RESUMO

Loxoscelism is the pathological condition triggered by a brown spider bite. The venom of these spiders is rich in phospholipases D (PLDs), which can induce virtually all local and systemic manifestations. Recombinant mutated PLDs from clinically relevant Loxosceles species in South America have been investigated as potential antigens to develop novel therapeutic strategies for loxoscelism. However, certain gaps need to be addressed before a clinical approach can be implemented. In this study, we examined the potential of these recombinant mutated PLDs as antigens by testing some variations in the immunization scheme. Furthermore, we evaluated the efficacy of the produced antibodies in neutralizing the nephrotoxicity and sphingomyelinase activity of brown spider venoms. Our findings indicate that the number of immunizations has a greater impact on the effectiveness of neutralization compared to the amount of antigen. Specifically, two or three doses were equally effective in reducing dermonecrosis and edema. Additionally, three immunizations proved to be more effective in neutralizing mice lethality than one or two. Moreover, immunizations mitigated the signs of kidney injury, a crucial aspect given that acute renal failure is a serious systemic complication. In vitro inhibition of the sphingomyelinase activity of Loxosceles venoms, a key factor in vivo toxicity, was nearly complete after incubation with antibodies raised against these antigens. These findings underscore the importance of implementing an effective immunization scheme with multiple immunizations, without the need for high antigen doses, and enhances the spectrum of neutralization exhibited by antibodies generated with these antigens. In summary, these results highlight the strong potential of these antigens for the development of new therapeutic strategies against cutaneous and systemic manifestations of loxoscelism.

Assuntos

Fosfolipase D , Proteínas Recombinantes , Venenos de Aranha , Animais , Fosfolipase D/imunologia , Fosfolipase D/genética , Venenos de Aranha/imunologia , Camundongos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Picada de Aranha/imunologia , Aranha Marrom Reclusa/imunologia , Feminino , Antígenos/imunologia , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/imunologia , Anticorpos Neutralizantes , Antivenenos/imunologia , Antivenenos/administração & dosagem , Modelos Animais de Doenças , Imunização , Diester Fosfórico Hidrolases

RESUMO

In the original publication [...].

RESUMO

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.

Assuntos

Fosfolipase D , Venenos de Aranha , Aranhas , Animais , Camundongos , Diester Fosfórico Hidrolases , Venenos de Aranha/química , América do Sul

RESUMO

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.

Assuntos

Antineoplásicos , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/metabolismo , Sertralina/farmacologia , Sertralina/uso terapêutico , Biomarcadores Tumorais/genética , Antineoplásicos/uso terapêutico , Células MCF-7

RESUMO

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.

Assuntos

Fosfolipase D , Venenos de Aranha , Aranhas , Animais , Esfingomielinas/metabolismo , Diester Fosfórico Hidrolases/química , Fosfolipase D/metabolismo , Venenos de Aranha/química , Fosfolipídeos/metabolismo , Lisofosfatidilcolinas , Aranhas/metabolismo

RESUMO

Bites of Loxosceles spiders can lead to a set of clinical manifestations called loxoscelism, and are considered a public health problem in many regions. The signs and symptoms of loxoscelism are divided into cutaneous and systemic forms. The former is more frequent and includes signs of envenoming at the bite site or neighboring regions. Systemic loxoscelism, although much less frequent, is associated with complications, and can even lead to death. It may include intravascular hemolysis, acute renal failure, and thrombocytopenia. Loxosceles venoms are enriched with phospholipases D (PLDs), which are a family of isoforms found at intra-species and inter-species levels. Under experimental conditions, these enzymes reproduce the main clinical signs of loxoscelism, including an exacerbated inflammatory response at the bite site and dermonecrosis, as well as thrombocytopenia, intravascular hemolysis, and acute renal failure. The role of PLDs in cutaneous loxoscelism was described over forty years ago, when studies identified and purified toxins featured as sphingomyelinase D. More recently, the production of recombinant PLDs and discoveries about their structure and mechanism has enabled a deeper characterization of these enzymes. In this review, we describe these biochemical and functional features of Loxosceles PLDs that determine their involvement in systemic loxoscelism.

