Evaluation of mesenchymal cells and dapsone for the treatment of dermonecrotic wounds caused by Loxosceles laeta venom in rabbits / Avaliação de células mesenquimais e dapsona para o tratamento de feridas dermonecróticas causadas pelo veneno de Loxosceles laeta em coelhos

This study aimed to evaluate the efficacy of mesenchymal stem cells (MSC), alone or associated with dapsone (DAP), in treating dermonecrotic wounds caused by Loxosceles laeta venom. Twenty-five male rabbits were distributed into five groups. Negative control received ultrapure water (C-), whilst all other groups were injected with 20 μg of L. laeta venom. After 4 hours, each group received one of the following treatments: PBS (C+), DAP, MSC, and DAP+MSC. Animals were evaluated daily and photographic records made for analysis of wound area. Twelve days after, animals were euthanized and skin samples removed for histological analysis. We observed that DAP showed the best percentage of wound contraction at day 3. In the treatments using MSCs, a negative value of wound contraction was observed for the isolated MSCs, as well as a lower contraction value for the association of the MSC + DAP when compared to PBS, probably, by the increase in initial infammation after the application of stem cells, due to the fact that MSCs secrete a broad spectrum of bioactive molecules such as cytokines and growth factors that favor regeneration. Histologically, it was observed that animals of C+ showed extensive areas of necrosis, ulcers, neutrophilic infiltrate, and mineralization. Collagen deposition showed increase in MSC+DAP treatment, however vascularization remained unchanged. This is the first report using MSC and MSC+DAP as a treatment for cutaneous loxoscelism and more studies are needed to determine its use as an alternative therapy for dermonecrotic lesions caused by Loxosceles spider.

Este estudo teve como objetivo avaliar a eficácia das células-tronco mesenquimais (CTMs), isoladas ou associadas à dapsona (DAP), no tratamento de feridas dermonecróticas causadas pelo veneno de Loxosceles laeta. Vinte e cinco coelhos machos foram distribuídos em cinco grupos. O controle negativo recebeu água ultrapura (C-), enquanto todos os outros grupos foram injetados com 20 μg de veneno de L. laeta. Após 4 horas, cada grupo recebeu um dos seguintes tratamentos: PBS (C+), DAP, CTMs e DAP + CTMs. Os animais foram avaliados diariamente durante 12 dias, e feitos registros fotográficos para análise da ferida e no 12º dia, foram eutanasiados e, retiradas amostras de pele para análise histológica. Observou-se que a DAP apresentou o melhor percentual de contração da ferida no terceiro dia. Nos tratamentos com CTMs, observou-se uma contração negativa da ferida tanto para as CTMs isoladas, bem como a associação CTMs + DAP em relação ao PBS, possivelmente, pelo aumento da infamação inicial após a aplicação de células-tronco. Isso é devido ao fato de que as CTMs secretam um amplo espectro de moléculas bioativas como citocinas e fatores de crescimento que favorecem a regeneração. Histologicamente, observou-se que os animais de C+ apresentaram extensas áreas de necrose, úlceras, infiltrado neutrofílico, além de mineralização. Houve aumento de deposição de colágeno no tratamento CTMs + DAP, no entanto, a vascularização permaneceu inalterada. Este é o primeiro relato usando CTMs e CTMs + DAP como tratamento para loxoscelismo cutâneo e mais estudos são necessários para determinar seu uso como terapia alternativa para lesões demonecróticas causadas pela aranha Loxosceles.

Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins?

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.(AU)

Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins?

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.(AU)

Spider bites of medical significance in the Mediterranean area: misdiagnosis, clinical features and management

Despite the disrepute spiders have had for centuries, their bite is a rare occurrence. In the Mediterranean area, only two of the numerous known species are considered of medical significance: Latrodectus tredecimguttatus and Loxosceles rufescens. Spider bites have no pathognomonic signs or symptoms, therefore most diagnoses are presumptive; a spider bite can only be diagnosed when a spider (seen at the time of the bite) is collected and identified by an expert, since most physicians and patients are unable to recognize a certain spider species or distinguish spiders from other arthropods. Skin lesions of uncertain etiology are too often attributed to spider bites. In most cases, these are actually skin and soft-tissue infections, allergic reactions, dermatoses etc. Misdiagnosing a wound as a spider bite can lead to delays in appropriate care, cause adverse or even fatal outcomes and have medical-legal implications. Concerningly, misinformation on spider bites also affects the medical literature and it appears there is lack of awareness on current therapeutic indications for verified bites.(AU)

