Role of the complement system in kidney cell death induced by Loxosceles venom Sphingomyelinases D.

Envenomation by Loxosceles spiders can result in local and systemic pathologies. Systemic loxoscelism, which can lead to death, is characterized by intravascular hemolysis, platelet aggregation, and acute kidney injury. Sphingomyelinase D (SMase D) in Loxosceles spider venom is responsible for both local and systemic pathologies, and has been shown to induce metalloprotease activity. As the complement system is involved in many renal pathologies and is involved in hemolysis in systemic loxoscelism, the aim of this study was to investigate its role and the role of complement regulators and metalloproteases in an in vitro model of Loxosceles venom induced renal pathology. We investigated the effects of the venom/SMase D and the complement system on the HK-2 kidney cell line. Using cell viability assays, western blotting, and flow cytometry, we show that human serum, as a source of complement, enhanced the venom/SMase D induced cell death and the deposition of complement components and properdin. Inhibitors for ADAM-10 and ADAM-17 prevented the venom induced release of the of the complement regulator MCP/CD46 and reduced the venom/SMase D induced cell death. Our results show that the complement system can contribute to Loxosceles venom induced renal pathology. We therefore suggest that patients experiencing systemic loxoscelism may benefit from treatment with metalloproteinase inhibitors and complement inhibitors, but this proposition should be further analyzed in future pre-clinical and clinical assays.

Utilization of dapsone and hemoglobin in the epithelial skin regeneration therapy of cutaneous loxoscelism: A case report and integrative literature review

ABSTRACT BACKGROUND: Loxosceles spp are arthropods found worldwide. Its bite may produce cutaneous loxoscelism (necrotic or edematous) or cutaneous-visceral loxoscelism. Depending on their severity and location, cutaneous forms are managed with local cold application and systemic administration of antihistamines, corticosteroids, antibiotics, polymorphonuclear inhibitors, and analgesics. OBJECTIVE: This study aimed to report a case of cutaneous loxoscelism and to identify the main dermatological manifestations associated with the Loxosceles spp bite. DESIGN AND SETTING: This case report and literature review was conducted in a Mexican university. METHODS: A detailed report on the medical management of a patient with cutaneous loxoscelism treated at the emergency department of a public hospital was published. Scopus, PubMed, Web of Science, and Google Scholar databases were searched to identify articles reporting cutaneous loxoscelism. The following keywords were used during the database search: "loxoscelism" OR "spider bite," OR "loxosceles" OR "loxosceles species" OR "loxosceles venom" OR "loxoscelism case report" AND "cutaneous" OR "dermonecrotic arachnidism." RESULTS: A 62-year-old female patient with cutaneous loxoscelism was treated with systemic dapsone and local heparin spray. Eighteen studies with 22 clinical cases were included in this systematic review. Of the 22 patients, 12 (54.5%) were men. L. rufescens was the predominant spider species. CONCLUSIONS: The administration of dapsone and heparin for the management of cutaneous loxoscelism demonstrated success in this case, with no sequelae observed. In general, the literature review indicated favorable outcomes in patients treated with antimicrobials and corticosteroids, with continuous healing of skin lesions. SYSTEMATIC REVIEW REGISTRATION: PROSPERO ID CRD42023422424 (https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023422424).

Role of the complement system in kidney cell death induced by Loxosceles venom Sphingomyelinases D

Envenomation by Loxosceles spiders can result in local and systemic pathologies. Systemic loxoscelism, which can lead to death, is characterized by intravascular hemolysis, platelet aggregation, and acute kidney injury. Sphingomyelinase D (SMase D) in Loxosceles spider venom is responsible for both local and systemic pathologies, and has been shown to induce metalloprotease activity. As the complement system is involved in many renal pathologies and is involved in hemolysis in systemic loxoscelism, the aim of this study was to investigate its role and the role of complement regulators and metalloproteases in an in vitro model of Loxosceles venom induced renal pathology. We investigated the effects of the venom/SMase D and the complement system on the HK-2 kidney cell line. Using cell viability assays, western blotting, and flow cytometry, we show that human serum, as a source of complement, enhanced the venom/SMase D induced cell death and the deposition of complement components and properdin. Inhibitors for ADAM-10 and ADAM-17 prevented the venom induced release of the of the complement regulator MCP/CD46 and reduced the venom/SMase D induced cell death. Our results show that the complement system can contribute to Loxosceles venom induced renal pathology. We therefore suggest that patients experiencing systemic loxoscelism may benefit from treatment with metalloproteinase inhibitors and complement inhibitors, but this proposition should be further analyzed in future pre-clinical and clinical assays.

