RESUMO
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.
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Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and exudate samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. Envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP.
Assuntos
Venenos de Aranha , Aranhas , Animais , Colorimetria , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Diester Fosfórico Hidrolases/genética , Coelhos , Sensibilidade e Especificidade , Venenos de Aranha/genética , Venenos de Aranha/toxicidade , Aranhas/genéticaRESUMO
Viral inactivation for antibody induction purposes, among other applications, should ensure biosafety, completely avoiding the risk of infectivity, and preserving viral immunogenicity. ß-propiolactone (BPL) is one of the most used reagents for viral inactivation, despite its high toxicity and recent difficulties related to importation, experienced in Brazil during the SARS-CoV-2 pandemic. In this context, the main objectives of this work were to test different inactivation procedures for SARS-CoV-2 and to evaluate the induction of neutralizing antibodies in mice immunized with antigenic preparations obtained after viral treatment with formaldehyde (FDE), glutaraldehyde (GDE), peroxide hydrogen (H2O2), as well as with viral proteins extract (VPE), in parallel with BPL. Verification of viral inactivation was performed by subsequent incubations of the inactivated virus in Vero cells, followed by cytopathic effect and lysis plaques observation, as well as by quantification of RNA load using reverse transcription-quantitative real time polymerase chain reaction. Once viral inactivation was confirmed, cell culture supernatants were concentrated and purified. In addition, an aliquot inactivated by BPL was also subjected to viral protein extraction (VPE). The different antigens were prepared using a previously developed microemulsion as adjuvant, and were administered in a four-dose immunization protocol. Antibody production was comparatively evaluated by ELISA and Plaque Reduction Neutralization Tests (PRNT). All immunogens evaluated showed some level of IgG anti-SARS-CoV-2 antibodies in the ELISA assay, with the highest levels presented by the group immunized with FDE-inactivated viral antigen. In the PRNT results, except for VPE-antigen, all other immunogens evaluated induced some level of neutralizing anti-SARS-CoV-2 antibodies, and the FDE-antigen stood out again with the most expressive values. Taken together, the present work shows that FDE can be an efficient and affordable alternative to BPL for the production of inactivated SARS-CoV-2 viral antigen.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Antivirais , Antígenos Virais , Chlorocebus aethiops , Modelos Animais de Doenças , Peróxido de Hidrogênio , Camundongos , Células VeroRESUMO
The recent emergence of a coronavirus (SARS-CoV-2), first identified in the Chinese city of Wuhan in December 2019, has had major public health and economic consequences. Although 61,888 confirmed cases were reported in Brazil by 28 April 2020, little is known about the SARS-CoV-2 epidemic in this country. To better understand the recent epidemic in the second most populous state in southeast Brazil - Minas Gerais (MG) - we sequenced 40 complete SARS-CoV-2 genomes from MG cases and examined epidemiological data from three Brazilian states. Both the genome analyses and the geographical distribution of reported cases indicate for multiple independent introductions into MG. Epidemiological estimates of the reproductive number (R) using different data sources and theoretical assumptions suggest the potential for sustained virus transmission despite a reduction in R from the first reported case to the end of April 2020. The estimated date of SARS-CoV-2 introduction into Brazil was consistent with epidemiological data from the first case of a returned traveller from Lombardy, Italy. These findings highlight the nature of the COVID-19 epidemic in MG and reinforce the need for real-time and continued genomic surveillance strategies to better understand and prepare for the epidemic spread of emerging viral pathogens..
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Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Genoma Viral , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Adulto , Idoso , Brasil/epidemiologia , COVID-19 , Feminino , Geografia , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , SARS-CoV-2 , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
Epitope identification is essential for developing effective antibodies that can detect and neutralize bioactive proteins. Computational prediction is a valuable and time-saving alternative for experimental identification. Current computational methods for epitope prediction are underused and undervalued due to their high false positive rate. In this work, we targeted common properties of linear B-cell epitopes identified in an individual protein class (metalloendopeptidases) and introduced an alternative method to reduce the false positive rate and increase accuracy, proposing to restrict predictive models to a single specific protein class. For this purpose, curated epitope sequences from metalloendopeptidases were transformed into frame-shifted Kmers (3 to 15 amino acid residues long). These Kmers were decomposed into a matrix of biochemical attributes and used to train a decision tree classifier. The resulting prediction model showed a lower false positive rate and greater area under the curve when compared to state-of-the-art methods. Our predictions were used for synthesizing peptides mimicking the predicted epitopes for immunization of mice. A predicted linear epitope that was previously undetected by an experimental immunoassay was able to induce neutralizing-antibody production in mice. Therefore, we present an improved prediction alternative and show that computationally identified epitopes can go undetected during experimental mapping.