Spider bites of medical significance in the Mediterranean area: misdiagnosis, clinical features and management

Despite the disrepute spiders have had for centuries, their bite is a rare occurrence. In the Mediterranean area, only two of the numerous known species are considered of medical significance: Latrodectus tredecimguttatus and Loxosceles rufescens. Spider bites have no pathognomonic signs or symptoms, therefore most diagnoses are presumptive; a spider bite can only be diagnosed when a spider (seen at the time of the bite) is collected and identified by an expert, since most physicians and patients are unable to recognize a certain spider species or distinguish spiders from other arthropods. Skin lesions of uncertain etiology are too often attributed to spider bites. In most cases, these are actually skin and soft-tissue infections, allergic reactions, dermatoses etc. Misdiagnosing a wound as a spider bite can lead to delays in appropriate care, cause adverse or even fatal outcomes and have medical-legal implications. Concerningly, misinformation on spider bites also affects the medical literature and it appears there is lack of awareness on current therapeutic indications for verified bites.(AU)

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)

Alien spiders: First record of Loxosceles gaucho Gertsch, 1967 (Araneae: Sicariidae) in the Amazon region, Brazil

We present the first record for Loxosceles gaucho Gertsch, 1967 in the Amazonian region of Brazil. Four males, fifteen females and forty-nine immatures were collected in different places in Manaus, Amazonas, Brazil. This is the third species of Loxosceles reported in the Amazon region along with L. amazonica Gertsch, 1967, and L. similis Moenkhaus, 1898. This is the first record of an invasive species of a venomous animal in the state of Amazonas, Brazil, which is noteworthy due to its synanthropic habit, which increases the risk to the local population.

Alien spiders: First record of Loxosceles gaucho Gertsch, 1967 (Araneae: Sicariidae) in the Amazon region, Brazil

We present the first record for Loxosceles gaucho Gertsch, 1967 in the Amazonian region of Brazil. Four males, fifteen females and forty-nine immatures were collected in different places in Manaus, Amazonas, Brazil. This is the third species of Loxosceles reported in the Amazon region along with L. amazonica Gertsch, 1967, and L. similis Moenkhaus, 1898. This is the first record of an invasive species of a venomous animal in the state of Amazonas, Brazil, which is noteworthy due to its synanthropic habit, which increases the risk to the local population.(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)

Lesão dermonecrótica em um gato atribuída a envenenamento por Loxosceles - relato de caso / Dermonecrotic lesion in a cat attributed to Loxosceles envenomation – case report

Os acidentes causados por aranhas do gênero Loxosceles têm importância na clínica de pequenos animais, mesmo não havendo dados epidemiológicos desses ataques em animais de estimação, dada a gravidade da lesão e possíveis complicações sistêmicas resultantes, tornando indispensável maior conhecimento sobre a espécie e as consequências do envenenamento. O presente trabalho relata a presença de uma lesão dermonecrótica em um felino atribuída a aranha-marrom (Loxosceles sp). O atendimento ocorreu na clínica Empório de Bicho em Caxias do Sul, no estado do Rio Grande do Sul. O animal apresentava uma lesão necrótica em região perineal, com fibrina e secreção purulenta. O tratamento consistiu no uso clorexidina para limpeza, hidrogel, açúcar, Fitofix® e Dersani® como promotores de cicatrização. Após um mês de tratamento, o animal apresentava uma melhora satisfatória.

Accidents caused by spiders of the genus Loxosceles are important in the small animal clinic, even though there are no epidemiological data on these attacks in pets, given the severity of the lesion and possible systemic complications, it becomes necessary to know more about the species and the consequences of the poisoning. The present work reports the presence of a dermonecrotic lesion in a cat attributed to brown spider (Loxosceles sp). The patient had a necrotic lesion in the perineal region with fibrin and purulent secretion. The treatment consisted of using chlorhexidine for cleaning, hydrogel, sugar, Fitofix® and Dersani® as healing promoters. After one month of treatment, the animal showed a satisfactory improvement.