Assuntos

Fosfolipase D , Picada de Aranha , Venenos de Aranha , Aranhas , Trombocitopenia , Animais , Hemólise , Diester Fosfórico Hidrolases/toxicidade , Fosfolipase D/química , Venenos de Aranha/toxicidade , Venenos de Aranha/química , Isoformas de Proteínas , Aranhas/química , Picada de Aranha/complicações

RESUMO

Loxoscelism is the clinical condition triggered after the bite of spiders of the genus Loxosceles. The main species involved in accidents in South America are L. intermedia, L. laeta, and L. gaucho. The only specific treatment is the anti-Loxosceles serum produced with crude venoms. As phospholipases D (PLDs) trigger most of the effects observed in accidents, we developed and evaluated second-generation sera using mutated PLDs as antigens. Three isoforms of PLDs with site-directed mutations without biological activities were used for rabbit immunizations: D32A-E34A (L. gaucho), W230A (L. intermedia), and H12A-H47A (L. laeta). Sera were produced using crude venoms of three species of Loxosceles enriched with mutated recombinant PLDs (MIX) or using only mutated PLDs (REC). Immunizations stimulated the immune system from the second immunization with higher antibody production in the REC group. In vivo neutralization assays demonstrated that both sera reduced edema and dermonecrosis caused by Loxosceles intermedia crude venom. Follow-up of animals during the immunization protocols and in the neutralization assays demonstrated that the mutated proteins and the sera are safe. Results demonstrate the potential of using mutated recombinant PLDs in total or partial replacement of Loxosceles venoms in animal immunizations to produce anti-Loxosceles sera for treatments of Loxoscelism.

RESUMO

Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism.

Assuntos

Aranha Marrom Reclusa , Proteínas Mutantes/imunologia , Fosfolipase D/imunologia , Picada de Aranha/imunologia , Picada de Aranha/terapia , Vacinas/imunologia , Acidentes , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Antivenenos/sangue , Antivenenos/imunologia , Biomarcadores , Modelos Animais de Doenças , Imunogenicidade da Vacina , Contagem de Leucócitos , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Testes de Neutralização , Fosfolipase D/química , Fosfolipase D/genética , Coelhos , Picada de Aranha/diagnóstico , Picada de Aranha/prevenção & controle , Venenos de Aranha/imunologia , Relação Estrutura-Atividade , Resultado do Tratamento , Vacinação , Vacinas/administração & dosagem

RESUMO

Accidents caused by the bites of brown spiders (Loxosceles) generate a clinical condition that often includes a threatening necrotic skin lesion near the bite site along with a remarkable inflammatory response. Systemic disorders such as hemolysis, thrombocytopenia, and acute renal failure may occur, but are much less frequent than the local damage. It is already known that phospholipases D, highly expressed toxins in Loxosceles venom, can induce most of these injuries. However, this spider venom has a great range of toxins that probably act synergistically to enhance toxicity. The other protein classes remain poorly explored due to the difficulty in obtaining sufficient amounts of them for a thorough investigation. They include astacins (metalloproteases), serine proteases, knottins, translationally controlled tumor proteins (TCTP), hyaluronidases, allergens and serpins. It has already been shown that some of them, according to their characteristics, may participate to some extent in the development of loxoscelism. In addition, all of these toxins present potential application in several areas. The present review article summarizes information regarding some functional aspects of the protein classes listed above, discusses the directions that could be taken to materialize a comprehensive investigation on each of these toxins as well as highlights the importance of exploring the full venom repertoire.

RESUMO

Brown spider (genus Loxosceles) venoms are mainly composed of protein toxins used for predation and defense. Bites of these spiders most commonly produce a local dermonecrotic lesion with gravitational spread, edema and hemorrhage, which together are defined as cutaneous loxoscelism. Systemic loxoscelism, such as hematological abnormalities and renal injury, are less frequent but more lethal. Some Loxosceles venom toxins have already been isolated and extensively studied, such as phospholipases D (PLDs), which have been recombinantly expressed and were proven to reproduce toxic activities associated to the whole venom. PLDs have a notable potential to be engineered and converted in non-toxic antigens to produce a new generation of antivenoms or vaccines. PLDs also can serve as tools to discover inhibitors to be used as therapeutic agents. Other Loxosceles toxins have been identified and functionally characterized, such as hyaluronidases, allergen factor, serpin, TCTP and knottins (ICK peptides). All these toxins were produced as recombinant molecules and are biologically active molecules that can be used as tools for the potential development of chemical candidates to tackle many medical and biological threats, acting, for instance, as antitumoral, insecticides, analgesic, antigens for allergy tests and biochemical reagents for cell studies. In addition, these recombinant toxins may be useful to develop a rational therapy for loxoscelism. This review summarizes the main candidates for the development of drugs and biotechnological inputs that have been described in Brown spider venoms.