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Anticorpos Neutralizantes/biossíntese , Biologia Computacional/métodos , Epitopos de Linfócito B/imunologia , Venenos de Serpentes/imunologia , Algoritmos , Sequência de Aminoácidos , Aminoácidos/química , Animais , Árvores de Decisões , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Feminino , Imunização , Metaloproteases/metabolismo , Camundongos Endogâmicos BALB C , Modelos Moleculares , Peptídeos/química , Curva ROC , Reprodutibilidade dos TestesRESUMO
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.
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Animais , Coelhos , Picada de Aranha/terapia , Dapsona/uso terapêutico , Células-Tronco Mesenquimais , Aranha Marrom Reclusa , Modelos AnimaisRESUMO
BACKGROUND: Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage. METHODS AND FINDINGS: In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay. CONCLUSION: Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.
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Antivenenos/imunologia , Antivenenos/farmacologia , Elapidae/imunologia , Venenos de Serpentes/imunologia , Tecnologia Farmacêutica/métodos , Vacinas de DNA/imunologia , Animais , Modelos Animais de Doenças , Elapidae/genética , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Feminino , Camundongos Endogâmicos BALB C , Testes de Neutralização , Mordeduras de Serpentes/terapia , Venenos de Serpentes/genética , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
Animal venoms are a mixture of bioactive compounds produced as weapons and used primarily to immobilize and kill preys. As a result of the high potency and specificity for various physiological targets, many toxins from animal venoms have emerged as possible drugs for the medication of diverse disorders, including cardiovascular diseases. Captopril, which inhibits the angiotensin-converting enzyme (ACE), was the first successful venom-based drug and a notable example of rational drug design. Since captopril was developed, many studies have discovered novel bradykinin-potentiating peptides (BPPs) with actions on the cardiovascular system. Natriuretic peptides (NPs) have also been found in animal venoms and used as template to design new drugs with applications in cardiovascular diseases. Among the anti-arrhythmic peptides, GsMTx-4 was discovered to be a toxin that selectively inhibits the stretch-activated cation channels (SACs), which are involved in atrial fibrillation. The present review describes the main components isolated from animal venoms that act on the cardiovascular system and presents a brief summary of venomous animals and their venom apparatuses.
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Fármacos Cardiovasculares/química , Fármacos Cardiovasculares/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Sistema Cardiovascular/efeitos dos fármacos , Descoberta de Drogas , Peçonhas/química , Peçonhas/uso terapêutico , Sequência de Aminoácidos , Animais , Bradicinina/metabolismo , Fármacos Cardiovasculares/farmacologia , Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/fisiopatologia , Descoberta de Drogas/métodos , Humanos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Sistema Renina-Angiotensina/efeitos dos fármacos , Peçonhas/farmacologiaRESUMO
BACKGROUND: Scorpionism is a public health problem in Brazil, and Tityus serrulatus (Ts) is primarily responsible for severe accidents. The main toxic components of Ts venom are low-molecular-weight neurotoxins; however, the venom also contains poorly characterized high-molecular-weight enzymes. Hyaluronidase is one such enzyme that has been poorly characterized. METHODS AND PRINCIPAL FINDINGS: We examined clones from a cDNA library of the Ts venom gland and described two novel isoforms of hyaluronidase, TsHyal-1 and TsHyal-2. The isoforms are 83% identical, and alignment of their predicted amino acid sequences with other hyaluronidases showed conserved residues between evolutionarily distant organisms. We performed gel filtration followed by reversed-phase chromatography to purify native hyaluronidase from Ts venom. Purified native Ts hyaluronidase was used to produce anti-hyaluronidase serum in rabbits. As little as 0.94 µl of anti-hyaluronidase serum neutralized 1 LD50 (13.2 µg) of Ts venom hyaluronidase activity in vitro. In vivo neutralization assays showed that 121.6 µl of anti-hyaluronidase serum inhibited mouse death 100%, whereas 60.8 µl and 15.2 µl of serum delayed mouse death. Inhibition of death was also achieved by using the hyaluronidase pharmacological inhibitor aristolochic acid. Addition of native Ts hyaluronidase (0.418 µg) to pre-neutralized Ts venom (13.2 µg venom+0.94 µl anti-hyaluronidase serum) reversed mouse survival. We used the SPOT method to map TsHyal-1 and TsHyal-2 epitopes. More peptides were recognized by anti-hyaluronidase serum in TsHyal-1 than in TsHyal-2. Epitopes common to both isoforms included active site residues. CONCLUSIONS: Hyaluronidase inhibition and immunoneutralization reduced the toxic effects of Ts venom. Our results have implications in scorpionism therapy and challenge the notion that only neurotoxins are important to the envenoming process.