Lesão dermonecrótica em um gato atribuída a envenenamento por Loxosceles - relato de caso / Dermonecrotic lesion in a cat attributed to Loxosceles envenomation case report

Os acidentes causados por aranhas do gênero Loxosceles têm importância na clínica de pequenos animais, mesmo não havendo dados epidemiológicos desses ataques em animais de estimação, dada a gravidade da lesão e possíveis complicações sistêmicas resultantes, tornando indispensável maior conhecimento sobre a espécie e as consequências do envenenamento. O presente trabalho relata a presença de uma lesão dermonecrótica em um felino atribuída a aranha-marrom (Loxosceles sp). O atendimento ocorreu na clínica Empório de Bicho em Caxias do Sul, no estado do Rio Grande do Sul. O animal apresentava uma lesão necrótica em região perineal, com fibrina e secreção purulenta. O tratamento consistiu no uso clorexidina para limpeza, hidrogel, açúcar, Fitofix® e Dersani® como promotores de cicatrização. Após um mês de tratamento, o animal apresentava uma melhora satisfatória.(AU)

Accidents caused by spiders of the genus Loxosceles are important in the small animal clinic, even though there are no epidemiological data on these attacks in pets, given the severity of the lesion and possible systemic complications, it becomes necessary to know more about the species and the consequences of the poisoning. The present work reports the presence of a dermonecrotic lesion in a cat attributed to brown spider (Loxosceles sp). The patient had a necrotic lesion in the perineal region with fibrin and purulent secretion. The treatment consisted of using chlorhexidine for cleaning, hydrogel, sugar, Fitofix® and Dersani® as healing promoters. After one month of treatment, the animal showed a satisfactory improvement.(AU)

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Background Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.(AU)

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Background Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.

Long time not seen: Expanding the records of Loxosceles amazonica (Araneae: Sicariidae) in the Amazonas state, Brazil / Muito tempo sem ver: Expansão dos registros de Loxosceles amazonica (Araneae: Sicariidae) para o estado do Amazonas, Brasil

Spiders of the genus Loxosceles, commonly known as brown recluse spiders, can cause serious accidents in humans. Their venom has a powerful proteolytic and hemolytic action. Each year these spiders are the cause of a great number of araneism in Brazil. This work presents new records of Loxosceles amazonica for the municipal districts of Manaus and Iranduba, Amazonas, Brazil.(AU)

As aranhas do gênero Loxosceles, conhecidas como aranhas marrons, podem causar sérios acidentes em humanos. O veneno destas aranhas possui potente ação proteolítica e hemolítica. A cada ano estas aranhas são responsáveis pelo maior número de araneísmos no Brasil. Este trabalho apresenta novos registros de Loxosceles amazonica para o estado do Amazonas, Brasil.(AU)

Long time not seen: Expanding the records of Loxosceles amazonica (Araneae: Sicariidae) in the Amazonas state, Brazil / Muito tempo sem ver: Expansão dos registros de Loxosceles amazonica (Araneae: Sicariidae) para o estado do Amazonas, Brasil

Spiders of the genus Loxosceles, commonly known as brown recluse spiders, can cause serious accidents in humans. Their venom has a powerful proteolytic and hemolytic action. Each year these spiders are the cause of a great number of araneism in Brazil. This work presents new records of Loxosceles amazonica for the municipal districts of Manaus and Iranduba, Amazonas, Brazil.

As aranhas do gênero Loxosceles, conhecidas como aranhas marrons, podem causar sérios acidentes em humanos. O veneno destas aranhas possui potente ação proteolítica e hemolítica. A cada ano estas aranhas são responsáveis pelo maior número de araneísmos no Brasil. Este trabalho apresenta novos registros de Loxosceles amazonica para o estado do Amazonas, Brasil.