Clinical Evolution After Administering Antivenom in Patients With Loxoscelism.

Spiders are the most numerous arthropods of the arachnid class. More than 45 thousand species of spiders have been identified, and only a few are dangerous to humans. Among them, the "violin spider" or "brown spider" of the genus Loxosceles (family Sicariidae) has a worldwide distribution, and its bite can cause loxoscelism. Initial treatment of a Loxosceles spider bite includes application of local cold, rest, elevation of the extremity if possible, and systemic pharmacotherapy with antihistamines, corticosteroids, antibiotics, polymorphonuclear inhibitors, and analgesics or nonsteroidal anti-inflammatory drugs. During cutaneous or systemic loxoscelism, administration of Loxosceles antivenom (immunoglobulin (Ig)G F(ab')2 fragments) may be indicated to prevent progression to severe systemic phases. In this manuscript, we present three cases of patients with loxoscelism treated with the fabotherapeutic Reclusmyn®, developed and manufactured in Mexico. Two patients had a satisfactory outcome without severe skin or systemic damage. Only one patient with loxoscelism, despite early initiation of antivenom, had extensive skin lesions that healed satisfactorily, leaving only a non-disabling scar. Due to the global presence of this clinical problem, further studies are needed to establish local and general guidelines for the treatment and prevention of loxoscelism. This will allow health professionals to provide more efficient and higher quality medical care and feel supported in their decisions.

A prokaryote system optimization for rMEPLox expression: A promising non-toxic antigen for Loxosceles antivenom production.

Loxoscelism is the most dangerous araneism form in Brazil and antivenom therapy is the recommended treatment. Antivenom is produced by horse immunization with Loxosceles spider venom, which is toxic for the producer animal. Moreover, due to the high amount of venom required for horse hyperimmunization, new strategies for antigens obtention have been proposed. In this sense, our research group has previously produced a non-toxic recombinant multiepitopic protein derived from Loxosceles toxins (rMEPLox). rMEPLox was a successful immunogen, being able to induce the production of neutralizing antibodies, which could be used in the Loxoscelism treatment. However, rMEPLox obtention procedure requires optimization, as its production needs to be scaled up to suit antivenom manufacture. Therefore, an effective protocol development for rMEPlox production would be advantageous. To achieve this objective, we evaluated the influence of different cultivation conditions for rMEPLox optimum expression. The optimum conditions to obtain large amounts of rMEPlox were defined as the use of C43(DE3)pLysS as a host strain, 2xTY medium, 0.6 mM IPTG, biomass pre induction of OD600nm = 0.4 and incubation at 30 °C for 16 h. Following the optimized protocol, 39.84 mg/L of soluble rMEPLox was obtained and tested as immunogen. The results show that the obtained rMEPLox preserved the previously described immunogenicity, and it was able to generate antibodies that recognize different epitopes of the main Loxosceles venom toxins, which makes it a promising candidate for the antivenom production for loxoscelism treatment.

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.

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.

Microcirculation abnormalities provoked by Loxosceles spiders' envenomation.

Loxoscelism is caused by envenomation by spiders from Loxosceles genus. Clinical symptoms only appear a few hours after envenomation and can evolve in local reactions, such as dermonecrosis, and systemic reactions, including intravascular haemolysis, intravascular coagulation and renal failure. Considering that alterations in the microcirculatory network are involved in the pathogenesis of different diseases, including the inflammatory process, the aim of this study was to investigate the action of venoms of males and females of Loxosceles intermedia and Loxosceles laeta on the microcirculatory network and examine the systemic production of inflammatory mediators in a murine model of loxoscelism. We observed that during systemic envenomation, the alterations in the microcirculation include increase in the number of rolling cells, which was more intense in animals injected with female Loxosceles spider venoms. This positively correlated with increase in TNF-α and NO serum levels, induction of which was higher by female venoms when compared with male venoms. The increase of leukocytes rolling was not accompanied by increase of cell adhesion. The absence of leukocyte extravasation may explain why in mice, in contrast to humans, no cutaneous loxoscelism occurs. Thus, targeting the neutrophil adhesion and extravasation in Loxosceles envenomed patients may prevent cutaneous pathology.

Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms.

We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.