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Hialuronoglucosaminidase/imunologia , Venenos de Escorpião/imunologia , Escorpiões/genética , Sequência de Aminoácidos , Animais , Anticorpos/sangue , Sequência de Bases , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/genética , Imunoensaio , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Venenos de Escorpião/química , Venenos de Escorpião/enzimologia , Venenos de Escorpião/genética , Escorpiões/química , Alinhamento de SequênciaRESUMO
Scorpion stings cause human fatalities in numerous countries. Serotherapy is the only specific means to try to circumvent the noxious effects of venom toxins. TsNTxP is a natural anatoxin from the venom of the scorpion Tityus serrulatus that may be useful to raise therapeutic anti-venom sera. Linear epitopes recognized by anti-TsNTxP antibodies have previously been mapped. Here, we attempted to identify discontinuous epitopes in TsNTxP since neutralizing epitopes are often associated with such complex entities. One hundred and fifty-three octadecapeptides with the general formula (P1)-(Gly-Gly)-(P2) were synthesized by the Spot method on cellulose membranes. P1 and P2 were octapeptides from the TsNTxP N-terminal and C-terminal sections, respectively. Each sequence of eight amino acids was frameshifted in turn by three residues, in order to cover TsNTxP entire sequence. Binding of neutralizing anti-TsNTxP rabbit antibodies to spotted peptides revealed GREGYPADGGGLPDSVKI as the more reactive peptide sequence. This epitope was made from the first eight residues of the protein (GREGYPAD) and from residues 47 to 54 (GLPDSVKI) of the C-terminal part of TsNTxP. BALB/c mice were immunized with synthetic GREGYPADGGGLPDSVKI peptide conjugated to ovalbumin. One week after the last immunization, in vivo protection assays showed that immunized mice could resist a challenge by an amount of T.serrulatus whole venom equivalent to 1.75 LD(100), a dose that killed all control non-immune mice. Based on molecular models of TsNTxP and related Tityus toxins, we found that the above peptide matches with a discontinuous epitope, well exposed at the toxin molecular surface which contains residues known to be important for the bioactivity of toxins.
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Antitoxinas/imunologia , Epitopos/imunologia , Oligopeptídeos/imunologia , Venenos de Escorpião/antagonistas & inibidores , Venenos de Escorpião/imunologia , Venenos de Escorpião/toxicidade , Animais , Antitoxinas/uso terapêutico , Mordeduras e Picadas/imunologia , Mordeduras e Picadas/prevenção & controle , Epitopos/farmacologia , Humanos , Camundongos , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Coelhos , EscorpiõesRESUMO
In this work, polyclonal antibodies anti-human Factor IX were produced in New Zealand rabbits by immunization with commercial pure human FIX (hFIX) (Octanyne®, Octapharma, USA). The serum containing immunoglobulins anti-hFIX was useful to detect hFIX antigen in human plasma fractions submitted to anionic exchange chromatographic process and with a large yield. Immunoassays (ELISA) using bovine serum albumin, trypsin and peptides generated by cleavage assays with trypsin as digestion enzyme was performed and revealed adequate specificity of the polyclonal antibodies produced.
Neste trabalho foram produzidos anticorpos policlonais anti-fator IX humano em coelhos New Zealand imunizados com FIX humano (hFIX) comercial puro (Octanyne®, Octapharma, EUA). O soro contendo as imunoglobulinas anti-hFIX foi útil para a detecção do antígeno hFIX em frações do plasma humano submetido a cromatografia de troca iônica. Imunoensaios (ELISA) usando soro-albumina bovina, tripsina e peptídeos gerados por ensaios de clivagem com tripsina com enzima de digestão foram realizados e revelaram especificidade adequada dos anticorpos policlonais produzidos.