Highlights in the knowledge of brown spider toxins

Abstract Brown spiders are venomous arthropods that use their venom for predation and defense. In humans, bites of these animals provoke injuries including dermonecrosis with gravitational spread of lesions, hematological abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins (440 kDa). Characterization of the venom confirmed the presence of three highly expressed protein classes: phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels of expression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins), hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D family (also known as the dermonecrotic toxin) is the most studied class of brown spider toxins. This class of toxins single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure. The functional role of the hyaluronidase toxin as a spreading factor in loxoscelism has also been demonstrated. However, the biological characterization of other toxins remains unclear and the mechanism by which Loxosceles toxins exert their noxious effects is yet to be fully elucidated. The aim of this review is to provide an insight into brown spider venom toxins and toxicology, including a description of historical data already available in the literature. In this review article, the identification processes of novel Loxosceles toxins by molecular biology and proteomic approaches, their biological characterization and structural description based on x-ray crystallography and putative biotechnological uses are described along with the future perspectives in this field.

Highlights in the knowledge of brown spider toxins

Abstract Brown spiders are venomous arthropods that use their venom for predation and defense. In humans, bites of these animals provoke injuries including dermonecrosis with gravitational spread of lesions, hematological abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins (4-40 kDa). Characterization of the venom confirmed the presence of three highly expressed protein classes: phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels of expression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins), hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D family (also known as the dermonecrotic toxin) is the most studied class of brown spider toxins. This class of toxins single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure. The functional role of the hyaluronidase toxin as a spreading factor in loxoscelism has also been demonstrated. However, the biological characterization of other toxins remains unclear and the mechanism by which Loxosceles toxins exert their noxious effects is yet to be fully elucidated. The aim of this review is to provide an insight into brown spider venom toxins and toxicology, including a description of historical data already available in the literature. In this review article, the identification processes of novel Loxosceles toxins by molecular biology and proteomic approaches, their biological characterization and structural description based on x-ray crystallography and putative biotechnological uses are described along with the future perspectives in this field.(AU)

Highlights in the knowledge of brown spider toxins

Brown spiders are venomous arthropods that use their venom for predation and defense. In humans, bites of these animals provoke injuries including dermonecrosis with gravitational spread of lesions, hematological abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins (4-40 kDa). Characterization of the venom confirmed the presence of three highly expressed protein classes: phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels of expression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins), hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D family (also known as the dermonecrotic toxin) is the most studied class of brown spider toxins. This class of toxins single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure. The functional role of the hyaluronidase toxin as a spreading factor in loxoscelism has also been demonstrated. However, the biological characterization of other toxins remains unclear and the mechanism by which Loxosceles toxins exert their noxious effects is yet to be fully elucidated. The aim of this review is to provide an insight into brown spider venom toxins and toxicology, including a description of historical data already available in the literature. In this review article, the identification processes of novel Loxosceles toxins by molecular biology and proteomic approaches, their biological characterization and structural description based on x-ray crystallography and putative biotechnological uses are described along with the future perspectives in this field.(AU)

Secretoma no tratamento de lesão dermonecrótica induzida pelo veneno de Loxosceles intermedia

