RESUMO
The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production.
Assuntos
Venenos Elapídicos/química , Elapidae , Epitopos de Linfócito B/genética , Fosfolipases A2/genética , Toxinas Biológicas/genética , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Brasil , Técnicas de Química Sintética , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/metabolismo , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/genética , Peptídeos/imunologiaRESUMO
In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production.
Assuntos
Antivenenos/imunologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Venenos de Aranha/química , Animais , Antivenenos/farmacologia , Western Blotting , Brasil , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Cavalos , Testes de Neutralização , Peru , Diester Fosfórico Hidrolases/análise , Especificidade da Espécie , Venenos de Aranha/enzimologiaRESUMO
This manuscript describes the general biochemical properties and immunological characteristics of Peruvian spider Loxosceles laeta venom (PLlv), which is responsible for the largest number of accidents involving venomous animals in Peru. In this work, we observed that the venom of this spider is more lethal to mice when compared with L. laeta venom from Brazil (BLlv). The LD50 of PLlv was 1.213 mg/kg when the venom was intradermally injected. The venom displayed sphingomyelinase activity and produced dermonecrotic, hemorrhagic and edema effects in rabbits. 2-D SDS-PAGE separation of the soluble venoms resulted in a protein profile ranging from 20 to 205 kDa. Anti-PLlv and anti-BLlv sera produced in rabbits and assayed by ELISA showed that rabbit antibodies cross-reacted with PLlv and BLlv and also with other Brazilian Loxosceles venoms. Western blotting analysis showed that bands corresponding to 25-35 kDa are the proteins best recognized in every Loxosceles spp venoms analyzed. The immunized rabbits displayed protective effect after challenge with PLlv and BLlv. In vitro assays with horse anti-loxoscelic antivenoms produced in Brazil and Peru demonstrated that these commercial antivenoms were efficient to inhibit the sphingomyelinase activity of PLlv and BLlv.
Assuntos
Antivenenos/farmacologia , Diester Fosfórico Hidrolases/toxicidade , Venenos de Aranha/toxicidade , Aranhas/metabolismo , Animais , Western Blotting , Brasil , Reações Cruzadas , Edema/induzido quimicamente , Edema/patologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Cavalos , Imunização , Dose Letal Mediana , Masculino , Camundongos , Testes de Neutralização , Peru , Coelhos , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismoRESUMO
A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.
Assuntos
Proteínas Recombinantes de Fusão/química , Venenos de Aranha/química , Animais , Anticorpos Neutralizantes/imunologia , Antivenenos/imunologia , Edema/imunologia , Edema/prevenção & controle , Epitopos de Linfócito B/genética , Hemorragia/imunologia , Hemorragia/prevenção & controle , Necrose/imunologia , Necrose/prevenção & controle , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Pele/imunologia , Pele/patologia , Esfingomielina Fosfodiesterase/química , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/imunologia , Venenos de Aranha/genética , Venenos de Aranha/imunologia , AranhasRESUMO
This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD50 determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.
Assuntos
Venenos de Escorpião/química , Venenos de Escorpião/imunologia , Venenos de Escorpião/intoxicação , Animais , Western Blotting , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Hialuronoglucosaminidase/metabolismo , Soros Imunes/imunologia , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos C57BL , Peru , Fosfolipases A2/metabolismo , Proteólise , Coelhos , Ratos , Ratos Wistar , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32 percent) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.
Assuntos
Animais , Masculino , Ratos , Antitireóideos/farmacologia , Fator B do Complemento/metabolismo , Via Alternativa do Complemento/efeitos dos fármacos , Propiltiouracila/farmacologia , Tiroxina/sangue , Tri-Iodotironina/sangue , Via Alternativa do Complemento/fisiologia , Hipertireoidismo/sangue , Hipertireoidismo/induzido quimicamente , Hipertireoidismo/imunologia , Hipotireoidismo/sangue , Hipotireoidismo/induzido quimicamente , Hipotireoidismo/imunologia , Medições Luminescentes , Ratos Wistar , TireoidectomiaRESUMO
Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32%) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.
