreensão de suas estratégias reprodutivas e, possivelmente, no desenvolvimento de um programa de controle da sua população. As fórmulas usadas continham glutaraldeído com ou sem paraformaldeído, tampão cacodilato ou fosfato, e NaCl ou sacarose como osmólitos. As soluções fixadoras compostas por 2.5% glutaraldeído e 2% paraformaldeído, em tampão fosfato com adição de sacarose ou em 200 mM cacodilato de sódio, preservaram bem estruturas como mitocôndrias, aparelho de Golgi e membranas em geral. Os tampões são praticamente isosmóticos (439 mOsm/kg H2O e 455 mOsm/kg H2O, respectivamente) à hemolinfa da aranha marrom (478 mOsm/kg H2O). Ainda, com relação aos agentes fixadores, a combinação do glutaraldeído e paraformaldeído levou a uma melhor preservação das células. Para outras espécies de aranhas, a osmolalidade da hemolinfa deve ser medida e considerada, mas as fórmulas acima citadas podem ser testadas, com chance de sucesso.

Tropical House Gecko (Hemidactylus mabouia) predation on brown spiders (Loxosceles intermedia)

Brown spiders (Loxosceles spp.) are venomous arachnids, successfully adapted to urban habitats in Brazil. Loxoscelism became a serious public health problem in Paraná State, especially at the capital Curitiba, where the most abundant species is Loxosceles intermedia. Hemidactylus mabouia (Gekkonidae) lizards are synanthropic predators of arthropods. In this paper, we describe the predatory behavior of the Tropical House Gecko H. mabouia on L. intermedia under laboratory conditions. Twelve geckos were observed, and all of them fed on brown spiders (n=123 observations). The attack consisted of a fast run followed by one bite on the spiders abdomen or legs. The geckos did not attack L. intermedia anterior body parts, probably due to the fangs present in this region. Two Hemidactylus individuals were killed by L. intermedia bites: during a predatory encounter, and by an induced bite on a restrained lizard. The observations summarized in this paper show that H. mabouia could be used in the biological control of Loxosceles populations in human dwellings. However, additional field studies are necessary to quantify the impact of H. mabouia predation on urban populations of L. intermedia and other species of the same genus.