Objetivou-se avaliar os efeitos do secretoma, em feridas dermonecróticas em coelhos submetidos à injeção de veneno de Loxosceles intermedia. Foram utilizados 16 coelhos machos, adultos, Nova Zelândia, com peso médio de 2,0 kg, distribuídos em quatro grupos (n=4). À exceção do grupo controle (grupo I), que foi submetido apenas à aplicação de secretoma (60g de secretoma diluído em tampão fosfato-salina a 0,5%), todos os outros grupos foram submetidos à administração de 10g de veneno de Loxosceles intermedia, diluído em NaCl 0,9%, via intradérmica (ID) na região interescapular e, tratados 30 minutos após a injeção do veneno, da seguinte forma: grupo II (NaCl 0,9%, via ID); grupo III (60g de secretoma diluído em tampão fosfato-salina 0,5%, via ID) e, grupo IV (60g de secretoma diluído em tampão fosfato-salina 0,5% via endovenosa - EV). Os animais foram avaliados diariamente e realizados registros fotográficos em altura pré-definida de 30 cm para posterior análise da evolução da área da ferida por morfometria. Amostras de sangue foram coletadas imediatamente antes da aplicação do veneno (tempo 0) e 3, 9 e 15 dias após, para avaliação e monitoração de parâmetros hematológicos e bioquímicos séricos e plasmáticos. Após 15 dias os animais foram eutanasiados, submetidos a necropsia e amostras de pele ao redor da lesão foram coletadas para posterior análise histológica. Os resultados demonstraram que os animais do GI não apresentaram edema, eritema, halo ou necrose. No primeiro dia da injeção do veneno, após tratamentos com secretoma, os animais do GIII e GIV, apresentaram maior grau de edema, quando comparado aos animais do GII. Todavia, no 15ª dia de avaliação, o edema foi menor nos animais do GIII e GIV e, de forma inversa, maior no GI. O eritema foi observado nos grupos que receberam veneno de L. intermedia (GII, GIII e GIV), e comparativamente, no primeiro dia, nos grupos II e III foram similares entre si, e diferentes do GIV que apresentou menor diâmetro de eritema (p<0,05). O halo hemorrágico não foi observado no GI e nos animais que receberam veneno (GII, GIII e GIV), nos tempos 1 e 3, não houve diferença (p>0,05). Deve ser salientado que, no nono dia, somente nos animais do GIV, ainda havia halo hemorrágico. Todavia, macroscopicamente, no GIV, apenas um animal apresentou evolução para ferida dermonecrótica. Na avaliação microscópica, não foram observadas alterações na pele dos animais do GI, entretanto, apesar de todos animais que foram desafiados com o veneno, apresentarem alterações muito semelhantes, como necrose e infiltração heterofílica, no GIV, os animais apresentaram ativação fibroblástica, desenvolvimento precoce de tecido conjuntivo, neovascularização e reepitelização tecidual, conferindo alternativa comprovadamente eficaz em relação ao processo de cicatrização. Em relação a hematologia não houve alteração digna de nota e na bioquímica sérica, somente houve aumento na concentração de CK no tempo 3, nos grupos GIII e GIV e, posterior redução a partir do nono dia. Esses mesmos grupos também apresentaram aumento de LDH e ureia, porém os valores permaneceram dentro dos parâmetros fisiológicos para a espécie leporina. Conclui-se que a terapia com o secretoma pode ser utilizada na cicatrização da ferida dermonecrótica no loxoscelismo.

The objective was to evaluate the effects of the secretome, in dermonecrotic wounds in rabbits subjected to injection of Loxosceles intermedia. Sixteen male, adult, New Zealand rabbits, with a mean weight of 2.0 kg, were distributed in four groups (n=4). Except for the control group (group I), which was subjected only to the application of secretome (60g of secretome diluted in 0.5% phosphate-saline buffer), all other groups were subjected to the administration of 10g of L. intermedia venom, diluted in 0.9% NaCl, via intradermal (ID) in the interscapular region and, treated 30 minutes after the venom injection, as follows: group II (NaCl 0.9%, via ID); Group III (60g of secretome diluted in 0.5% phosphate-saline buffer, via ID) and, group IV (60g of secretome diluted in 0.5% phosphate-saline buffer intravenous - IV). Animals were evaluated daily and photographic records were taken at a predefined height of 30 cm for later analysis of the evolution of the wound area by morphometry. Blood samples were collected immediately before venom application (time 0) and 3, 9 and 15 days after, for evaluation and monitoring of hematological and serum and plasma biochemical parameters. After 15 days, the animals were euthanized, submitted to necropsy, and skin samples around the lesion were collected for subsequent histological analysis. The results showed that the animals in GI did not have edema, erythema, hemorrhagic halo or necrosis. On the first day of venom injection, after secretome treatments, the animals in GIII and GIV showed a more significant degree of edema, when compared to the animals in GII. However, on the 15th day of evaluation, the edema was lower in the animals of GIII and GIV, and conversely, higher in GI. Erythema was observed in the groups that received L. intermedia venom (GII, GIII and GIV), and comparatively, on day 1st, groups II and III were similar to each other, and different from GIV which showed smaller erythema diameter (p<0.05). The hemorrhagic halo was not observed in GI and in the animals that received venom (GII, GIII, and GIV), at times 1 and 3, there was no difference (p>0.05). It should be noted that on the 9th day, only in the animals of GIV, there was still a hemorrhagic halo. However, macroscopically, in GIV, only one animal showed evolution to a dermonecrotic wound. In the microscopic evaluation, no changes were observed in the skin of animals of GI, however, although all animals that were challenged with the venom presented very similar changes, such as necrosis and heterophilic infiltration, in GIV, the animals showed fibroblastic activation, early development of connective tissue, neovascularization, and tissue reepithelialization, conferring a proven effective alternative in relation to the healing process. Regarding hematology, there was no noteworthy change, and in biochemistry serum profile, there was only an increase in CK concentration at time 3, in groups GIII and GIV, and a subsequent reduction from day 9. These same groups also showed an increase in LDH and urea, but the values remained within the physiological parameters for the leporine species. It is concluded that secretome therapy can be used in dermonecrotic wound healing in loxoscelism.