Assuntos
Antitireóideos/farmacologia , Fator B do Complemento/metabolismo , Via Alternativa do Complemento/efeitos dos fármacos , Propiltiouracila/farmacologia , Tiroxina/sangue , Tri-Iodotironina/sangue , Animais , Via Alternativa do Complemento/fisiologia , Hipertireoidismo/sangue , Hipertireoidismo/induzido quimicamente , Hipertireoidismo/imunologia , Hipotireoidismo/sangue , Hipotireoidismo/induzido quimicamente , Hipotireoidismo/imunologia , Medições Luminescentes , Masculino , Ratos , Ratos Wistar , TireoidectomiaRESUMO
Propylthiouracil (PTU) was employed in a fixed quantity to evaluate the effect of the period of treatment with this drug on the antibody response to sheep red blood cells in Wistar rats. Animals were treated for 8, 16, 30 and 90 days by intragastric route with 5 mg/day, and immunized 24 h following the end of treatment; other groups were treated for 21 days, and immunized on the 17th day of treatment. Animals were sacrificed 5 or 6 days following immunization; the primary response was evaluated by the number of plaque-forming spleen cells and in some cases also by enzyme-linked immunosorbent assay (ELISA). Secondary response of PTU-treated rats, immunized and boostered after 15 days, was evaluated by ELISA 3 days following the booster. RIA performed the measurement of T3 in animals treated for 16 days, immunized, and sacrificed after 1, 2, 3, 4 and 5 days following immunization. Results showed that treatment for 8 and 16 days increased production of antibodies, and for 30 and 90 days decreased this response. Thus, according to the period of treatment, the same dose of PTU stimulates or suppresses the antibody response. This biphasic effect of a single dose of PTU was independent of alterations of serum levels of T3 during the buildup of the immune response. These results contributes towards the understanding of the literature controversy regarding the effects of this drug on the immune response, and could be of interest for studies involving autoimmune processes in thyroid.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Antitireóideos/farmacologia , Propiltiouracila/farmacologia , Administração Oral , Animais , Antitireóideos/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Técnica de Placa Hemolítica , Imunização , Masculino , Propiltiouracila/administração & dosagem , Ratos , Ratos Wistar , Baço/imunologia , Fatores de Tempo , Tri-Iodotironina/sangueRESUMO
The effect of propylthiouracil (PTU) on the lytic activity of complement in rat serum was investigated in vivo. Rats (180+/-10 g) were treated daily by gavage with PTU doses of 1-50 mg/200 g body weight for time intervals ranging from 1 to 30 days. Serum classical pathway (CP) and alternative pathway (AP) activities were determined 24 h after the last dose. A single dose of 50 mg/200 g body weight was administered to additional groups and the animals were sacrificed after periods of 1-48 h. The results showed a relatively small reduction ( approximately 30%) in CP activity, evident only in animals treated with 50 mg of PTU for three weeks. However, a clear and opposite effect of PTU, an increase in lytic activity reaching values up to 180% of controls, was observed on AP activity. This effect was seen at all PTU doses used, and occurred within 4 days of treatment with the highest dose. Maximum activity was observed at intermediate intervals, depending on the PTU dose, with a return to control levels occurring after the longer periods of treatment. The lytic activity of serum from animals treated with a single PTU dose of 50 mg/200 g body weight and sacrificed 1-48 h after dosing did not differ from controls. Serum levels of thyroid hormone (triiodo L-thyronine, T3, and thyroxine, T4) were determined in representative groups of treated animals (injected with 5 mg of PTU/200 g body weight/day). These were either undetectable or considerably lower than those of controls. The serum PTU levels of these rats increased for up to 22 days, reaching values of 2-4 microg/ml.PTU is described in the literature as a modulator of both cellular immune responses and antibody production. Upon complement activation fragments of complement components bind to immune complexes and to specific receptors on cells of the immune system. Thus, alteration in AP activity caused by PTU treatment suggests a possible mechanism by which the drug exerts its modulatory effect. Increased complement AP activity might affect events as antigen presentation and hence the onset and course of the immune response.