CLINICAL AND LABORATORIAL EFFECTS AND IMMUNOGENICITY OF Loxosceles spp. VENOM PROTEINS IN HORSES PARTAKING IN ANTIVENOM PRODUCTION

O envenenamento por aranhas detém importância mundial no âmbito da saúde pública e alcança números cada vez mais significativos a cada ano. Acidentes envolvendo Loxosceles spp. detêm destaque nessa estatística, especialmente no Brasil. A soroterapia permanece sendo o único tratamento efetivo contra esse agravo e o seu processo produtivo enfrenta diversos problemas, uma vez que o mesmo é diretamente dependente da imunização de animais (principalmente cavalos) para o posterior processamento do plasma, o que possui implicâncias éticas referentes à bem estar animal. Dessa forma, o objetivo do presente trabalho é avaliar os efeitos clínicos causados pelo veneno de Loxosceles spp. em equinos submetidos a imunização para produção de antiveneno, assim como o de coelhos submetidos a um procedimento de imunização semelhante. No experimento 1, 11 equinos hígidos, nunca antes imunizados, foram avaliados em três momentos experimentais distintos: T0 (antes da imunização); T1 (após o primeiro ciclo de imunização); e T2 (após a primeira sangria comercial). Os animais foram avaliados clinicamente, submeteram-se à coleta de sangue e ao exame eletrocardiográfico (ECG). Achados clínicos de destaque recaíram sobre a presença de abscessos subcutâneos supurados atribuídos ao emprego de adjuvantes de Freund e tromboflebites devido às venopunções sistemáticas. Equinos em T2 apresentaram as alterações hematimétricas mais relevantes, com redução do hematócrito (PCV), contagem de células vermelhas (RBC) e de hemoglobina. Funções hepática e renal permaneceram normais. A hiperproteinemia diagnosticada foi atribuída ao aumento do teor de globulinas, O ECG ilustrou arritmias em poucos equinos em T2, como bloqueio átrio-ventricular de segundo grau (BAV-2) e aumento em amplitude de ondas T e R. No experimento 2, sete equinos, os quais já haviam sido submetidos à seis ou mais ciclos completos de imunização para produção de antiveneno, foram avaliados e denominados grupo imunizado (GI). Onze equinos, sob o mesmo manejo, foram dispostos como grupo controle (GC). GI apresentou menor PCV e RBC, apesar de mantê-los dentro dos índices de referência para a espécie. A função renal não se apresentou comprometida, mas as enzimas hepáticas apresentaram-se elevadas em relação ao GC, provavelmente devido ao alto requerimento de produção de imunoglobulinas. O ECG ilustrou anormalidades de traçado no GI, corroboradas com o aumento na enzima creatina quinase em sua fração MB (CK-MB). No experimento 3, 11 coelhos machos Nova Zelândia foram utilizados. Cinco foram alocados no grupo controle (GC), recebendo adjuvante (montanide) e tampão salina-fosfato. Os seis coelhos restantes receberam 21g de veneno de Loxosceles spp. empregando como adjuvante o montanide (GV). Após cinco ciclos de imunização, um desafio foi realizado com 7g de veneno de L. intermedia, com posterior mensuração das lesões dermonecróticas. Os coelhos foram eutanasiados e amostras de órgãos e tecidos foram coletados para análise histopatológica. Nenhum parâmetro sanguíneo referente ao eritrograma mostrou-se alterado significativamente, mas a contagem total de leucócitos foi maior em GV, corroborando com a ação quimiotáxica neutrofílica já descrita para o veneno. Fígado e rins mantiveram a função preservada de acordo com a análise de bioquímica sérica. O ECG não demonstrou alterações entre grupos e momentos experimentais. A histopatologia elucidou uma tendência do veneno loxoscélico em induzir cardio/reno e hepatotoxicidade, tanto de forma direta como indireta. Conclui-se que apesar de o veneno possui ação cardio/reno e hepatotóxica, ambos coelhos e equinos mantiveram-se em condições clínicas adequadas. Uma maior deve ser dada aos equinos no período pós-sangria, devendo os mesmos serem acompanhados. A reinfusão de papa de hemácias é uma sugestão para esses animais.

Spider envenomation holds worldwide importance in public health and reaches significant and increasing numbers every year. Loxosceles spp. plays an important role in these statistics, especially in Brazil. Antivenom remains the only effective treatment against this ailment, and its production faces several hindrances since it depends on immunizing animals (mainly horses) and later processing their plasma, which leads to increasing animal welfare concerns. Therefore, the aim of this study is to evaluate the general clinical effects of Loxosceles spp. venom in horses that underwent immunization protocols for loxoscelic antivenom procurement, as well as those in rabbits who underwent a shorter immunization protocol. In experiment 1, eleven healthy horses, never immunized, were evaluated on three different periods: T0 before immunization; T1 after their first loxoscelic antivenom immunization; and T2 after their first commercial bleeding. Horses were clinically evaluated, sampled for blood, and underwent electrocardiographic (ECG) recordings. Significant clinical findings were the several suppurated subcutaneous abscesses due to the use of Freunds adjuvants and thrombophlebitis due to systematic venipunctures for commercial bleeding procedures. Horses at T2 presented the most blood alterations, including reduced packed cell volume (PCV), red blood cells (RBC), and hemoglobin. Liver and renal functions were unaffected. Hyperproteinemia occurred due to increase in globulin levels. ECG showed arrhythmias in few horses in T2, such as second-degree atrioventricular block (AVblock), as well as an increase in T and R waves. On experiment 2, seven crossbred horses, who had partaken in six or more complete antivenom-producing cycles, were used and established as the immunized group (IG). Eleven horses, under the same handling and general management, were established as the control group (CG). IG presented lower red blood cell count and packed cell volume, despite keeping values within inferior limits for the species. Renal function was not impaired, but liver-related enzymes were higher when compared to CG, probably due to liver exertion from immunoglobulin synthesis. ECG showed some abnormalities in IG, corroborated by increase in creatine kinase/isoenzyme MB fraction (CK-MB). In experiment 3, eleven male New Zealand rabbits were used. Five were allocated as a control group (CG), that received adjuvant (montanide) and phosphate-buffer saline. The six remaining rabbits received 21g of Loxosceles spp. venom using montanide as adjuvant (VG). After five immunization cycles, a trial with 7g of L. intermedia was performed, and dermonecrotic lesions were measured. Rabbits were then euthanized, and their organs harvested for histopathology analysis. No erythrocyte-related parameter showed significance, but white blood cell count was higher in VG, corroborating with venoms neutrophil chemotaxis. Liver and kidney functions were also preserved according to blood biochemical panel. ECG showed no alteration between experimental groups and evaluation periods. Histopathology showed a tendency for loxoscelic venom to produce a direct and indirect cardiotoxicity, renal toxicity, and hepatotoxicity. It was concluded that despite loxoscelic venom exerts cardiotoxicity, hepatotoxicity, and renal toxicity, both rabbits and horses were kept within acceptable clinical conditions. A crucial point to be highlighted is bleeding for industrial antivenom production, when horses should receive greater attention and perhaps reinfusion with suspended red blood cells.